ScienceSampler

Actin-dependent nuclear movement in neurons

2009-10-12T08:08:00.003-04:00

I know it's been a while since I post here. But I cannot resist to quickly tell you about this paper:

"Actomyosin Is the Main Driver of Interkinetic Nuclear Migration in the Retina",
Caren Norden, Stephen Young, Brian A. Link and William A. Harris
Cell, Volume 138, Issue 6, 18 September 2009, Pages 1195-1208

Not only the authors show very nicely that interkinectic movement is actomyosin-dependent (while until now the model suggest an microtubule-dependent movement), but most interesting is the analogy they describe in the discussion:

"....in our opinion, IKNM is reminiscent of movements of people at a crowded party held in a room with a bar at one end. When people get thirsty, they go to the bar to get a drink. Then they move or are pushed away to make room for others at the bar. Between drinks the partygoers jostle around the room, but when they get thirsty again, they return to the bar in a fast and directed manner."

Great analogy!

Dynein and neuronal dendrites

2008-10-30T11:00:00.003-04:00

Two interesting papers were published in Nature Cell Biology:

Zheng, Y. et al. Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons. Nat Cell Biol 10, 1172-1180(2008).

Satoh, D. et al. Spatial control of branching within dendritic arbors by dynein-dependent transport of Rab5-endosomes. Nat Cell Biol 10, 1164-1171(2008).

Both papers identified that mutations in dynein subunits in Drosophila neurons (da neurons) reduce the extension of dendrites and their branching occurs more proximal to the cell body.

Curiously, Zheng et al observed that in dynein mutants, golgi apparatus markers (ManII-GFP) are localized in the axons, in contrast with wild-type neurons. Furthermore, using two interesting probes for plus-ends (EB1-GFP) and minus-ends (Nod-B-gal, which comprise of the Nod motor domain fused to kinesin-1 coiled-coiled domain, followed by B-galactosidase), these authors show that microtubule organization is changed. In WT axons, microtubules are organized with their plus-ends pointing away from the cell body. In dynein mutants, they observed some microtubules pointing in the opposite direction.

How dynein regulates these processes is still unknown so we will have to wait to find that out.

Robots and wound healing

2008-09-11T16:33:00.009-04:00

OK, so the title is a bit of a stretch. But the paper is kind of about wound healing. And they use a robot. And given the current political climate of massive misrepresentation, it's not all that bad. At least I didn't call it Lipstick on a Pig.

Identification of genes that regulate epithelial cell migration using an siRNA approach
Simpson et al., Nature Cell Biology, September 2008.

As part of the efforts of the multi-laboratory Cell Migration Consortium, the Brugge lab published the results of an siRNA screen to identify genes involved in epithelial cell migration. Although not a genome-wide screen, they used libraries of all known human kinases (576) and phosphatases (192), as well as a custom library targeting 313 genes with known or predicted roles in migration or adhesion.

The technique they used is elegantly simple, based on the "scratch-wound" assay performed in numerous laboratories. Basically, cells are grown to confluency, and then a region of cells in the middle is "scratched" away. Cells will then migrate into the scratched area in an attempt to close the "wound". The Brugge laboratory grew human MCF-10A (breast epithelial) cells to confluency, incubated cells with the siRNA, then used a robotic pinning device to create predictable scratch in the monolayer. Cells were allowed to migrate into the wound for 12 hours and the size of the resulting wound was compared to control. siRNA that caused increased rates of migration had smaller wounds, while siRNA that caused lower rates of migration had larger wounds.

Unlike many other screening papers, the final product of this paper was not simply a list of "hits" - positive results from the screen - with a bit of follow-up on their favorite hit. Instead, the group repeated the experiments of all hits and performed time-lapse, video microscopy of the 12 hour migration period for each. This allowed them to begin to parse the mechanistic basis for the change in migration rate for each. For example, knocking down p120-catenin and MLCK caused similar increases in migration rate (as assayed by the extent to which the wound closed). However, time-lapse microscopy showed that the increased rates were for completely different reasons; p120-catenin knockdown decreased cell-cell adhesion allowing cells to migrate more freely into the wound, while knockdown of MLCK had normal adhesion but an increase in wound-directed protrusion.

All hits from the inital screen were measured on the basis of four parameters:
1) extent and nature of adhesion impairment
2) directionality of movement
3) alterations in cell polarity
4) leading edge morphology and dynamics

As an added bonus, all of the videos from their analysis, as well as their annotation of each parameter can be found at www.cellmigration.org/pubs/wound_rnai.htm.

Seeing three colors in one channel

2008-09-11T07:50:00.006-04:00

Sometimes you would like to be able to image two or more different proteins in the same channel, and be able to separate them? For instance, wouldn't be great to see GFP-actin and GFP-mitochondria in separate channels? That is now possible with the publication of two new reversible switchable fluorescent proteins (RSFPs).

Nature Biotechnology 26, 1035 - 1040 (2008)| doi:10.1038/nbt.1493
Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopyMartin Andresen¹, Andre C Stiel¹, Jonas Fölling¹, Dirk Wenzel², Andreas Schönle¹, Alexander Egner¹, Christian Eggeling¹, Stefan W Hell¹ & Stefan Jakobs¹

What is a reversible switchable fluorescent protein (RSFP)? These fluorescent proteins have excitation and emission properties similar to GFP, but they can be turned ON and OFF reversibly using a lower wavelength light source. Up to now all the RSFP identified, such as Drompa and rsFastLime (Drompa-Val157Gly), are turned OFF with irradiation of blue light (around 500nm) and turned ON with UV light (405 nm).
In this paper the authors describe a new variant of rsFastLime that behaves inversily regarding turning ON and OFF. rsFastLime is turned OFF with UV light and turned ON with blue light. they called it "Padron".

Using this two fluorofores (rsFastLime and Padron) to label mitochondria and actin cable in yeast, they were able to image the dynamics of both these structures over time, using a single "GFP fluorescent channel". They achieved this by alternatively turning ON and OFF rsFastLime and Padron (see image).

The other advantage of distinguishing two proteins in the same imaging channel is to avoid chromatic aberrations present in the different microscope components. This is particulary important when imaging at nanoscopic scale, as shown in this paper.

In addition, the authors identified another mutant with similar ON-OFF properties of rsFastLime, but with a broader absorption peak towards the UV. This allow them to excite and detect this new fluorofore (called bsDronpa) with UV light even in the presence of active rsFastLime. Furthermore, bsDronpa has better photobleaching properties than EBFP2, making it a good alternative for a UV-exciting genetically encoded fluorofore.

Overall, these new fluorofores are very usefull addition to the growing list of genetically encoded fluorofores.

actin, actin, actin

2008-03-04T12:02:00.001-05:00

here is a nice review on actin role at the plasma membrane. For those of you who are not familiar with this protein and what it can do at the plasma membrane, I strongly recommend reading this review. Even if you don't read it, keep it in mind because it is a good source of definitions such as lamellipodia and lamella. you never know the day you will need to find out that these are two different structures...sometimes.

The many faces of actin: matching assembly factors with cellular structures

Ekta Chhabra and Henry Higgs

Nat Cell Biol 9 (10), 1110-21 (Oct 2007)

info:doi/10.1038/ncb1007-1110

The dark side...

2008-01-30T08:55:00.000-05:00

Nothing like a day on the uScope (dark) room stuck between nuclei chasing and wound scannings to get one’s readings up to date. Today I`ve come across two interesting reports on the other (also dark and increasingly visible) side of science:

1 – How deep is the Impact?
A recent JCB editorial [1] was dedicated to the infamous black box of publishing science - the Thomson/ISI impact factor (IF). Debuting with an excellent sum up of the “IF curiosities and peculiarities”, editors report on how they (Rockefeller University Press) decided to buy the data from Thomson Scientific concerning their three journals (JEM, JCB, JGP) to do the simple math of impact factors… Guess what? The results did not match the published impact factors!
From wrong article-type designations to incorrect numbers of citations, it appears that anything goes… Confronted with these results, Thomson replied that the database they provided was a different “version” than the one used for JCR… A new database was provided and still… 2+2=5. Bottom line, if authors provide unreliable data to publishers, their articles are not accepted or can be retracted. So why should they be judged by an “irreproducible factor”(if)? (Might just be because the funding guys never read these editorials…)
*Thomson Scientific has issued an official reply [2] to this editorial and JCB editors have further commented [3] on it.

1. Rossner, M., E. Hill, and H. Van Epps. 2007. Show me the data. J. Cell Biol. 179:1091–1092 info:doi/10.1083/jcb.200711140
2. Pendlebury, D.A. 2007. Article Titled "Show me the Data", Journal of Cell Biology, Vol. 179, No. 6, 1091–1092, 17 December 2007 is Misleading and Inaccurate. http://scientific.thomson.com/citationimpactforum/8427045/ (accessed January 4, 2008).
3. Rossner, M., Van Epps, H., Hill, E. (2008). Irreproducible results: a response to Thomson Scientific. J. Cell Biol. 180: 254-255 info:doi/10.1083/jcb.200801036

2 – Seeing double…
Picking up on that last sentence, not always are the rotten apples left out. Claims of article duplication (either by the same authors or others) or plagiarism have increased in recent years. But are scientists really publishing more duplicate papers? In a commentary to Nature [1], Errami and Garner report on how they used eTBLAST, a freely available text similarity software they created, to probe a subset of more than 62.000 Pubmed abstracts from the last 12 months for possible duplicates. The resulting 421 hits were deposited online in Déjà vu, with a rate of false positives estimated to 1%. Manual evaluation of the results was made difficult in many cases due to the unavailability of full-text (for example articles published by the same authors in non-English, national level journals). Extrapolating this number to the whole Pubmed database size would give 117,500 duplicates with the same authors, against the 739 records currently marked as duplicate in Pubmed!! Expanding their approach, the authors have now come to approximately 70,000 candidate duplicates on their database. Although these numbers must be seen with caution and distinctions between duplications by the same authors (legitimate or elegitimate) from pure plagiarism (that was rarely detected) must be made, they do leave a clear message!
The steady increase on the number of publications for the past few years constitutes a growing opportunity for this practice and no country appears to lead the way, since the number of duplicates was roughly proportional to the countries’ Pubmed contribution. It is up to publishers and also authors to fight this apparently growing epidemic…

1.Mounir Errami and Harold Garner, “A Tale of Two Citations,” Nature 451, no. 7177 (January 24, 2008). info:doi/10.1038/451397a

de novo formation of centrosomes

2007-05-23T16:43:00.000-04:00

A big question in cell biology has been if centrioles can be formed de novo or they required a template. A recent paper in Science from the Glover's and Bettencourt-Dias's labs addressed this controversy.

Revisiting the Role of the Mother Centriole in Centriole Biogenesis
A. Rodrigues-Martins, M. Riparbelli, G. Callaini, D. M. Glover, M. Bettencourt-Dias

Science 316 (5827), 1046-50 (18 May 2007)

info:doi/10.1126/science.1142950

In Drosophila, the unfertilized egg does not contain centrioles since they are ejected during oocyte formation. Thus, the centrioles are provided by the sperm after fertilization. What the authors found in this study was that overexpression of SAK/PLK4 induced an abnormal number of centrioles in the eggs, even in unfertilized eggs. So, de novo formation of centrioles can occur and it is triggered by SAK kinase.

Interestingly the authors went further on their study and identified downstream players. SAK homolog in C. Elegans is Zyg-1 which has been sh own to be involved in centriole formation in a SAS-5 and SAS-6-dependent manner. The authors showed that the same players act downstream of SAK in the formation of centrioles in the Drosophila egg.

I wounder what happen if SAK is expressed in plant cells since these cells don't have centrioles!
Also, It would be cool to rescue flies without centrioles, by overexpression of SAK...

note: this work was also commented in the daily transcript

Organizing PDF papers in your computer using iTunes

2007-04-22T06:30:00.000-04:00

This post is a little different from all the previous posts. I though of sharing my new experience of using iTunes to organize the PDF papers in my computer. This should work both on Macs and PCs, although I only tested in PCs.

It has been a long time since I've been trying to find a way to organize the PDFs in my computer. I want to find the PDF file of papers stored in my computer in a fast and accurate way. And also to quickly and efficiently file a PDF after download. Initially, I had my papers organized in folders where each folder corresponds to a theme. But I quickly started having the problem of saving the same paper in two folders and even sometimes in the same folder. My other issue was to find papers. When I was looking for a paper from a certain author, published in year X and in journal Y, I always ran into problems. Even If I named the file with the author and journal name, and publication year, sometimes I couldn't find the paper. Also, I could not sort the papers by year or by author.
I tried bibliographic software such as endnote and reference manager but I hate their search interface and, for instance, I never found a way of setting a column with the last author.

