What’s up with the Stephens Lab blog?

Nothing is up – that’s the problem. I think that having the blog has taught me one key thing.

I don’t have time to blog!

One day there will be another update!


Comments on: Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport

Vicky Miller (Postdoc in the Stephens lab) and I (David) have written the following comments on this paper that also appear on the Faculty of 1000 site. Vicky is also on Twitter @Dr_VickyMiller

Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport.

Cutrona MB, Beznoussenko GV, Fusella A, Martella O, Moral P, Mironov AA.

Traffic 2013; DOI: 10.1111/tra.12060 PMID: 23433038

This paper describes studies of trafficking of secretory cargo from the ER in cells depleted of the small GTPase Sar1, an essential component of the COPII coat. A COPII-independent mechanism of secretory trafficking would mark a step-change in our understanding of secretory protein trafficking in cells. While we do not think that this paper requires such a re-evaluation of our thinking, it does provide some thought provoking data. By disrupting the normal mechanism of transport the authors seek to reveal COPII-independent mechanisms and challenge the primacy of COPII-dependent transport. The most important point to bear in mind when reading this paper is that siRNA knock-downs are unable to generate a complete depletion of any protein and that residual Sar1 may influence some of the results seen. Indeed in our opinion, one cannot conclusively draw the conclusion that COPII-independent ER export pathways exist in cells from this work alone.

The authors confirm that Sar1-depletion effectively inhibits COPII vesicle formation (demonstrated by EM and reduced immunofluorescence staining of COPII components). They see changes to both ER-Golgi intermediate compartment (ERGIC) formation (the compartment between the Golgi and the ER) and Golgi organisation (resulting in formation of mini-stacks), also in agreement with a reduction in secretory trafficking. Despite this, pulse-chase experiments show no reduction in the total amount of protein secretion in Sar1-depleted cells. From this, the authors conclude that alternative transport mechanisms are being used. The virus glycoprotein VSV-G also passes from the ER to the plasma membrane in Sar1-depleted cells, even when additionalCOPII subunits (Sec23A and B) are depleted in addition to Sar1A and B. Based on these and other experiments, the authors propose a COPI-dependent replacement pathway that takes VSV-G to the Golgi. Export of procollagen transport is inhibited by Sar1-depletion, but as collagens require specialized COPII-coated vesicles to accommodate their large size, they therefore are not representative of typical COPII trafficking (Jin et al., 2012). Furthermore, many other studies support the notion that procollagen secretion is exquisitely sensitive to perturbation of the early secretory pathway (Smits et al., 2010; Townley et al., 2008; Venditti et al., 2012). 

These data do support the concept of a “short-loop” ER-to-Golgi trafficking pathway that involves juxtanuclear ER and possible even ER-Golgi contact sites. It is as yet unclear how possible mechanisms such as kiss and run could retainsufficient selectivity to prevent non-selective transport. It remains possible however that the small remaining amount of COPII proteins in the RNAi experiments described here is sufficient to direct COPII-dependent selectivity.

In summary the paper presents some interesting findings. Complete removal of Sar1 from cells by gene deletion is required to examine fully the existence ofCOPII-independent trafficking pathways and the relative importance of COPII-trafficking in cells. 


Jin, L., K.B. Pahuja, K.E. Wickliffe, A. Gorur, C. Baumgärtel, R. Schekman, and M. Rape. 2012. Ubiquitin-dependent regulation of COPII coat size and function. Nature 482(7386):495-500. doi: 10.1038/nature10822

Smits, P., A.D. Bolton, V. Funari, M. Hong, E.D. Boyden, L. Lu, D.K. Manning, N.D. Dwyer, J.L. Moran, M. Prysak, B. Merriman, S.F. Nelson, L. Bonafe, A. Superti-Furga, S. Ikegawa, D. Krakow, D.H. Cohn, T. Kirchhausen, M.L. Warman, and D.R. Beier. 2010. Lethal skeletal dysplasia in mice and humans lacking the golginGMAP-210. N. Engl. J. Med. 362:206-216.

Townley, A.K., Y. Feng, K. Schmidt, D.A. Carter, R. Porter, P. Verkade, and D.J. Stephens. 2008. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. J. Cell Sci. 121:3025-3034.

