Actin-dependent nuclear movement in neurons

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

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

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.