So, the solution I found was to use iTunes to organize my PDFs. Why iTunes? Well, it turns out that it is a great database and it can organize PDFs (although it is better known for organizing music...). If you want to give it a try, use these instructions:

- Download itunes here and install it.
- If you already have iTunes and use it to organize your music, it is better to create a separate database to organize your PDFs. To do that, hold down "shift" while double-click the iTunes icon to open it. The following menu will pop up:

- Click "create library" and select the folder where you want to create your itunes PDF folder (e.g. my documents/itunes PDF)

- iTunes will open:

- ITunes has two main areas: the Browser and the Files area as indicated in the figure. If you don't see the browser area with the three columns named "genre","artist" and "album", press CTRL+"B" to show the browser.

- Now, you must configure iTunes in order to keep all the PDF files you add to the database in the same place, preferentially the same folder you selected to put your database file. Follow this instructions that I got from this tutorial:

First, open iTunes’ Preferences window. Choose the “Advanced” Tab.
Ensure that “Keep my iTunes Music Library Organized” AND “Copy New files to the iTunes Music Library Folder” are enabled.
Click OK, and exit Preferences.

I have not figured out an way to change the names of the fields in itunes, so you have to memorize the following:

Genre = Publication date
Artist = Last author
Album = Journal

the reason I select these fields is because "publication date", "last author" and "journal" are normally the bibliographic information that I remember about a paper and therefore I use it when I am looking for a specific paper. The other reason is related to the fact that iTunes organizes the files in the computer by folders with the "artist" names and subfolders with the "album" names. Similarly, your PDFs will be organized in your PDF folder by "last author" and "journal name".

- you can change which columns appear in the columns that appear in the "files area" by clicking with the right button on the title of the column. you can also drag the columns to change their order. I found useful to use the field "album artist" as "first author". Currently I have the following columns in my database:

Name, Album artist, artist, album, genre and date added

which correspond to:
paper title, first author, last author, journal, year and date added

I found the field "date added" useful to find recently added files.
There are a lot of other fields you can use, such as "comments" where you can use for comments...

- Now, to add a PDF into you library you simply drag the PDF from any folder into iTunes "files area". Note: you can change the filename after, so don't worry about the strange filenames you get when you download a PDF to your computer.

- your PDF is now in the library and the name of the file appears in the "name" field

- You must add the info regarding the journal name, publication year and first and last author. To do that, click on the column corresponding to the field and then insert the info. You can do the same if you want to change the name of the paper.

- at this point, when you start adding papers to your library, you realize the advantage of itunes. For instance, when you start typing a name of an author itunes will give you suggestions based on previously entered authors. So you can just press "enter". This is also true for other fields making the process of adding PDFs to the library faster and prevents spelling errors.

- another great advantage is that you can click on a column name in the "files area" to sort the records by that column.

- The three columns of the browser become populated with the "publication date" in the "genre" column, the "last author" in the "author" column and the "journal name" in the "album" column. If you click on an author name, itunes automatically shows in the "files area" the PDFs from that author:

- the "album" column contains only the journals associated with this author records. So, to find a PDF from author X published in journal Y, just click on the author X in "author" column and the journal Y in "album" column. You will see the PDF you were looking for in the files area.

- to open the PDF, just double click the record.

So these are the basic features to setup and start using itunes to organize your PDFs. Now I will described some other advantages.

Searching:
In the top right corner of itunes you have a search box. You can use it to search any field in the database. Useful for quick searches and to search the "comment" field.

Playlists:
you can use the playlist features in itunes to organize your papers in themes or collections. just create the playlist in file>new playlist and then drag the PDFs from the files area into the playlist on the left side.

Sharing PDFs:
I like this feature a lot. Imagine you want to give to a colleague a collection of PDFs. What you need to do is to create a playlist containing the PDF files you want to send to him/her. Then select the playlist and click file>burn playlist to disk. You will have your PDF files from that playlist in a CD or DVD and then you can give it to your colleague. Now it is the cool part: If your colleague is using itunes to organize the PDFs, once he inserts the CD/DVD into his computer, iTunes will detect the disk and also the database so he will have all the PDFs already organized!!! To add the papers into his own database he just has to drag and drop the PDFs from the disk into his database (this has to be done within itunes).

Backup or move your database to a new computer:
Just follow this tutorial I mentioned aboved

Managing your music and PDF database:
Unfortunately you can only have one itunes library/database open at the same time in one computer. This is a problem if you also use itunes to listen to music while you are working and need to access your PDF database. To go around this problem, you can install the shareware libra, that will help you in switching between libraries (music and PDFs). Backup your library (see above) before you play around with this software.

Please comment on how you organize your PDFs and if you start using iTunes please share with us your tips and tricks.

Myosin II regulation of cell adhesion

2007-03-23T06:02:00.000-04:00

A curious paper was published recently on JCB from Alan Horwitz lab:

Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells
The Journal of Cell Biology 176 (5), 573 (2007)

info:doi/10.1083/jcb.200612043
They propose an independent role for non-muscle myosin-IIA (MIIA) and IIB (MIIB) in adhesion dynamics, both at the leading edge and at the trailing edge. Using RNAi against each of the isoforms they show that both isoforms are required for proper formation and maturation of focal contacts at the leading edge, although these motors are not localized at focal contacts. Interestingly, only MIIA is required for adhesion disassembly at the trailing edge.
How these motors regulate adhesion is still unknow, but likely myosin II exert its regulation through actin fibers that emanate (or end) and focal contacts and focal adhesions. Let's see if future works validate this hypothesis.

Another asymmetry in stem cells

2007-02-03T19:23:00.000-05:00

A nice paper published in the January 26 issue of science by Yukiko M. Yamashita, Anthony P. Mahowald, Julie R. Perlin, and Margaret T. Fuller titled Asymmetric Inheritance of Mother versus Daughter Centrosome in Stem Cell Division adds a new wrinkle to the study of stem cell maintenance. As the title suggests they have identified a new and interesting aspect of stem cell biology. Specifically they use Drosophila male germ line stem cells (GSC) to show that the mother stem cell retains the mother centrosome while the daughter cell takes with it the newly replicated daughter centrosome. This is shown quite eloquently by pulse-chase experiments where centrosomes were labeled by GFP-PACT expression during early embryogenesis with the expression of GFP-PACT being shut off after germband extension (right). After two cell cycles they found that the GSC still retained a labeled centrosome indicating that the newly replicated centrosomes are taken by the daughter cell. Interestingly, the mother centrosome is maintained adjacent to the stem cell niche hub. The authors use EM to show that several microtubules emanate from the mother but not daughter centrosome and speculate that the microtubules might provide a mechanism for maintaining the mother centrosome adjacent to the hub. While much more analysis is needed to build support for this hypothesis, the authors do add one piece of mechanistic data to their intriguing observation. They find that the maintenance of the mother centrosome in the stem cell is dependent on centrin as knocking out centrin makes centrosome inheritance random. While the ultimate importance of centrosome inheritence in stem cell maintenance remains to be seen this observation seems to be too elegant to not be essential.

How proteins cross the Nuclear Pore Complex

2006-12-04T09:05:00.000-05:00

Over this past summer I saw Dirk Görlich give a talk about how the multitude of FG repeats found within the nuclear pore complex (NPC), form a gel like matrix. This "elastic hydrogel" acts as the major barrier within the NPC. Although the gel can prevent the passage of most large molecules (>30kD), it is permeable to nuclear transport receptors (NTRs). Note that all this "story" was published in the November 3rd edition of Science Magazine (link). In that paper there's a nice diagram in the that explains it all:

An "FG repeat" is a long stretch of amino acids that form non-covalent cross linking type interactions to form a gel like substance within the pore. FG repeats are found in many NPC proteins, and each of these proteins contains many of these motifs. (Shown here are the FG repeats found in Nsp1, a single NPC protein). Proteins that are too big to diffuse within the holes of the matrix are barred from crossing the gel. NTRs on the other hand can displace the FG-FG interactions by forming FG-NTR interactions and thus participating in the gel's scaffold. This property allows the NTRs (and associated proteins) to cross the matrix while maintaining the integrity of the gel scaffold (see cartoon above).

Gorlich's lab has even been able to form this gel in the lab. They show that if enough of the FG repeats are mutated, then the interactions are broken and the gel can't form. These mutations also make the NPC more leaky in yeast.

So it turns out that the core of the NPC is like a gummy bear that can absorb like molecules and exclude everything else.

Ref:
Steffen Frey, Ralf P. Richter, Dirk Görlich
FG-Rich Repeats of Nuclear Pore Proteins Form a Three-Dimensional Meshwork with Hydrogel-Like Properties
Science (06) 314:815-817

Proks have dynamin like molecules!

2006-11-30T08:46:00.000-05:00

When I was a grad student, eukaryotes had all the neatest toys ... actin, microtubules, kinesins, dynein, myosin, dynamin, SNAREs ...

OK that's not totally true - bacteria had their version of tubulin (the constituent of microtubules), and it's called FtsZ. Then others found that bacteria had a version of actin, the most known is called MreB. The latest is that prokaryotes have dynamin. (Click here for previous dynamin entries.)

From the paper:

Given the presence of large GTPases with predicted dynamin-like domain organization in many members of the Eubacteria such as E. coli and Bacillus subtilis, it is likely that bacterial dynamins, or BDLPs as we term this class, are not restricted to cyanobacteria. Such observation, combined with our results, suggests a bacterial ancestry for the dynamin superfamily.

And yes, it forms radial tubes in vitro.

And yes, it also it tubulates membranes in vitro.

And yes, they have a crystal structure.

And yes, it's totally cool.

Ref:
Harry H. Low and Jan Löwe
A bacterial dynamin-like protein
Nature advance online publication 22 November 2006 doi:10.1038/nature05312

PS: What's next? Will we find that proks have membrane trafficking?

Eating Lipids to Fuse Mitos

2006-11-16T17:03:00.000-05:00

A couple of weeks back I wrote about dynamins and mitochondrial fusion. Well the latest piece of the puzzle came in ... I just saw a paper in the latest Nature Cell Biology on this very topic. Apparently a mitochondrial version of phospholipase D (MitoPLD) may act downstream of the dynamin like molecule Mfn1 to promote fusion of the outer mitochondrial membrane.

Now remember mitos have two membranes. If two mitos want to get together they must fuse the outer membranes. This requires a dynamin protein (Mfn1 in mammals). After this fusion the two inner mitochondrial membranes can come together and fuse (this second fusion requires yet another dynamin like molecule). Also remember that some dynamins can form spirals and/or rings in vitro. This is weird as dynamins are thought to form spirals the can pinch and thus break off membranes (just like spiral collar around your neck would decapitate you by squeezing your neck and pushing your head away from your torso).

Back to the paper ...

Most of the assays in the paper are at the cellular level (looking for mito aggregates and/or fragmentation), although the authors do confirm that MitoPLD converts cardiolipin to phophatydic acid (PA). Also it looks like Mfn1 first tethers two mitos together and then mitoPLD performs some key step that activates membrane fusion. Beyond this simple explanation things are still murky.

How can mitoPLD stimulate this activity? From the paper:

The requirement for production of phosphatidic acid is reminiscent of the requirement for classic PLD-dependent generation of phosphatidic acid in SNARE-mediated fusion of secretory vesicles with the plasma membrane during many types of regulated exocytosis3, 4 and during sporulation in yeast11. Taken together, these findings reveal a common requirement for a specific manipulation of the lipid environment despite the lack of conservation of the associated protein machinery and mechanism of action. The role of phosphatidic acid remains undetermined -- it may facilitate fusion by generating negative curvature in the opposing bilayers, by recruiting or activating other key proteins or enzymes, or by being further converted to other fusogenic lipids (such as diacylglycerol). Interestingly, phosphatidic acid has been shown to recruit Spo20, the yeast homologue of the mammalian t-SNARE 25, to sites of vesicle fusion through direct interaction28, and to accelerate the rate of mammalian SNARE-driven vesicle fusion in vitro29.

So PA may just promote fusion and may act in several fusion events in cells. Or here is a crazy idea, maybe mitoPLD dissolves some mito membrane (by digesting cardiolipin) and acts as a hole puncher on the mitochondrial outer membrane. This hole puncher activity might be locally contained within Mfn1 rings (see the pic above and the dynamin entry). After the holes are punched, the Mfn1 rings could serve as connector between the two mitos fixtures thus serving as a temporary channel between mitos. The outer membranes of adjacent mitos could then mix ... and presto fusion is complete!

Now of course there are lots of problems. Mfn1 is not known to form rings. There isn't that much cardiolipin in mito membranes and this would be a very messy way of fusing the outer mito membranes ... in any case I'm sure we're missing many components that will shed new light to this process.

(Also there is a discussion as to how this process of mito fusion may be connected to apoptosis [i.e. regulated cell death]. Didn't have time to reread up the background, but I'd thought I'd mention it.)

ref:

Seok-Yong Choi, Ping Huang, Gary M. Jenkins, David C. Chan, Juergen Schiller & Michael A. Frohman
A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis
Nature Cell Biology (06) 8:1255-1262
doi:10.1038/ncb1487

How Doa10p gets into the nucleus, or another freaky experiment done in yeast

2006-11-07T07:42:00.000-05:00

I heard about this paper (Deng and Hochstrasser. Nature (06) 443:827-831) and took a look at it over the weekend. Wow! There are lots of goodies in there. And it showcases how manipulable yeast are. (As you can tell I am really jealous of researchers who use yeast as a model system.)