Venditti, R., T. Scanu, M. Santoro, G. Di Tullio, A. Spaar, R. Gaibisso, G.V. Beznoussenko, A.A. Mironov, A. Mironov, Jr., L. Zelante, M.R. Piemontese, A. Notarangelo, V. Malhotra, B.M. Vertel, C. Wilson, and M.A. De Matteis. 2012. Sedlin controls the ER export of procollagen by regulating the Sar1 cycle. Science. 337:1668-1672.

Full disclosure: David is a member of the “Traffic” editorial board but had not role in the editing or reviewing of this paper.

These comments are also published on F1000 Prime

Art competition entries 2012

I entered a few of my images into our Faculty Art Competition this year. They didn’t win but I thought I would post them here anyway.

Click on the images for a larger view.

You can see the winners here.

3D rendering of ciliated cells

3D rendering of ciliated cells. LLC-PK1 (pig kidney epithelial) cells were grown on a round micropattern to constrain growth. Cilia are in green with the Golgi in magenta and nuclei in blue. The image is a 3D rendering of a deconvolved z-series acquired using widefield microscopy.

Image of Aequorea victoria.

A pseudocoloured image of the jellyfish Aequorea victoria from which Green Fluorescent Protein was isolated. The pseudocolouring illustrates the diverse colour palette of GFP variants that we now have available.
The image is a photograph taken by me at Monterey Bay aquarium in 2003. There is only one jellyfish in the original photo, this image is a montage.

Evalutaion of “SUMOylation of the small GTPase ARL-13 promotes ciliary targeting of sensory receptors”

I evaluated this article for F1000 Prime

Y Li, Q Zhang, Q Wei, Y Zhang, K Ling and J Hu (2012)
SUMOylation of the small GTPase ARL-13 promotes ciliary targeting of sensory receptors.

J Cell Biol. 2012 Nov 12; 199(4): 589-98. PMID: 23128241 DOI: 10.1083/jcb.201203150

This paper is intriguing because it shows that SUMOylation of a very small pool of the small GTPase Arl13 is required for trafficking of some receptors into primary cilia. Arl13 is essential for cilia function, with mutations in Arl13 leading the Joubert syndrome. Arl13 is required for the formation of primary cilia (ciliogenesis) and also for the trafficking of certain receptors into the cilium. Here, the authors show that SUMOylation of Arl13 is not required for ciliogenesis itself, but is required for the trafficking of cargo, including polycystin-2, to the cilium once formed. The mechanistic basis of this role remains to be determined. The fact that only a very small pool of Arl13 is SUMOYlated at any one time will complicate analysis, but also hints at either a highly specialized pathway or, perhaps more likely, a very dynamic SUMOylation/deSUMOylation pathway being in operation.

Movies of primary cilia: GFP-Rab8A

I made some time-lapse movies this week of cilia in pig kidney epithelial cells (LLC-PK1) and saw some interesting things that I can’t explain and/or don’t really understand. I thought this would give a good chance to explore this as a route to getting some feedback. I would be very interested in any comments on these videos, especially those labelled 1 and 2. The major caveat is that this is from transient transfection so is at a relatively high level of expression. I absolutely do not rule out that we are inducing artefacts by doing this but it is just possible that we are highlighting structures better. Lots more we could do of course but in particular I am keen to know whether anyone knows of a similar tubular network to that in Movie 1 or to the odd “Trafficking” event seen in Movie 2.

Experimental detail:

Cells plated 24h before transfection and imaged 18h after that (so a total of 42h from plating to imaging).

Transfection was done using 1 microgram of DNA and Lipofectamine 2000 (standard protocol, premix LF2000 with Optimem for 5 mins then mix with DNA and 15 mins later drop carefully on to cells). GFP-Rab8a cDNA was a kind gift from Johan Peränen (Helsinki, Finland; construct described in Hattula, K., Furuhjelm, J., Tikkanen, J., Tanhuanpaa, K., Laakkonen, P., and Peranen, J., 2006. Characterization of the Rab8-specific membrane traffic route linked to protrusion formation. J. Cell Sci. 119, 4866-4877).

Cells were grown in complete medium throughout and imaged in DMEM supplemented with HEPES and sodium bicarbonate but in the absence of serum or phenol red. Over these short times, serum withdrawal does not seem to change ciliation of these cells – they grow cilia in complete medium on reaching confluence. Imaging was done on a wide-field microscope and hopefully these movies include scale bars and time stamps.

The videos are hosted by YouTube and are available through the the links below.


