Comments for Stephens Lab Blog

Comment on Movies of primary cilia: GFP-Rab8A by stephenslab

stephenslab — Mon, 26 Nov 2012 14:10:46 +0000

Thanks Viki,
I agree – they just look wrong and anyway, why wouldn’t it spread more widely. My bet is on a sub-set of endosomes.
Thanks for taking the time to take a look!

Comment on Movies of primary cilia: GFP-Rab8A by Viki Allan

Viki Allan — Mon, 26 Nov 2012 13:53:36 +0000

Hi David, I’d say the tubular network definitely does not look like ER! The major difference is in the angles of the junctions between tubules.

Comment on Comments on “Structural basis for microtubule binding and release by dynein”. by stephenslab

stephenslab — Thu, 22 Nov 2012 09:39:42 +0000

Thanks for the comment Dynein Dude.
Perhaps this salt bridge doesn’t need to be regulated. Could one have a situation where the salt bridge provides the enhanced dynamic range and then processivity (or other features) are modulated by association of accessory subunits, post-translational modifications etc?
The cell biology of the mutant incapable of forming the salt bridge could be an interesting one to look at.

Comment on Comments on “Structural basis for microtubule binding and release by dynein”. by Dynein dude

Dynein dude — Wed, 21 Nov 2012 18:42:06 +0000

Any thoughts on how the cell might tune the dynamic salt bridge, other than local changes in ionic strength (which seems unlikely)?

Comment on Movies of primary cilia: GFP-Rab8A by Dave Bridges (@dave_bridges)

Dave Bridges (@dave_bridges) — Mon, 19 Nov 2012 16:32:43 +0000

in our experience, the gtp loading state seems to vary… some gtpases are mostly active (GTP) and others are mostly inactive (GDP). I think because of that usually the CA/DN mutants can end up being more informative functionally. I wouuld guess that the wt would roughly phenocopy the DN if the gtpase is mostly GDP (i doubt much is empty).

Comment on Movies of primary cilia: GFP-Rab8A by stephenslab

stephenslab — Mon, 19 Nov 2012 15:33:05 +0000

Great points Dave.
1. Tubular network rare, clearly expression level dependent phenotypes here but all likely revealing I’d say.
2. No apparent correlation between phenotypes and expression level.
3. Its wild-type Rab8a so I would expect most to be empty (?).
Always unclear in these transient expression experiments as to how much the probe reveals of a “naturally occurring process” and how much the probe itself induces. More work needed, just hoping someone might have seen the same….
Thanks again for comments.

Comment on Movies of primary cilia: GFP-Rab8A by stephenslab

stephenslab — Mon, 19 Nov 2012 15:29:01 +0000

Great idea- would love to do that …..

Comment on Movies of primary cilia: GFP-Rab8A by Dave Bridges (@dave_bridges)

Dave Bridges (@dave_bridges) — Mon, 19 Nov 2012 14:31:56 +0000

I am far from an expert in cilliogenesis but here are my thoughts. If Rab8 subfamily GTPases both cause the formation of cilia, and also positively mark them, it might be easier if a secondary marker was present, to see whether the primary effect are on ciliogenesis or on Rab8 localization.

1) What is the releative distribution of these? For example I can really only see the tubular network in the one image.
2) Are these roughly equivalent expression levels, or for example do you get the multiple cilia when you have really high Rab8A expression?
3) Do you have any sense whether the transfected Rab8A is mostly GTP or GDP in these cells?

Comment on Movies of primary cilia: GFP-Rab8A by manuel thery

manuel thery — Fri, 16 Nov 2012 19:34:26 +0000

video 1: if you bleach the cilia, will you empty the network? and vice-versa?

Comment on Dynein alone? by Dynein dude

Dynein dude — Wed, 10 Oct 2012 14:47:08 +0000

The proteomics work sounds very interesting, looking forward to reading the paper! The LIC’s are mysterious subunits.

Also looking forward to more blog posts (about dynein hopefully!)

Best,
Dynein dude