The premise of the paper is not bad either.

There had been some rumours that proteins could get degraded within the nucleus through the ubiquitin/proteosome pathway. Now to some this idea was heretical but this new paper gives some mechanistic info into how this process occurs.

Doa10p is an E3 ligase, that is it is the enzyme that attaches ubiquitin to the protein destined to be degraded. It's also a membrane bound ubiquitin ligase that is involved in the extraction of endoplasmic reticulum (ER) proteins that have a misfolded cytoplasmic domain (i.e. ERAD-C, see this note on ERAD and this note on the 3 types of ERAD). Previously Doa10p had also been implicated in the degradation of mat-alpha2 transcription factor, a nuclear protein involved in turning on genes in response to mating factor. So what's happening?

Doa10p had been localized to the peripheral ER but it was unclear if it diffused into the inner-nuclear membrane. Remember that the ER extends over the nucleus and is continuous with the outer-nuclear membrane (ONM) and inner-nuclear membrane (INM), however to get to the INM proteins have to pass through the nuclear pore complex (NPC). To degrade nuclear proteins, a part of Doa10p must thus pass through the NPC. To confirm this, Deng looked in cells that over express Nup53 and have an over abundance of INM (in these cells the INM looks wrinkled while the ONM is normal). Indeed Doa10p was enriched in the nucleus of these cells indicating that Doa10 does partition into the INM. How is Doa10p crossing the NPC to get to the INM? Well if you remember I wrote an entry on how the Blobel lab discovered that some INM proteins use a nuclear localization sequence (NLS) to cross the nuclear pore, so is this true for Doa10p? At first approximation it doesn't seem like Doa10p has an NLS. Now this may not be a problem, the nuclear pore complex only filters particles that are larger than ~25kDa, anything smaller can freely diffuse across. The authors first speculate that Doa10's cytoplasmic domain may be small enough to passively diffuse in, but if they make Doa10's cytoplamsic domain larger, it still can cross in. So what does it take to get in? Just like the Blobel paper, they screen cells that have mutant forms of each NPC component (called Nups) to figure out how the pore regulates the import of Doa10. Incredibly they find that Doa10 requires different Nups than what had been shown for other INM proteins! This would indicate that membrane proteins travel across the NPC via different mechanisms.

Then they perform a really crazy experiment. The authors want to prove that mat-alpha2 is indeed degraded by nuclear Doa10p. The problem is forcing Doa10p to remain out of the nucleus. To accomplish this feat the authors fuse Doa10p to coronin, an actin binding protein. The result is that Doa10p is retained in the cortical ER where it binds to actin filaments and thus sequestered away from the nucleus. Not surprisingly they find that these cells don't effectively degrade mat-alpha2. And then if cells are treated with actin depolymerizing drug (latrunculin), Doa10p is released from the peripheral ER, enters the nucleus and degrades Mat-alpha2.

Holly crap! Talk about showcasing the manipulability of yeast ... and also the idea of combining genetic manipulation with pharmacology.

So there you have it - the newest twist on how proteins reach the inner-nuclear membrane.

Ref:
Deng, M. and Hochstrasser M.
Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase.
Nature (06)443:827-831

You can't control me, but you can control my actin...

2006-10-20T15:20:00.000-04:00

The presence of actin in the nucleus has been known for a long time, and although it has been implicated in transcriptional regulation, chromatin remodeling, and structural support, little is known about what it is doing there.

Goley et al. describes that upon infection, baculovirus induces nuclear actin polymerization. After viral infection, cytoplasmic Arp2/3 complex is recruited into the nucleus where the presence of the viral capsid-associated protein, p78/83, activates Arp2/3 to induce nuclear actin polymerization. Surprisingly, the ability of p78/83 to activate Arp2/3 is required for viral replication.

Examination of conserved residues found in other Arp2/3 nucleators led the authors to make a DE384-5AA mutation of p78/83, which yields a reduced rate of Arp2/3 dependent actin nucleation in vitro. The same mutation shows aberrant viral morphology and decreased viral replication in vivo, due to the inability of p78/83 to activate the Arp2/3 complex.

All I can say is, this is the bee's knees.

Science Vol. 314. no. 5798, pp. 464 - 467

New Technique to Control Protein Expression

2006-10-13T16:04:00.000-04:00

One of the problems in modern day biomedical research is turning on/off protein expression.

In order to control in vivo protein levels, many researchers have reverted to genetically tractable organisms such as yeast and worms. In the September issue of Cell, there's a cool paper by Banaszynski et al., who developed reagents so that you can pharmacological manipulate the expression of any protein in the cell.

They take advantage of the FKBP-rapamycin-FRB system, where the addition of a drug (rapamycin) promotes the association of two proteins (FKBP and FRB). Using Yellow fluorescent protein-FKBP chimeras and error-prone PCR they screened cells (each expressing a mutant version of the FKBP chimera) by FACS analysis and isolated mutant versions of the FKBP protein that are inherently unstable but become stable in the presence of a rapamycin derivative (Shld-1).

Take home: you can fuse mutant FKBP to any protein. Your protein of interest is now stable only in the presence of Shld-1.

Step 1: Transfect cells with a plasmid encoding the fusion protein (FKBP-your favorite protein). Want to see what your protein does to cells over short periods of time? Step 2: Add Shld-1 and your favorite protein can be made. Want to get rid of your protein? Step 3: Wash out the Shld-1 and your favorite protein is degraded.

Another neat new reagent.

Ref:
Laura A. Banaszynski, Ling-chun Chen, Lystranne A. Maynard-Smith, A. G. Lisa Ooi, and Thomas J. Wandless
A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules
Cell (06) 126:995-1004

Take care of your actin so you can drink more

2006-10-09T22:26:00.000-04:00

Two great and curious papers came out last week in Cell.

They found that interfering with pathways that regulate the actin cytoskeleton prevent effects induced by ethanol. In other words, if you prevent actin remodeling, you can drink more without getting drunk.

In one paper (Rothenfluh et al.) a RhoGAP was identified in a drosophila screening for ethanol-induced behavior changes. They found that inhibition of this protein, that negatively regulates Rho and Rac, originate flies that are more resistant to ethanol.

In the other paper (Offenhäuser et al.), the authors revisited Eps8 knockout mice to find out that these mice are more resistant to ethanol than their wild-type neighbours. Eps8 is involved in NMDA-dependent remodeling of the actin cytoskeleton, through regulation of cofilin, an F-actin severing protein, however the intermediate players are not known.

ref:
Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Nina Offenhäuser, Daniela Castelletti, Lisa Mapelli, Blanche Ekalle Soppo, Maria Cristina Regondi, Paola Rossi, Egidio D'Angelo, Carolina Frassoni, Alida Amadeo, Arianna Tocchetti, Benedetta Pozzi, Andrea Disanza, Douglas Guarnieri, Christer Betsholtz, Giorgio Scita, Ulrike Heberlein, and Pier Paolo Di Fiore
Cell, Vol 127, 213-226, 06 October 2006

Distinct Behavioral Responses to Ethanol Are Regulated by Alternate RhoGAP18B Isoforms

Adrian Rothenfluh, Robert J. Threlkeld, Roland J. Bainton, Linus T.-Y. Tsai, Amy W. Lasek, and Ulrike Heberlein
Cell, Vol 127, 199-211, 06 October 2006

mRNA expression in mammalian cells

2006-10-03T08:21:00.000-04:00

Newest from PLoS Biology:

Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S
Stochastic mRNA Synthesis in Mammalian Cells.
PLoS Biol (2006) 4(10): e309

The authors genomically incorporated a gene with

32 tandem copies of a 43-base-pair probe-binding sequence at the 3′ end of a coding sequence for a fluorescent protein

into CHO (chinese hamster overy) cells and probed fixed cells with fluorescent oligos (in other words they used FISH). The high signal (32 oligos/transcript) allowed the group to see individual mRNAs. The incorporated gene was under an inducible promoter.

What did they find? RNA is transcribed in bursts. When you look at two genes, the transcription burst is uncorrelated except if the two genes are right next to eachother. Interpretation?

The fact the mRNA is produced in bursts points to new means by which the cell may control transcription. There are three apparent means by which a cell would be able to upregulate a gene's transcription: it could (i) increase the rate of gene activation, (ii) increase the rate of transcription when the gene is in the active state, or (iii) decrease the rate of gene inactivation (the opposite behaviors, of course, apply should a cell decide to downregulate a gene's transcription). These mechanisms, while all resulting in the same average increase in transcription, differ markedly in the nature of the cell-to-cell variations induced. Our data indicate that in our system, either case (ii) or (iii) applies, whereas case (i) does not; in other words, the average burst size is being modulated rather than their frequency. The observation that altering the level of transcriptional activator does not reduce the rate of gene activation supports this hypothesis. Furthermore, the fact that altering the level of transcriptional activator does not reduce the rate of gene activation again argues for the intrinsic nature of the variations observed: if the primary source of cell-to-cell variation is the infrequent events of gene activation and those events are independent of the level of transcriptional activator, then the variations are likely due to some intrinsic fluctuations gene activation that do not depend on transcriptional activators. If gene activation does indeed correspond to chromatin remodeling, this points to the possibility that the nucleation of chromatin decondensation at a gene locus may be an inherently random event that does not require the presence of transcription factors but, once initiated, requires those factors to sustain the decondensed state.

Translation (excuse the pun): You can regulate how often the burst occur (i.e. turning on a gene), the rate of transcription during a burst, or the length of a burst (i.e. turning off a gene). Their data argues that the later two effects play important roles. Furthermore the correlation between the activation of two nearby genes suggests that local chromatin remodeling may play a significant role in regulating groups of genes in one chromosomal neighborhood.

An mRNA Nuclear Export Factor Regulates Itself

2006-09-18T17:19:00.000-04:00

Biology is filled with feedback loops and other natural buffers to promote homeostasis. In the latest Nature, there is a ... cute ... paper about how the RNA export factor Tap (aka NXF1) mediates the nuclear export of an alternatively spliced form of it's own mRNA transcript. (For more background on the mechanism of nuclear export of mRNA, click here).

Viruses like the Mason-Pfizer monkey virus can exploit our mRNA export pathway by having their transcripts bind directly to export factors such as Tap. The RNA elements that bind Tap are called constitutive export elements (CTEs). In a hunt for CTEs in transcripts endogenous to the mammalian genome, the authors came up with a CTE in Tap's own transcript. Turns out that this CTE is only present in an alternatively spliced (and shorter) form of Tap. This small Tap has a RNA binding domain but lacks a domain that interacts with the nuclear pore complex thus disabling the protein's nuclear export capability.

This feedback loop may regulate Tap activity -- the more Tap activity one has, the more mRNA that encodes inactivated Tap is exported. Once synthesized, inactivated Tap may suppress Tap activity, however the exact mechanism remains unclear. One problem is that the small Tap remains cytosolic. Any ideas? From the paper:

... Although the function of the small Tap protein is unclear, it retains the RNA-binding domain but lacks the nucleoporin and NXT1-binding domains, and locates mainly to the cytoplasm. Thus, it may regulate the function of Tap at the translational level.

We'll have to see how the loose ends get tied up in this story.

Ref:
Ying Li, Yeou-cherng Bor, Yukiko Misawa, Yuming Xue, David Rekosh and Marie-Louise Hammarskjöld
An intron with a constitutive transport element is retained in a Tap messenger RNA
Nature (2006) 443:234-237

Physics versus Signaling

2006-09-08T14:35:00.000-04:00

This is a very simple paper that attempts to answer a simple question – does physics play any role in regulating cell siganling. As cell biologists we generally like to think of signaling cascades as regulating cellular processes. We consider that an extracellular ligand binds a receptor triggering a cascade that results in some cellular behavior. The Odde lab however takes a different approach and proposes that physics, specifically cell size and shape play an important role in regulating cell signaling. Using mathematical models they show that if two signals are spatially segregated, there will be a sharp drop off in activity. In the theoretical portion of the paper they use the example of a protein that is phosphorylated by a membrane bound kinase and dephosphorylated by a cytosolic phosphatase under a variety of situations (a migrating cell is depicted to the left). While it seems obvious that if you bring the membrane close together you will increase the local kinase concentration/activity and decrease the local phosphatase concentration/activity, I don’t remember it being said before. Finally they use their model to recapitulate Cdc42 activity as was experimentally shown by Nalbant et al. (right), but unfortunately this is the only experimental evidence they site.

This is certainly an interesting take on signaling however, it raises a chicken or the egg type of question. Do we know as a matter of fact that Cdc42 does not contribute to cell “flattening” where it is active? Similarly they use a migrating cell in their models as an example of a situation where one would have spatially segregated signaling but as far as I can tell it is difficult to ascertain whether cellular physics lead to signaling or if signaling precedes the physics.