1. Tubular network visible below the primary cilium.

Is this a subset of endosomes? Part of the ER? Part of the cilium itself e.g. an expanded pre-ciliary vesicle? I wonder if this could be an expanded Rab11/Rab8 compartment as implicated by many papers including those of Chris Westlake and colleagues (. ).

2. Unusual looping out from base of cilia.

This looks odd, 3D reconstructions suggests that this “object” is indeed emerging from the base of the cilium and then seemingly heading back in again. Could this be something related to intraflagellar transport (IFT)? I need to read more on the way that Rab8 is trafficked to, and within, the cilium.

The next three are more just observations of things that one might expect to see.

3. Long wavy cilia.

This is a maximum intensity projection of a z-stack where the cilium is showing some substantial movements – not entirely sure how to describe it – we aren’t doing anything to these cells so this is really just diffusive/random motion. These are of course non-motile cilia so it isn’t beating. Just looks good! A key caveat is of course the speed of imaging versus the speed of movement.

4. 3-part cilia?

This video shows some retraction of a ling cilium, presumably induced by us imaging it (i.,e. photodamage). The interesting parts are the apparent 3-part labelling of the structure at the start of the movie suggesting a difference in thickness or accumulation of GFP-Rab8a along the length. The final structure is something we often see around the dish of cells – presumably “damaged” cilia. They are long and fragile in these in vitro cultures.

5. Cilia wrapping around one another

Here we see several cilia on adjacent cells interacting with one another. This seems similar to what the Lippincott-Schwartz lab has recently described in the journal Cilia:

Primary cilia utilize glycoprotein-dependent adhesion mechanisms to stabilize long-lasting cilia-cilia contacts Carolyn Ott, Natalie Elia, Suh Jeong, Christine Insinna, Prabuddha Sengupta, Jennifer Lippincott-Schwartz Cilia 2012, 1:3

It is hard from our video to define individual cilia or to determine the extent or effect of any interaction. As always it is hard to capture this to video – looking down the microscope, these structures clearly look like cilia and are certainly on top of the cells so do not represent the tubular network seen in video 1.

So, thoughts please. Comments here preferred but I’ll check YouTube as well.

PS Any alternative to this method of posting or problems people encounter would be welcome. Would this be better on Figshare? I haven’t explored that yet and these aren’t really figures, just observations on which I would welcome feedback.

If you got this far, thanks for checking my first contribution to the brave new world of Open Science also known as “crowd sourcing better understanding”!


Evaluated: The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation.

The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation.
McMahon C, Studer SM, Clendinen C, Dann GP, Jeffrey PD, Hughson FM. (2012)
J Biol Chem. in press. PMID: 23109340 DOI: 10.1074/jbc.M112.420141

This article caught my eye for two reasons: first, it maps at high resolution the likely interface between the guanine nucleotide exchange factor Sec12 and the small GTP binding protein Sar1. This interaction is critical for Sar1 activation, the essential precursor to COPII vesicle formation at the endoplasmic reticulum. Second, and where this paper is really worth a look is in the finding that this “K loop” on Sec12 that contacts Sar1 also binds a potassium ion. Furthermore, this K+ ion is essential for the catalytic activity of Sec12 towards Sar1. This paper provides a great example of the value of crystallography on proteins for which there is a very strongly predicted structure.

Dynein alone?

I evaluated the following article for F1000:

Dynein light chain 1 and a spindle-associated adaptor promote dynein asymmetry and spindle orientation.
AK Dunsch, D Hammond, J Lloyd, L Schermelleh, U Gruneberg and FA Barr J Cell Biol. 2012 Sep 17; 198(6): 1039-54
PMID: 22965910 DOI: 10.1083/jcb.201202112

The thing that fascinates me the most about this is the possibility that dynein containing light chain-1 acts without dynactin…..hence the crap title for the post.

This paper is highly intriguing from a dynein motor perspective as it strongly suggests that, in mitosis, the binding of dynein light chain 1 (DYNLL1) and the major dynein regulator dynactin are mutually exclusive. The work details very clearly how the adaptor proteins CHICA and HMMR target dynein to the mitotic spindle. This serves to control spindle positioning in cells, at least in part through promoting dynein asymmetry. It will be fascinating to determine whether DYNLL1 and dynactin can bind to dynein at the same time in interphase cells; this would of course have major implications for those studying dynein and dynactin function.