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

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

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

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

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.