IP6 and mRNA Export

2006-09-01T08:44:00.000-04:00

See this entry for background on inositols. Inositol-6-phosphate (aka Inositol hexaphosphate, phytic acid, phytate) is a strange compound.

Apparently plants make loads of it, and it is thought that they use this molecule to store phosphate. Also it would seem that lots of cancer researchers have been throwing this compound onto oncogenic cell lines. Apparently IP6 works to inhibit cell growth ... but as to it's effectiveness in vivo, I don't know. Phytate is also sold as a dietary supplement. But lets talk about its known cellular functions. Now it turns out that IP6 is a co-factor required for mRNA export.

mRNA export is a strange field. Some background ... mRNA is synthesized in the nucleus and then exported to the cytoplasm where it is translated into protein. It was originally thought that mRNA utilized karyopherins and the Ran cycle to get exported from the nucleus (click here for more on nuclear transport), but it turned out that mRNA used its very own transport machinery. In vertebrates, UAP56 is recruited to the mRNA during transcription. Then during splicing, a second factor Aly associates with UAP56. Finally Aly recruits a heterodimer of TAP and NXT1 which have to ability to interact with components of the Nuclear Pore Complex (NPC)and thus facilitate nuclear export. Now obviously mRNA export must be powered by an energy utilizing process. Enter Dbp5, an RNA helicase that is situated in the NPC. Helicases are enzymes that use ATP derived energy to walk along DNA or RNA chains (conceptually similar to a myosin motor using ATP to walk along an actin filament). As the helicase move along the DNA/RNA it acts to either separate double stranded nucleic acid chains or strip off proteins that are bound to the single stranded RNA. OK a simple story. Dbp5 is a motor that drags mRNA out of the nucleus and strips off all these factors that got it to the NPC in the first place.

Now some weird stuff. In yeast, Inositol kinases mutants had mRNA export defects. As I explained a couple of days ago, many Inositol derivatives act as signalling molecules. So what gives? Well it turns out that Dbp5 requires IP6 as a co-factor. This small molecule stimulates Dbp5's helicase activity and it's ability to bind to NPC components such as Gle1. This is illuatrated in this diagram from a recent review:

Now why would IP6 be involved in mRNA export? There must be something big behind this. And I'm sure we will get more information soon. But it sounds like IP6 could act as a regulatory molecule ... want to bump up mRNA export? Make IP6. Want to turn it off? Degrade IP6. Now when and why global mRNA export should be regulated is completely unknown, but that's also true of 90% of cellular behavior ...

ref:
Cole CN, Scarcelli JJ. Related Articles, Links
Transport of messenger RNA from the nucleus to the cytoplasm.
Curr Opin Cell Biol. (2006) 18:299-306.

Weirich CS, Erzberger JP, Flick JS, Berger JM, Thorner J, Weis K.
Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.
Nat Cell Biol. (2006) 8:668-76

Alcazar-Roman AR, Tran EJ, Guo S, Wente SR
Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export.
Nat Cell Biol. (2006) 8:711-6.

Inner Nuclear Membrane Proteins are Actively Imported

2006-08-27T13:17:00.000-04:00

This is the newest from the Blobel lab.

Note to all "they've discovered everything" types: this finding shows how much we know about how cells operate.

Background: As I've described before the nucleus and the cytoplasm are two cellular compartments that are kept apart by the Nuclear Pore Complex (NPC). This mega-assembly of proteins is the gate (or the bouncer) of the nucleus - pass it and you can gain access to the nucleus from the cytoplasm (or vice versa). NPCs sit in the nuclear envelope, an extension of the ER that covers the chromosomes.

Nuclear proteins are synthesized in the cytoplasm. To cross the NPC and thus gain access to the nucleoplasm, these proteins are equiped with a nuclear localization sequence (NLS) that recruits special proteins called "Karyopherins" (alpha/beta importin in the diagram). Karyopherins are like VIPs, they can side step the bouncer and thus escort the proteins into the nucleus. Once inside, the importins release their guest, bind to the small G-protein Ran in it's GTP bound configuration, and are exported back out of the nucleus. Once out Ran hydrolyses its GTP into GDP + phosphate.

The whole import pathway is thus maintained by a Ran-GTP gradient across the membrane and requires energy (in the form of GTP).

But there are exceptions to this active import process. Smaller proteins (< 30 KDa) freely diffuse through the NPC, and it was thought that proteins found in the inner-nuclear-membrane could also diffuse across this barrier.

But now a new paper from the Blobel lab demonstrates that at least some inner-nuclear membrane proteins in yeast can only enter the nucleus by recruiting karyopherins. Since these karyopherins require a Ran-GTP gradient, this import process requires energy. So inner-nuclear-membrane proteins (in this diagram the red blob) may act just like most proteins - they can't just waltz into the nucleus without a VIP pass.

The NPC may not let just any old membrane protein into the inner-nuclear-envelope, and thus the composition of the inner-nuclear-membrane is more tightly regulated than we once believed. In other words this component of the ER (the inner-nuclear-membrane) may be more differentiated than we one thought.

Ref:

Megan C. King, C. Patrick Lusk and Günter Blobel
Karyopherin-mediated import of integral inner nuclear membrane proteins
Nature (06) AOP

Crossposted at The Daily Transcript.

Something in the tubes, and Dynein!

2006-08-22T14:36:00.000-04:00

A new paper from Dick McIntosh's group appeared in Science this week that is similar to the paper in Nature I highlighted last week. In fact, the title is almost identical (The Molecular Architecture of Axonemes Revealed by Cryoelectron Tomography vs. Molecular Architecture of Axonemal Doublets Revealed by Cryo-electron Tomography). However, in my opinion, the paper by the McIntosh group covers a great deal more ground than the paper by Downing's group. But, read them both and decide for yourself.

Flagella are composed of 9 microtubule doublets surrounding the central pair of single microtubules. Movement of flagella takes place as a result of pulling forces exerted by the microtubule-dependent motor, dynein. Nicastro et al. performed cryoelectron tomography of flagella from Chalmydomonas and sea urchin sperm and did 3D reconstruction of the entire network. The resolution level of their images of dynein provide insight not only into the localization of dynein relative to the microtubules, but also possible mechanisms of dynein activity as a result of the orientation of different parts of the dynein heavy chain. Summarizing models are shown below.

Of interest to me (you might have guessed) is that they also saw significant densities on the inner lumen of the microtubule doublets, which they call MIPs (Microtubule Inner Proteins). Three significant MIPs are observed in this study which all exhibit 8 or 16 nm periodicity along the MT lattice. They suggest that these unknown proteins may provide stability to the long-lived microtubules in the flagellum - just as taxol, which binds to the inner surface ot microtubules, also stabilizes microtubules. However, they make no strong statements as to the identity of these molecules.

I find it interesting to compare the results from the two EM studies (if you compare them side by side, flip one of them 180 degrees because they present their data in opposite orientations). Although they both picked up similar densities in the A-tubule, the McIntosh group does not see the density suggested to be Tektins by the Downing group. Conversely, the prominent density in the B-tubule seen by the McIntosh group is not reported in the work from the Downing group. Instead, they see a density on the other side of the B-tubule. Oddly, neither group see the "ponticulus" identified by Vaughan et al. Since they both used sea urchin sperm for at least part of their work, why wouldn't they see exactly the same thing? Someone more familiar with cryo-EM methods may be able to show where the two preparations are different. If that is true, does it mean that they both have incomplete models and there is more inside the MT lumen than either suggest individually?

LINK:
Nicastro, D., Schwartz, C., Pierson, J., Gaudette, R., Porter, M.E., and McIntosh, R. 2006. The Molecular Architecture of Axonemes Revealed by Cryoelectron Tomography. Science 313: 944-948.

Rho and polarity establishment

2006-08-21T20:49:00.000-04:00

CYK-4/GAP Provides a Localized Cue to Initiate Anteroposterior Polarity upon Fertilization
Noah Jenkins, Jennifer R. Saam, and Susan E. Mango
Published online 27 July 2006, Science
[DOI: 10.1126/science.1130291]

Establishment of Cell Polarity is key in several cellular contexts. Small GTPases, in particular Cdc42, are known regulators of cell polarity. It was known from previous works that the sperm determines the posterior pole of the C. elegans embryo. Righ after fertilization, during pronuclear migration, a wave of cortical actin (cortical actin flow) and myosin II moves away from the site of sperm entry, which becomes the posterior pole, and is involved in polarization of PAR-3 and -6 to the anterior pole, and PAR-2 to the posterior.

The identity of the sperm protein responsible for setting the cue remained unanswered until now. Schonegg et al. identified a RHO-GAP (CYK-4) that is required for the establishment of polarity. CYK-4 is present in the sperm centrosome and no actin or myosin polarization is observed in the absence of paternal CYK-4. CYK-4 is a GAP for RHO, i.e. it inhibits RHO activation. The authors propose that CYK-4 breaks down the symmetry in the actin cytoskeleton, resulting in cortical actin flow away from sperm entry site.

So how does Rho regulates actin cytoskeleton? The obvious candidate is Rho Kinase (ROCK), which activates myosin II and originates actin bundling. So a model can be proposed where local inhibition of Rho by CYK-4, leads to inhibition of ROCK and myosin II at the future posterior pole. The C. elegans homologue of ROCK is Let-502 and it is not know to be involved in the establishment of C. elegans embryo polarity, so probably we will soon find out...

Oligomers of tubulin can polymerize!

2006-08-16T11:44:00.000-04:00

This is an important step for studying microtubule dynamics and is in my humble opinion among the most important papers on the microtubule front since Mitchison first described dynamic instability (Mitchison, 1984). As the title suggests this paper provides insight into microtubule polymerization at a molecular level. Prior to this work by the Dogterom laboratory we have only been unable to study how individual tubulin molecules polymerize within a microtubule. Many researchers (including myself) have speculated that tubulin under certain circumstances (i.e. in the presence of microtubule plus-end tracking proteins - +TIPs) might add as an oligomer that might then act as a stabilizing cap to facilitate further polymerization. This paper uses optical tweezer technology to show that MTs polymerized exclusively from tubulin grows in step sizes of up to 30 nm (a tubulin dimer is only 8 nm so this is clearly oligomers of some sort can polymerize). They go on to show that XMAP215 increases the step size to 60 nm thus supporting those hypotheses that +TIPs can add tubulin oligomers. Finally, they go the extra step using physics to demonstrate that the steps are not the result of open sheet closure but instead actual addition of tubulin oligomers. This work is clearly only a stepping stone leaving many unresolved questions. For example are the oligomers a single protofilament of tubulin or does a tubulin oligomer add within multiple protofilaments. Additionally this work provides a method to study the effects of +TIPs on MT assembly as opposed to the reckless hypothesizing with which we are familiar.

Something in the tubes, Part 2

2006-08-15T11:37:00.000-04:00

Biology is first and foremost driven by observations. Much of the current field of cell biology involves working out the details of phenomena that were observed a hundred years ago or more using the naked eye, shaped glass, and rudimentary microscopes. Today's biologists are armed with ever more powerful tools to peer deeper into the molecular level of the cell and see things we had never seen before. Even subtle changes in the methods of preparing samples enable us to see things we hadn't previously seen. The paper I highlight today is one such example.

Like yesterday, I am presenting an electron microscopy study of axonemal microtubules. Yesterday's paper was the flagellum of sea urchin sperm; today's is the flagellum of the African trypansome Trypanosoma brucei (responsible for African sleeping sickness). Although the two organisms are separated by millions of years of evolution, the inherent ultrastructure of their flagella are the same (another example of nature's elegant simplicity) - but I digress. Previously, tannic acid was used in the fixation of samples for transmission electron microscopy (a relatively simpler technique than yesterday's cryo-EM study) to enhance the contrast of the resulting image (as seen in panel B of the figure below compared to panel A). However, Vaughan and colleagues noted that in the absence of tannic acid a structure is retained in the lumen of the B-tubule of the microtubule doublet that is not apparent in the presence of tannic acid. Here's the image:

They call this structure the "ponticulus" (little bridge) and note that it is present in all nine of the outer doublet microtubules of the flagellum (see lower portion of panel A, above). They go on to point out that newly formed flagella do not contain this structure, but that it is instead modified later - after the flagellum has formed. They also note (although no data is shown) that this structure is also present in the flagellum of other trypanosomes, namely Leishmania and Chrithidia.

What is this ponticulus? Since it is formed after the flagellum is made, how does it get access to the outer tubule of the doublet? Is it necessary for the structural integrity of the trypanosome flagellum? Also, why was it not seen in the study by Sui and Downing of sea urchin spem flagella (see below)? Is it unique to trypanosomes? Like early observations of biological phenomena, these observations will drive future work in cell biology.

Reference:
Vaughan, S., Shaw, M., and Gull, K. (2006). A post-assembly structural modification to the lumen of flagellar microtubule doublets. Curr. Biol. 16, R449-50.

Something in the tubes

2006-08-14T20:22:00.000-04:00

For my inaugural post in the blog, I wanted to share a paper that covers a topic that I am really excited about. It's something I became interested in as a graduate student hanging out in Gregg Gundersen's laboratory and hope to spend some time on now that I'm a post-doc. It gets at the fundamental difference between microtubules and the other cytoskeletal proteins (actin and intermediate filaments). Unlike the other two elements, microtubules (as their name implies) are indeed tubes. The protofilaments assemble in such a way as to create an inner core with a diameter of 16 nanometers (admitedly small but would support a globular protein somewhere in the range of 300 kiloDaltons). Although a great deal of work has gone into studying proteins that bind the outer surface of microtubules (motors, MAPs, plus-end tracking proteins, etc.), very few people are looking at the inside. A recent publication in the journal Nature may get a few more people thinking about it.

Haixin Sui and Kenneth Downing, working at the Lawrence Berkeley National Laboratory did a cryo-electron tomography (fancy high resolution imgaing) study of axonemal microtubules (the microtubules found in cilia and flagella). In these structures, a stereotypical doublet of microtubules forms as is shown in the picture here:

Upon careful examination of their images, they noticed densities in the lumen of the microtubule (the inner surface) that could not be accounted for by fitting the known crystal structure of tubulin to their images. As they analyzed the data along the length of the microtubules, they found that these densities exhibited periodicity consistent with the known distance between tubulin monomers. Through the analysis of thousands of images, they generated a 3D density map of the microtubule inner surface as shown here:

They suggest that the shape and size of one group of these proteins would identify them as Tektins, an intermediate filament-like family of proteins previously co-purified with axonemal microtubules. However, the identity of the other proteins in the microtubule lumen remains a mystery (although they suggest that the other proteins may be Sp77 and Sp83, proteins previously identified in sperm flagella).

Previous studies have suggested that there must be proteins on the inner lumen of microtubules. Indeed, acetylation of microtubules takes place on a lysine that is predicted to orient toward the lumenal surface. However, this is, to my knowledge, the most elegant description of microtubule lumenal proteins to date.

What are these proteins? Do they contribute to the structure of the axonemal microtubules? Are there similar proteins in the lumen of the interphase array of microtubules in other cells? These questions seem to be wide-open areas for future study.

Reference:
Sui, H. and Downing, KH. (2006) Molecular architecture of axonemal microtubule doublets revealed by cryo-electron tomography. Nature 442, 475-478.

New Evidence for Endosymbiotic Origin of the Centrosome

2006-08-12T08:12:00.000-04:00

A new paper provides evidence that certain RNAs associate with centrosomes and may represent a centrosomal RNA genome. Furthermore this potential genome includes an enzyme that could copy the centrosomal associated RNA. But first some background.

For many years, there have been scientists interested in the mysterious entity called the centrosome. Often refered to as the black hole of the eukaryotic cell, these intracellular structures (see red dots) are often located next to the nucleus (blue), where the microtubule cytoskeleton (green) converges (called the microtubule organizing center).

Another fact for you: the primary cilium, a long skinny cellular extension found in most cells, grows off of one of the centrosomes. This primary cilium is probably involved in some sensing mechanism. In fact in some cells (such as your rods and cones) the primary cilium is where most of the cellular sensory apparatus is located. Mutations that result in short primary cilliums cause kidney problems and probably involve a missensing of some property of fuid within the kidney.

Centrosome are composed of two centrioles, barrel like structures made mostly of tubulin + other more unique proteins (see image below the fold). The number of centrosomes per cell is tightly regulated and the duplication of centrosomes is quite bizarre. You start off with a single centrosome. At some point (S phase?) the two centrioles come apart and new (or "daughter") centrosomes seem to grow off of the old (or "mother") centrosomes. Very reminiscent of semi-conservative replication of DNA. But it get stranger.

If you watch the centrosomes throughout cell division, right before cytokinesis (the physical cleavage of two daughter cells) occurs, one of the two centrosomes from one of the two cells, migrates to the cleavage furrow and (in the words of Michel Bornens) gives it a "kiss". This event is thought to stimulate the final braking off of the two daughter cells.

Then about 5-6 years ago Alexey Khodjakov performed a remarkable experiment. He labeled the centrosomes with a green fluorescent protein (I think it was GFP-gamma-tubulin) then blasted them away with a laser. After centrosome duplication he blasted one of the two centrosomes and got cells with lopsided resulting mitotic spindles. The microtubules one side converged on a centrosome while the microtubules on the other side having no centrosome, became unorganized. Incredibly if he ablated both centrosome, the cells could form a spindle (mostly due to the fact that motor dynein can focus microtubules without centrosomes). The cells then progressed through mitosis and even divided (although sometimes they were trapped in cytokinesis - see Bornens' kiss). After division they got a huge surprize, the cells stoped dividing further. They discovered that centrosomes are needed to initiate a new round of DNA synthesis (or the G1-S transition). So just before DNA is copied, a cell asks itself "is my centrosome okay?" If the answer is yes => proceed with DNA copying. If not => stop cell division. You can advance through this "check point" if you get rid of p53, a key protein that regulates the cell cycle.

But why all the fuss? What is so special about the centrosome? Why are cells checking it? Why is it kissing stuff? Why?

Michel Bornens came up with this individuation theory. Centrosomes mark an individual cell. At certain points, cells check up on their centrosome to check on their individualistic identity. If something is wrong, such as improper cell division, cells can sense their non-individual state and activate programed death and/or other drastic measures. Screwed up cells (such as cancer cells) have all sorts of centrosome defects ... many have an overabundance of centrosomes and centrosome associated proteins suck as pericentrin.

And there's more! Many years ago, Lynn Margulis (famous for her theory of the endosymbiotic origin of mitochondria) proposed that centrosomes originated from the fusion of a spirochaete-like-organism (right, image of a spirochaete) and the prototypical eukaryotic cell. The spirochaete may have helped the host cell to move around (I never found this explanation very satisfying - the spirochaete must have offered some other benefit). In support of this idea, various publications in the 80s claimed that the centrosome contained its own nucleic acid, just like mitochondria and other products of symbiosis, but these experiments were never definitive.

Now out of ~~Bob Palazzo's lab (no relation ... see extra comment at the end)~~ Mark Alliegro's Lab (click here for a comment on this change) has come up with a truly remarkable finding. His group purified centrosomes from clam oocytes ... for some reason clams have huge centrosomes and thus makes this experiment a whole lot easier. Along with the centrosomes came ... certain specific RNAs. But wait, people have claimed this in the past, and one of the enriched RNA was 18s ribosomal RNA, perhaps it was contamination. On the other hand the enrichment was for only certain RNAs (five in this paper). Besides the 18s rRNA, the other four RNAs were not found in any genomic database. (For you RNA enthusiasts these have now been called cnRNAs - centrosomal RNAs.)

And one of these cnRNAs is QUITE intriguing. It encodes a protein that has similarity to 'RNA-dependent nucleotide polymerase'. Furthermore, this RNA probably gets translated into protein. (Warning: technical jargon comming up) The group maid antibodies against a peptide derived from the hypothetical protein, and found an immunoreactive band in lysates from oocytes and adult clam (end jargon).

So let me rephrase this. Clam centrosomes contain RNAs not found in any genome (so far), and one of these RNAs encodes an enzyme that could potentially copy RNA? Holly replication, Batman!

So could it be? Centrosomes have their own genome (an RNA genome at that?) Wow!

Ref:
Mark C. Alliegro, Mary Anne Alliegro, and Robert E. Palazzo
Centrosome-associated RNA in surf clam oocytes
PNAS (2006) 103:9034-9038

(Note: This whole Palazzo/centrosome thing is very quirky. He knows of me, I know of him, but we've never met. He has worked in the centrosome field for quite a while, I've published papers on a related structure, the microtubule organizing center. At meetings, some people think I have his long publication records, others who know Bob Palazzo well, ask me whether I'm his son -- he in fact does have a son named Alex. I almost went into the centrosome field by going to Steve Doxsey's lab (great guy!) but just couldn't live in Worcester. Now I'm studying (in part) RNA ... and Bob Palazzo is into cnRNA ... I know ... very strange.)

RNA classification

2006-08-05T09:24:00.000-04:00

Well two weeks ago in Science, two reports came out about yet another species of small RNA ... rasiRNA ... uhm i mean piRNA (OK they haven't harmonized their nomenclature yet).

So here is a brief review of the types of RNA:

- mRNA (messenger RNA). These are the RNAs that encode polypeptide chains.
- rRNA (ribosomal RNA). These form the core structure of the ribosome. The ribosome is the enzyme that translates the tri-nucleotides, to amino acids. In this way it synthesizes (or "translates") proteins from mRNAs.
- tRNAs (transfer RNA). These are used by the ribosome to translate the tri-nucleotie. On one end a loop displaying the complement of the tri-nucleotide is displayed, on the other end is the corresponding amino acids. So if you think about it tRNAs are responsible for coupling the tri-nucleotide to the amino acid.
- snRNA (small nuclear RNA). These form the core components of the splicesome, an enzyme that catalyses the splicing of mRNA.
- snoRNA (small nucleolar RNA). These strange critters help to process and assemble a variety of RNAs such as rRNAs.
- siRNA (small interfering RNA). These are processed from long double stranded RNAs, mostly exogenous in origin (exogenous = originates from outside the entity, in this case the cell) but occasionally are produced from endogenous transcripts (endogenous = originates from the same entity, in this case the cell's genome). The 21-23 nucleotide fragments are then used by the RNAi (RNA interference) pathway to turn off the expression of any single stranded RNA that the siRNA can base pair with. The machine that mediates RNAi is the RISC (RNAi Induced Silencing Complex), which includes the siRNA, a protein called Argonaute, and some other factors.
- miRNA (micro RNA). These are processed from short nearly double stranded small hairpin RNAs (shRNA) that are produced from endogenously transcribed RNAs. Like siRNAs, miRNAs are 21-23 nucleotides in length and are used by RNAi and the RISC machinery to inhibit mRNA transcription.
- rasiRNA (repeat associated small interfering RNA). These small RNAs (24-29 nucleotides in length) are produced from endogenous transcripts in fruit flies and seem to silence. Unlike siRNA, rasi RNA they are highly enriched in germline cells (sperm+oocyte) and form a complex similar to the RISC complex except the main protein involved is Piwi, a member of the Argonaute family of proteins. It seems like rasiRNAs function to inhibit bits of selfish DNA that copy themselves and multiply within the genome. These "selfish genes" are called retrotransposons.
- piRNA (piwi interacting RNA). Similar to rasiRNA, piRNAs are 25-31 nucleotides in length and produced from endogenous genes in mammalian cells. Along with Piwi, they form the piRNA complex (piRC) and act to. Unlike siRNA and miRNA, piRNA (and rasiRNA?) are not processed from a double stranded RNA precursor. For the moment the role of piRNAs is not clear, but like rasiRNA, these are highly enriched in germ cells. It is possible that they may regulate selfish genes, but there wasn't enough info in the paper ... I'm sure we'll find out soon what their targets are.

Ref:

rasiRNA
Vasily V. Vagin, Alla Sigova, Chengjian Li, Hervé Seitz, Vladimir Gvozdev, and Phillip D. Zamore
A Distinct Small RNA Pathway Silences Selfish Genetic Elements in the Germline
Science (2006) 313:320 - 324

piRNA
Nelson C. Lau, Anita G. Seto, Jinkuk Kim, Satomi Kuramochi-Miyagawa, Toru Nakano, David P. Bartel, and Robert E. Kingston
Characterization of the piRNA Complex from Rat Testes
Science (2006) 313:363 - 367

Let's inhibit Cdc42

2006-08-03T20:32:00.000-04:00

Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro.
HE Pelish, JR Peterson, SB Salvarezza, E Rodriguez-Boulan, JL Chen, M Stamnes, E Macia, Y Feng, MD Shair, and T Kirchhausen
Nat Chem Biol, Jan 2006; 2(1): 39-46

Cdc42 is a small GTPase associated with multiple cellular functions. It is "active" when is bound to GTP and membranes, and "inactive" when bound to GDP. This and other small GTPases are further regulated by RhoGDI, which regulates Cdc42 binding to membranes.

The paper I picked describes a small molecule - Secramine - that interferes with Cdc42-related activities, therefore it can be used as an inhibitor of Cdc42. The authors found that secramine does not interfere with the loading of GTP into Cdc42. Instead, secramine seems to interfere with membrane-dependent activation of Cdc42, probably by stabilizing the interaction between Cdc42 and RhoGDI. Further work is probably on the way to characterize the mechanism of action of secramine, but in the mean time the authors showed that secramine inhibited transport of NCAM from the golgi to the plasma membrane and golgi reorientation in migrating cells. These two cellular processes are known to be dependent on Cdc42. Secramine can be a very powerfull tool to use as a reversible inhibitor of Cdc42.

Protein folding is coupled to mRNA stability ... across a membrane

2006-07-28T20:20:00.000-04:00

Yes this is the surprising ~~result~~ interpretation of Jonathan Weissman's paper in Science. For non ER-aficionados, click here first, to get some background on the unfolded protein response (UPR) and ER associated degradation (ERAD). And to learn about some recent developments on ERAD, click here.

OK on to the HARDCORE cell biology ...

Remember under UPR conditions cells want to stop translating ER targeted proteins and instead synthesize chaperones and ERAD components. UPR inhibits translation through PERK (see the post on UPR) but what happens to the mRNA that encodes ER targeted proteins? Well it turns out that several of these mRNAs get degraded. This mRNA destruction requires IRE1 but not XBP1. If targeting of the mRNAs to the ER is disrupted, the mRNAs evade the UPR activated destruction.

So the question is why are certain ER targeted mRNAs destroyed (like those that encode plasma membrane or secreted proteins) while not others (like mRNAs that encode ERAD components)?

Well it could either be the mRNA that has a special code that makes it susceptible to UPR degradation, or the translational product may trigger the destruction of the mRNA.

To test the second idea, the Weissman group incorporate either a single, double or triple nucleotide insertion into the mRNA. Remember that a trinucleotide specifies an amino acid ... in the case of a single or double nucleotide insertion, the reading frame of every downstream trinucleotide is altered resulting in a completely different translational product. If a trinucleotide is inserted, the reading frame is conserved and all you've done is add an extra amino acid to your translational product. Well what they found was that when you altered the translational product, you abolished the mRNA's susceptibility to UPR mediated degradation. Yes the protein specifies the stability of the mRNA. The implication is that if a protein doesn't fold correctly as it is being translated and translocated into the ER, it targets the destruction of it's mRNA. But for the protein to direct the cleavage of the very mRNA it is coming from you would have to build a model where mRNA translation, translocation, and IRE1 dircted cleavage should all be happening at the same time (or in cis). Incredible! The model is nicely summarized in this figure from a David Ron review:

(click here for a larger version.)

One weird thing. By introducing a frame shift (i.e. insertion of 1-2 nucleotides in the coding region), the Weissman group transformed a natural protein into "junk" protein. This "junk" should have MORE problems folding than the original product, SO WHY IS IT NOT PROMOTING UPR MEDIATED mRNA DEGRADATION? (Very weird.)

This is a big finding, and there are some obvious experiments that could be done to give some more confidence to the model.

1) Alter an mRNA that isn't subjected to UPR mediated mRNA decay by adding a frame shift. The new mRNA will then encode "junk protein" that should have problems folding. By the new model, this new mRNA should be sensitive to UPR.
2) Test whether the mRNA destruction really operates in cis. This can be done by coexpressing two mRNAs, one with a frame shift and one without. If the expression of the frame shifted mRNA affects the stability of the unaltered mRNA then this process does not operate in cis (and hence in trans).

Overall a cool story.

Ref:
Julie Hollien and Jonathan S. Weissman
Decay of Endoplasmic Reticulum-Localized mRNAs During the Unfolded Protein Response
Science (2006) 313:104-107

Actin differentiation through Arginylation

2006-07-25T22:23:00.000-04:00

In cells, actin polymers dictate cell morphology.

Actin filaments can adopt several conformations, they can be bundled into large microfilaments (often called stress fibers; here "mf" - electron micrograph taken from the Borisy lab webpage) or arranged in a meshwork (as seen in the second electron micrograph). One actin isomer (Gamma-actin) predominates in the stress fibers that are present in the cell body, while another (beta actin) predominates in the actin meshwork found in the leading edge of migrating cells. In fact, the generation of this meshwork right at the tip of the cell (see arrows in the first figure) is thought to push the cell membrane forward. This leading edge polymerization provides the major force for membrane spreading and plays an important role in cellular locomotion.

So if gamma-actin makes up microfilaments, and beta-actin makes up the meshwork, how do the two isoforms differ? How is the differential distribution explained? Beta-actin mRNA is concentrated at the leading edge and is thought to be synthesized there, but most researchers thought that there must be more to the story than that. Gamma-actin and beta-actin differ by only 4 out of 100 residues, and both genes are EXTREMELY well conserved across different species (100% conserved at the amino acid level between birds and humans).

A couple of weeks ago a great paper came out in Science about actin arginylation that cleared things up a bit.

For many years it had been noted that tubulin, the building block of the microtubule cytoskeleton, undergoes many post-translational modifications (see my post on this topic). These modifications help to differentiate microtubules that are aligned along the cell's main axis from the bulk microtubules that are present through out the cell. Now a group from U Penn who were studying arginine tRNA protein transferase (Ate1), discovered that actin gets post-translationally modified too. Bottom line - Ate1 adds arginine onto beta-actin's N-terminal glutamate side chain. This modification (similar to what was seen in tubulin & microtubules) aids in differentiating a part of the actin network from bulk actin filaments.

Now comes the new stuff ...

In the recent Science paper, the authors report that beta-actin, but not gamma-actin, gets arginylated by Ate1. This is supported by the finding that beta-actin arginylation is abolished in cells lacking Ate1. Aha! Perhaps arginine may give beta-actin the required properties to form meshworks. In agreement with this idea, Ate1 -/- cells have reduced levels of actin meshwork, and show defects in cell spreading and cell migration (see the figure, right). So then the big question is: how does arginylation affect beta-actin?

(here is a good schematic I ripped off of Chloe Bulinski's nice commentary in the same issue of Science).

The authors speculate that the extra positive charge from arginine may help prevent actin from bundling through electrostatic repulsion. Quite honestly I do not believe that the answer will be so simple. The actin meshwork and filaments are formed by separate mechanisms. The meshwork is formed by the polymerization of actin "branches" by the arp2/3 complex. Microfilaments are formed (probably) by formins which wrap around actin, forcing it into long fibers. These fibers are then bundled by the motor protein myosin. It is then likely that arginylation affects one of these actin-modeling processes.

In conclusion this paper offers a neat new concept for the cytoskeletal field.

Ref:
Marina Karakozova, Marina Kozak, Catherine C. L. Wong, Aaron O. Bailey, John R. Yates, III, Alexander Mogilner, Henry Zebroski, Anna Kashina
Arginylation of ß-Actin Regulates Actin Cytoskeleton and Cell Motility
Science (2006) 313:192-196

Golgi Maturation

2006-07-06T11:53:00.000-04:00

No soccer today. So instead of spending time watching others run around, go read the two papers, published in last week's Nature on Golgi maturation.

Proteins that need to traverse, or be embedded within membranes are synthesized in the endoplasmic reticulum (ER) and are transported cotranslationaly for the most part through a pore called the translocon. Most of these proteins are then delivered, through vesicular transport to the Golgi complex, where they are post-translationally modified. Sugars are added, sugars are removed ... indeed these proteins undergo many different modification steps. The Golgi is divided into many pancake shaped organelles stacked on top of each other, every pancake (or cisterna) having a different modification enzymes. Like penalty kicks and red cards, the Golgi seems to be an endless source of controversy. Does the Golgi dissolve into the endoplasmic reticulum during mitosis? Do proteins travel via vesicular transport from cisterna to cisterna, or does each cisterna "mature"?

But first, what does "mature" mean? It is the act of replacing one set of modifying enzymes with another set of modifying enzymes. So that early (or cis) Golgi is gradually transformed into Golgi containing the next set of modification enzymes (medial Golgi) which in turn is gradually transformed into late (or trans) Golgi. Well actually there are many cisterni (in the neighborhood of 6) in each Golgi stack. From the Golgi vesicles full of modified proteins are transported to various intracellular compartments such as the endosome recycling compartment.

How to tell whether Golgi cisterni "mature"? you would think that it's easy. Just fluorescently label proteins that traverse the Golgi and watch whether they stay in one cisterna or whether they travel across a stack. Unfortunately the stacks are smaller than the resolution limit of light microscopy. One could also image Golgi modification enzymes, but the size issue remains. Another attempted method, is to load the Golgi with giant particles. These substrates being too large to fit in vesicles, would presumably be stuck in one cisterna. But such experiments never gave conclusive evidence.

So what to do? Two groups solved this by reverting to yeast. (It seems like yeast are just simply the best organism to get at tricky problems ... ) You see yeast are weird. For a long time it was even questioned whether they had Golgi as no Golgi stacks were ever seen by electron microscopy. It turned out that the lack of stacks was due to the unique organization of the Golgi in yeast. You see in yeast the different cisterni are physicaly separated ... there are no stacks. And that is no problem for budding yeast, cuz they are so damn small. Also Golgi organization in "higher eukaryotes", is dependent on the microtubule network, but yeast do not use microtubules to direct vesicular transport. Now most proteins that are important for Golgi function are conserved between yeast and other eukaryotes, so even if ultra-structurally the Golgi shape may vary, the core operating system should be conserved.

OK fine so lets use yeast and image each separate Golgi sac to see if each sac matures, or to see whether substrates jump from sac to sac. To answer this, both groups labeled early and late Golgi markers and imaged the distribution of these markers over time. Do the markers jump from cisterna to cisterna? Or is each cisterna static in it's composition of modification enzymes.

So the answer: Golgi stacks mature. The rate of Golgi maturation is compatible with the rate of secreted protein production. If you block vesicle transport (via COP I temperature sensitive mutation) Golgi maturation slows down, indicating that modification enzymes get transported to and from each cisterna ... but since cistetna still matured, other transport mechanisms may help "mature" any particular Golgi sac.

OK that's all I have to say. Go check out the papers, and watch the movies. The results are pretty clear, hopefully the game on Sunday will provide for as much excitement.

Refs:

Eugene Losev, Catherine A. Reinke, Jennifer Jellen, Daniel E. Strongin, Brooke J. Bevis and Benjamin S. Glick
Golgi maturation visualized in living yeast
Nature (06) 441:1002

Kumi Matsuura-Tokita, Masaki Takeuchi, Akira Ichihara, Kenta Mikuriya and Akihiko Nakano
Live imaging of yeast Golgi cisternal maturation
Nature (06) 441:1007

Crossposted at The Daily Transcript.

Stochastic expression of proteins in a single cell

2006-06-29T21:20:00.000-04:00

Every thought about how variable the expression of a particular gene is across an entire cell population?

That’s what the Weissman lab described in a manuscript in a recent issue of Nature. Anytime you want to take on such a project – take my advice, you turn to yeast. The yeast field has created a library of strains, each containing a copy of GFP (Green fluorescent protein) fused to a particular gene within the genome. If you measure the fluorescence, you can quantify the level of protein expression. The next trick is to use flow cytometry to rapidly measure the brightness individual cells in a population. Brightness per cell = GFP per cell = expression of the tagged gene, per cell.

Now you can record how a particular gene is expressed, in terms of protein levels, on a cell to cell level. In addition one can identify how cells alter their protein levels when exposed to various conditions. Note that this type of experiment has been done at the mRNA level using microarrays, yet until now no one has published any account of how to perform these measurements at the protein level.

So what did they find?

- For cells grown in rich media, 30% of their genes have elevated protein expression, 10% have decreased protein expression when compared to cells grown in minimal medium. Proteins with increaed levels in rich media, are those involved in cell division and cell wall biosynthesis. Proteins with increaed levels in minimal media, are biosynthetic genes (if the environment doesn’t have aminoacids and nucleotides, you’ve got to make them yourself.)
- Importantly, the researchers asked whether protein levels reflected mRNA levels, or whether other post-translational events (such as protein degradation) played significant roles. To the relief of many microarray manufactures, changes in protein levels largely correlated with changes in mRNA fir most genes. There were some exceptions (like ribosomal RNA processing enzymes and enzymes involved in the Kreb cycle).

But now comes the interesting part, proteins involved in core functions (i.e. ribosomal proteins, initiators of translcription, protein synthesis, protein degradation) have low variability in protein levels on a cell-to-cell basis. The output from these genes had low noise. Strangely, Golgi components have low noise as well, but mitochondrial and peroxisome proteins have high noise. Overall it seems like mRNA production is the greatest determinant of noise:

…variation most likely originates from the stochastic production and destruction of mRNA molecules. Indeed, the magnitude of the variation observed here (CV 30% for low–medium abundant proteins) is entirely consistent with that expected if protein variation results from Poisson noise owing to small mRNA numbers (1–2 per cell) and is mitigated by a filtering effect that arises because proteins are typically far longer-lived than their messages.

...

... high noise is likely to be due, at least in part, to the introduction of a slow step into the production of mRNA, making the process more prone to bursts.

Furthermore, noisy genes are regulated at the transcriptional level by similar transcription factors and chromatin remodeling enzymes. Stable genes tend to be regulated by another group of transcription factors. Stable genes are also less likely to be affected by fluctuating mRNA numbers. This is best achieved by have increased numbers of messages that turnover rapidly.

The authors point out that noise (or the variability of expression) is an important consideration in how genes are regulated. Cells may want certain genes, such a those that respond to environmental stress, to have “noisy outputs”.

for some proteins that permit cells to respond to environmental perturbations, excursions from the mean at the single-cell level might benefit populations. In the short term, such deviations might facilitate a cell's initial response to environmental variation. More generally, the capacity to vary might permit a population to sample multiple phenotypic states to maximize the chances of some, but not all, cells' survival in an adverse environment.

Other genes, such as those involved in ribosomal maintenance and cell cycle regulation, need to have stable levels in order to ensure cellular homeostasis.

The takehome message, forget about the average, the generation of variability or stability may be a crucial component to how a cell is hardwired.

Ref:
John R. S. Newman, Sina Ghaemmaghami, Jan Ihmels, David K. Breslow, Matthew Noble, Joseph L. DeRisi and Jonathan S. Weissman
Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise
Nature (2006) 441:840-846

Cross posted at The Daily Transcript.

Giving your mRNA a boost by increasing GC content

2006-06-26T10:51:00.000-04:00

Abstract from PLoS (the abstract says it all):

Mammalian genes are highly heterogeneous with respect to their nucleotide composition, but the functional consequences of this heterogeneity are not clear. In the previous studies, weak positive or negative correlations have been found between the silent-site guanine and cytosine (GC) content and expression of mammalian genes. However, previous studies disregarded differences in the genomic context of genes, which could potentially obscure any correlation between GC content and expression. In the present work, we directly compared the expression of GC-rich and GC-poor genes placed in the context of identical promoters and UTR sequences. We performed transient and stable transfections of mammalian cells with GC-rich and GC-poor versions of Hsp70, green fluorescent protein, and IL2 genes. The GC-rich genes were expressed several-fold to over a 100-fold more efficiently than their GC-poor counterparts. This effect was not due to different translation rates of GC-rich and GC-poor mRNA. On the contrary, the efficient expression of GC-rich genes resulted from their increased steady-state mRNA levels. mRNA degradation rates were not correlated with GC content, suggesting that efficient transcription or mRNA processing is responsible for the high expression of GC-rich genes. We conclude that silent-site GC content correlates with gene expression efficiency in mammalian cells.

Very Interesting. High GC content at codon position #3, led to higher protein content, higher mRNA content, but no change in mRNA degradation. Unfortunately the authors did not test whether the mRNAs were transcribed more efficiently. Other possibilities is that mRNA 3' processing is inefficient or mRNA export is compromised (with the unexported mRNA being degraded immediately).

It's something to think about when designing your own genes. This finding also has implications for biologists who track mutations at synonymous sites ... although these mutations may be "silent" in how they change the coding of a protein, they may not be so silent (different use of the term) with regards to how they affect protein expression. This may also be a way of generating variability with regards to protein expression between different alleles. On the other hand, the effects of one point mutation may be very small. Is it too small to be selected on? We'll see.

Kudla G, Lipinski L, Caffin F, Helwak A, Zylicz M (2006) High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells. PLoS Biol 4(6): e180

Cross posted at Daily Transcript.

Endosomal Transport in Fungi

2006-06-22T20:57:00.000-04:00

The recent paper by JH Lenz et al. describes the mechanism for early endosome trafficking in Ustilago maydis, a pathogenic fungus.

A dynein loading zone for reterograde endosome motility at microtubule plus ends
JH Lenz, I Schuchardt, A Straube and G Steinberg
The EMBO Journal (2006) 25, 2275–2286

The trafficking they are concerned with is that to the hypha tip because polarized growth of the fungus in this area is an important factor controlling its ability to invade the host cell. Not surprisingly the endosomal trafficking is microtubule dependent and occurs via dynein/kinesin cooperation. The take-home message is that kinesin3 carries early endosomes to the hyphal apex where they can contribute to cell expansion before being carried reterogradly by dynein. They find that when they knock out kin3 the hypa are smaller and the early endosomes cluster near the nucleus. This is not surprising given that they use the +TIP EB1 to show that almost 90% of MTs are oriented with their plus end toward the hyphoid tip. Conversely if dynein is knocked out (conditionally) the early endosomes cluster at the hyphoid tip/MT plus-ends. Probably the nicest piece of data in the paper is the microscopy showing that early endosomes move toward the regions of concentrated dynein before reve rsing directions. I’ve taken their Figure 3E (left) where dynein is in green and the early endosomes are in red and you can see the red migrate into the green, then turn yellow and move in the opposite direction. They take their studies a step further and address the question of how dynein is itself regulated during this process. They find that depleting lis1 causes dynein to accumulate at the hyphal tip while depleting dynactin reduces dynein at the tip. Finally they show that kin1 transports dynein/dynactin to the hyphoid tip by showing that knocking out kin1 blocks their accumulation. They go the extra mile and show that dynein does not colocalize with early endosomes moving to the tip and additionally show that the early endosomes still accumulate in the absence of dynein. This data together supports the following model taken from their paper. Inactive dynein is carried to the hypoid tip/MT plus end by kin1. Early endosomes are then transported to the tip at which time lis1 is activated by a yet undetermined mechanism. Lis1 then stimulates dynein dependent reterograde transport of the endosomes for recycling.

RNA decay Particles

2006-06-21T18:42:00.000-04:00

Ujwal Sheth from Roy Parker's lab details the molecular mechanism that targets RNAs with premature stop codons to processing-bodies (or p-bodies) via the non-sense mediated decay (NMD) pathway. P-bodies are dense cytoplasmic granule-like structures that serve as sites of mRNA storage/degradation. P-bodies contain decapping enzymes, RNAses and many other proteins of unkown function. In this paper the authors demonstrate that the NMD component, and RNA helicase, Upf1p, targets aberrant mRNA to granules. Upf1p's ATPase activity is then required to recruit Upf2p and Upf3p to p-bodies.

Ujwal Sheth and Roy Parker, Targeting of Aberrant mRNAs to Cytoplasmic Processing Bodies.
Cell (2006) 125:1095-1109

It remains unclear how premature stop codons are recognized in yeast. In higher eukaryotes, if a stop codon occurs before a splice site, ribosomes fall off the RNA before they can kick off exon junction complexes that mark sites along the RNA where splicing has occurred. The exon junction complex then recruits NMD components that target the mRNA for destruction.

There has been much fuss lately with these p-bodies and the related structures termed "stress granules". Both structures are seen in most eukaryotes and play several seemingly incompatible roles. In general non-translating cytosolic mRNAs are shuffled into these structures. But why?

Some facts about RNA bodies:
- these cytosolic structures do not contain membranes yet are very dense and exclude large proteins
- much of the maternal RNA in oocytes is stored in granules
- a related structure, termed simply "RNA granules", transport RNA up dendrites in neurons
- stress granules are thought to be formed by the aggregation of TIA1, a protein thought to have prion activities
- neuronal RNA granules are thought to be regulated by CPEB (Cytioplasmic Polyadenylation Element Binder), another protein thought to have prion properties
- RNAi components target RNAs to p-bodies, and proteins involved in RNAi are enriched in p-bodies

The question is, why pack RNA so tightly into dense structures? And why form these particles with aggregating prions? Prions can exist in several forms, so perhaps RNA granules must adapt several roles? Some RNA granules, such as those in neurons and oocytes store RNA (i.e. a precious cargo), but in other cases RNA granules act as trash compactors. I'm sure that this story will get more interesting in the coming months/years.

What are the side effects of your media?

2006-06-20T08:02:00.000-04:00

Just a brief mention of an interesting paper recently published in Neuromuscular Disorders. In this paper by Ian Holt in the laboratory of Glen Morris they explore the defects in nuclear morphology associated with lamin mutations resulting in emery dreifuss muscular dystrophy. The take home message is that nuclear morphology is more pronounced in growth media that allow for more rapid cell proliferation. This is clearly something to keep in mind for anyone studying the nucleus and probably macromolecular cellular assembly using standard tissue culture techniques.

Lamin A/C assembly defects in Emery–Dreifuss muscular dystrophy can be regulated by culture medium composition
Ian Holt, Nguyen thi Man, Manfred Wehnert , Glenn E. Morris

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T9T-4JXPS4K-3-3&_cdi=5123&_user=18704&_orig=search&_coverDate=06%2F30%2F2006&_qd=1&_sk=999839993&view=c&wchp=dGLbVtb-zSkzV&md5=dcf45a449ff42ce99e6813cd6e182e79&ie=/sdarticle.pdf

GPCR independent regulation of cell migration

2006-06-20T07:48:00.000-04:00

This is an interesting paper from the June 2006 issue of Developmental Cell by Dandan Shan et al. This is an important paper because it implicates G13 in regulating cell migration independent of the GPCR. They start by noting that G12/13 knockout MEFs do not migrate in response to PDGF like their wild type counterparts. Ectopic expression of G13 but not G12 rescues this defect. They find that this migration proceeds through Rac because constituitively active Rac induces migration of WT MEFs but not G13 knock-out MEFs. Although this suggests that Rac works upstream of G13, other data confuses this issue. Specifically, they find that constituitively active G13 does not induce migration and more importantly they find that Rac activation is normal in the G12/13 knockout cells with respect to wild type MEFs. The key experiment of this paper involves making a G13 construct that cannot couple to the GPCR. They find that this construct like wild type G13 can rescue cell migration in the knockout background indicating that this is a receptor independent process. Biochemical analysis indicates that Rac interacts with G13-GDP, PDGF induces complex formation, and if constituitively active Rac is used PDGF is not required to induce complex formation. These data lead to a mechanism where PDGF activates Rac (possibly through a GPCR) and this then stimulates the interaction between Rac and G13. The two proteins then together regulate cell migration.

The G Protein Ga13 Is Required for Growth Factor-Induced Cell Migration

Developmental Cell 10, 707–718, June, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.devcel.2006.03.014

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WW3-4K424R3-6-2&_cdi=7119&_user=18704&_coverDate=06%2F30%2F2006&_sk=%23TOC%237119%232006%23999899993%23624658%23FLA%23display%23Volume_10,_Issue_6,_Page_685-850_(June_2006)%23tagged%23Volume%23first%3D10%23Issue%23first%3D6%23date%23(June_2006)%23&view=c&_gw=y&wchp=dGLbVtz-zSkWA&md5=e5d9d737767fa0cee828776a0f11f954&ie=/sdarticle.pdf

ScienceSampler

2006-06-09T12:01:00.000-04:00

Myosin VI is an unusual motor

Myosin VI Stabilizes an Actin Network during Drosophila Spermatid Individualization

Tatsuhiko Noguchi, Marta Lenartowska, and Kathryn G. Miller
Mol. Biol. Cell 2006 17: 2559-2571

Similar to other myosins, myosin VI contains an ATP-dependent motor domain, a coiled-coil domain, and a globular tail domain. However, myosin VI moves towards the pointed end as opposed to the barbed end of an actin filament. Previous work has implicated myosin VI as a motor for endosomal movement due localization studies and in vitro assays showing the ability for myosin VI to form dimers and move processively. However, myosin VI can also act as an actin dependent molecular crosslinker. When expressed in baculovirus, a majority of myosin VI is monomeric and shows no processive movement.

This paper sheds light on the role of myosin VI in vivo during spermatogenesis in drosophila, specifically in the actin cone. The deletion of myosin VI decreases both the relative amount and density of F-actin as opposed to wild-type in actin cones, while overexpression of myosin VI has the opposite effect. These data combined with the effect of myosin VI deletion on actin cones by EM, and the persistence of GFP-myosin VI after FRAP, illustrates myosin VI's role as a crosslinker. The above cartoon shows, a role for myosin VI stabilization of the branched actin meshwork at the front of actin cones. No data obtained from this paper implicates myosin VI as a cargo transporter.

Additionally, the structure of the actin cone is unique as the filaments in the cone are oriented with their barbed ends facing away from the direction of movement. This mechanism is opposite of that found in Listeria comet tails and lamellipodia where barbed ends face toward the direction of movement. This difference causes the authors to speculate on the actin polymerization mechanism which is also depicted in the cartoon.

Why do baby neurons need LIS1?

2006-06-07T08:33:00.000-04:00

This time I picked an "old" paper. It was published last year on JCB and describes the effects of knocking down Lis1 during brain development.

LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.

Tsai JW, Chen Y, Kriegstein AR, Vallee RB.

J Cell Biol. 2005 Sep 12;170(6):935-45

Cortical neuron migrate from the ventricular region to the perifery of the brain during brain development. Cortical neurons originate from the division of radial glial cells. Prior to cell division, the nucleus from radial cells migrates, away from the ventricular region, then returns to the initial position. Only after this detour, known as nuclear oscilation, radial glial cells divide. The daugther cells migrate to the sub-ventrical zone where they stop and become multipolar. Then they start extending axonal processes, become bipolar, and resume migration.
Lis1 mutations originate Lyssencephaly. Pacients with this mutations have severe defects on brain development and die 1-2 years after birth. Lis1 mutations are thought to interfere with neuronal migration required for brain development.
In this paper, the authors provide clear evidence for a role of Lis1 in different stages of neuronal migration. They used a very powerfull technique (in utero electroporation) to introduce fluorescent siRNA or shRNA targeting Lis1 into cells at the ventricular region. Then they analised the position and shape of transfected cells, and most impressivelly, they followed neuronal migration in slices by timelapse microscopy, up to 18h.
Using these approached they show that Lis1 is required for nuclear oscilations and cell division in astral glial cells, bipolar migration and axonal extension.

Three Brief Papers on Nuclear Pore Complexes

2006-05-30T19:37:00.000-04:00

The nuclear membrane separates the nuclear space from the cytoplasm. This barrier is comprised of two membranes (Inner and Outer Nuclear Membrane) that are continuous with the endoplasmic reticulum. To cross the double membrane, molecules traverse the nuclear pore complex (NPC), a giant macromolecular complex that has an eight-fold symmetry and weighs over 100MDa. To date, only two components of the NPC have been identified: gp210 and Pom120. Interestingly many cells only express one of these proteins. Well it seems like Dirk Gorlich’s group have been able to knock out BOTH genes from Hela cells … and the cells are fine! They have NPCs, they can reform the nuclear membrane after mitosis … something strange is going on (On top of that another group previously claimed that gp210 was essential … in Hela cells!)

Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells
Fabrizia Stavru, Gitte Nautrup-Pedersen, Volker C. Cordes, and Dirk Görlich
JCB (2006) 173:477-83

In a second paper the Gorlich group solve the problem … there is a third integral membrane protein that is part of the NPC, called Ncd1. Turns out that yeast don’t have gp210 or Pom120 orthologues. Instead they have Ncd1 and two other non-conserved integral membrane proteins, Pom152 and Pom34. Of the three, Ncd1 is the only protein that is essential and conserved in other organisms. Interestingly Ncd1 is also part of the yeast spindle pole body, a structure related to our centrosomes … (Does this sound interesting Gomez?). The Gorlich lab show that there are Ncd1 orthologues that localize to NPCs in fly, frogs, worms, humans

The Gorlich lab knocked down Ncd1 in Hela and the NPCs were defective (lower staining of NPC components). Worm knockouts were mostly inviable.

NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes Fabrizia Stavru, Bastian B. Hülsmann, Anne Spang, Enno Hartmann, Volker C. Cordes, and Dirk Görlich JCB (2006) 173:509-19

Last is a paper from Martin Hetzer’s lab. Here they tackle the tricky question of how do you form NPCs. There are two times when you have to form these structures; after mitosis when the nuclear envelope reforms, and during interphase as the nucleus expands. Using complicated in vitro manipulations of nuclei formed from frog egg extracts, the Hertzer lab shows that active Ran is required both in the nuclear and cytoplasmic compartments to activate NPC formation in an expanding nucleus (an analogous situation to NPC formation in interphase). They offer evidence that ran acts to dissociate the Nup107complex, which is a major constituent of the NPC) from Importin-beta in both the nucleoplasm and cytoplasm. Interestingly they show that excess free Nup107 complex can stimulate NPC formation IN NUCLEI THAT DO NOT HAVE NPCs. That means that exogenously added Nup107 complex can stimulate the fusion of the Outer Nuclear Membrane and Inner Nuclear Membrane, to form a hole where the NPC sits. They then observe that gp210 form patches on the nuclear envelope and may act as landing pads for Nup107 complex in intact nuclei.

What this last paper shows is that within the lumen of the nuclear envelope threre is some machinery that acts to fuse the two membranes to form pores. What is the machine? Well it looks like of the integral membrane proteins, gp210 and Pom120 are dispensable. Ncd1? There are survivors of the Ncd1 knockouts in worms. These survivors also have NPCs. Thus it is likely that a whole fusion complex exists but is waiting for someone to find it …

Nuclear Pores Form de Novo from Both Sides of the Nuclear Envelope
Maximiliano A. D’Angelo, Daniel J. Anderson, Erin Richard, Martin W. Hetzer
Science (2006) 312:440-443

Cross posted at the Daily Transcript.

Focal Adhesion Turnover

2006-05-29T15:11:00.000-04:00

In The Journal of Cell Biology Anjana Nayal et al. published an article entitled Paxillin phosphorylation at Ser273 localizes a GIT1–PIX–PAK complex and regulates adhesion and protrusion dynamics. In this paper they explore how interactions between Paxillin, GIT1, PAK, and PIX – proteins already identified as regulators of focal adhesion turnover are regulated.

http://www.jcb.org/cgi/reprint/173/4/587

As all good cell biologists do, they immediately turned to phosphorylation and promptly identify serine 273 of paxillin as being phosphorylated. They go on to show that PAK catalyzes this phosphorylation and that GIT binds preferentially to phosphorylated Paxillin. Additionally an S273D phosphomimic Paxillin increases cell migration, membrane extension, and turnover of membrane protrusions.

The authors then notice that “small” focal adhesions that contain zyxin and vinculin are prevalent approximately 1 µm behind the band of actin at the leading edge in the cells expressing S273D paxillin. Importantly these turnover in less than 1 second and are not present in cells expressing S273A paxillin. GIT1 is also found in the small focal adhesions and siRNA against GIT1 results in a loss of the small focal adhesions.

Among the most important findings of the paper is that PAK acts both upstream and downstream of paxillin. This conclusion comes from the combined findings that PAK phosphorylates paxillin (upstream function) and that dominant negative PAK blocks the ability of S273K paxillin to induce small focal adhesions (downstream function). This downstream function is shown to be dependent on PAK interacting with GIT1 and PIX.

Unfortunately, there is no final figure to present their final model. It could have been particularly useful as this is a data rich paper much of which I have not discussed here. However the take-home message is that PAK activates (phosphorylates) Paxillin which in turn recruits GIT1 and Pix to the focal adhesion (for focal adhesion disassembly) and this complex in turn recruits PAK for downstream functions/reactivation of the pathway.

ER compartmentalization

2006-05-23T18:42:00.000-04:00

The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei
David Frescas, Manos Mavrakis, Holger Lorenz, Robert DeLotto, and Jennifer Lippincott-Schwartz
J. Cell Biol. 2006 173: 219-230.

Three Brief Papers on the ER

2006-05-23T07:17:00.000-04:00

Here are some cool ER papers I've seen recently:

Direct membrane protein-DNA interactions required early in nuclear envelope assembly
Sebastian Ulbert, Melpomeni Platani, Stephanie Boue, and Iain W. Mattaj
JCB (2006) 173:469-476

When the nuclear envelope reforms after mitosis, ER vesicles must bind to the condensed chromatin, but how does this occur? Well about half of the nuclear envelope (NE) proteins have basic luminal domains that mediate electrostatic interactions with the DNA itself. (In comparison about 4% of general ER and Golgi proteins have basic luminal domains.) To prevent ER/DNA association during mitosis, these basic NE proteins are phosphorylated.

ER-bound PTP1B is targeted to newly forming cell-matrix adhesions
Mariana V. Hernández, Maria G. Davies Sala, Janne Balsamo, Jack Lilien and Carlos O. Arregui
JCS (2006) 119:1233-1243

How does the ER remain extended in cells? This becomes a problem once you realize that actin, which is constantly polymerized at the cell's edge, is constantly being pushed towards the nucleus, and taking everything else along with it. Now it seems like the ER can interact with focal adhesions via protein tyrosine phosphatase 1B. This interaction may help anchor the ER at peripheral focal adhesion sites. Whether this is the only ER protein that can interact with focal adhesions remains unclear.

The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei
David Frescas, Manos Mavrakis, Holger Lorenz, Robert DeLotto, and Jennifer Lippincott-Schwartz
JCB (2006) 173:219-230

In Drosophila melanogaster embryogenesis, the first 13 nuclear division cycles are not accompanied by any cellularization. What you get is a giant single syncytium with over 6000 nuclei. At a specific time point all the nuclei migrate to the surface of the syncytium and then around each nuclei a cell membrane is constructed. In this paper the authors examined how the ER and Golgi of the syncytium are formed. After nuclear migration, the ER which previously was one giant cortical network, compartmentalizes around each nucleus. This even occurs prior to cellularization and requires a functional microtubule array.

The Hot Topic of Asymmetric Cell Division

2006-05-20T13:59:00.000-04:00

Two papers were recently released as early epub on the Nature Cell Biology website. Both of these papers the first by Izumi and collegues and the other from Siller and colleagues. Although the two papers provide a nearly identical story, they are each worth reading as you will notice some small details that differ between the two papers.

As asymmetric cell division is a hallmark of self renewing cells, this topic has garnered much interest in recent years. Although many of the molecualar components regulating asymmetric cell division have been identified (there are dozens of reviews - for example Knoblich 2001 in Nat. Rev. Mol Cell Biol.), the story is still not clear. These papers add more complexity to the story by identifying a new player. The take home message from each of these papers is that a protein called Mud, a NuMA related protein, plays an essential role in regulating spindle orientation during asymmetric cell division of drosophila neuroblasts.

Both labs show that Mud binds directly to the TPR region of Pins and further show that the endogeneous proteins interact by immunoprecipitation. Additionally both papers show that during interphase Mud localizes to the apical surface, and that this localization is dependent on Pins. During mitosis Mud maintains its cortical localization but is now also found at the centrosomes. The Izumi paper finds that this relocalization is microtubule dependent. Both papers find that Mud has no effect on Pins localization but the Izumi paper finds that in the absence of Mud neuroblasts will often contain more than two centrosomes.

Finally both papers show that Mud is essential for the spindle to properly align during asymmetric cell division. Although both papers provide convincing data to this end, the Siller paper takes things a step further using live cell imaging to show that not only does the spindle not align, but it also does not move.

Both papers are highly recommended!

http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb1409.html

http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb1412.html

Lamins are cool!!!

2006-05-08T22:09:00.000-04:00

Lamin A-Dependent Nuclear Defects in Human Aging

Paola Scaffidi ¹ and Tom Misteli ¹^*
Published online April 27 2006; 10.1126/science.1127168 (Science Express Reports)

Lamins are intermediate filament proteins and are the main component of the nuclear lamina. Although these proteins are expressed in different tissues, mutations in the LMN gene that encodes Lamin A and Lamin C, are associated with distinct genetic disorders such as muscular dystrophies, lypodystrophies and progerias.

Now it was found that the same mutation on lamin A protein associated with progeria, a rare disease were children suffer from premature aging, also accumulates in elderly human cells. This mutation originates a truncated version of lamin A.

Two key findings are reported:
- Traces of this truncated version are also observed in cells from young humans, although the protein does not accumulate, as observed in elderly human cells. This suggests that younger cells have mechanisms to destroy this truncated version.
- Cell nuclei from elderly humans have the same morphology and DNA-damage levels as cell nuclei from progeria patients. Impressively, this morphology is reversed when the production of the truncated protein is prevented, by expression of a morpholino targeting the "truncated mRNA".

Maybe in the future cell aging can be prevented by inhibiting the formation of truncated lamin A.... It will be cool to generate a trangenic mice expressing the morpholino that inhibits the formation of spontaneous truncated lamin A.

Breaking the diffraction barrier

2006-05-07T17:30:00.000-04:00

A recent paper from Stefan W. Hell group (Nature 440, 935-939, 2006) uses stimulation emission depleted microscopy - STED microscopy, to address the faith of synaptotagmin I, a vesicle protein, after stimulation of neurotransmitter release. This technology allows for resolutions well bellow the theoretical diffraction resolution limits of light microscopy (down to ~60nm).

"In a typical STED microscope the excitation beam is overlapped with a doughnut-shaped beam that is capable of de-exciting fluorophores by stimulated emission. Co-alignment of the beams ensures that fluorescence is allowed only in the central area of the excitation spot where the doughnut beam is close to zero" - see figure right

This is not the only current available technology where the diffraction barrier of visible light has been beaten. Others approaches such as "4Pi", "I³M" and "I5M" are also used. One key aspect of these technologies is the resolution which is obtained in the Z-axis, much better than any available confocal microscope - see figure left. This will revolutionize fields such as epithelial cell polarity.

Currently only Leica (Leica TCS 4PI) sells microscopes with this technology, although soon other companies will release equivalent products.

Further reading:
Blinded by the Light, Bio-IT world
Hell, S. W. Toward fluorescence nanoscopy. Nature Biotechnol. 21, 1347Â1355 (2003)

2006-05-07T14:34:00.000-04:00

Hi,

The idea of this blog came from long conversations with some other bloggers. Here is the deal: To describe what is currently being discussed in the 5 minute men journal club. What is the 5 min men? Me and some other people from Columbia Univ. get together every friday and present a paper in 5 min + 5 min discussion. We have been doing this for almost a year and it is great. Since one of the funding members is leaving to San Francisco, I thought on going virtual.

So I hope that this will work. Stay tuned.