Comments on: The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1.

I evaluated the following article for F1000 Prime.

The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1.

Barlan K, Lu W, Gelfand VI.

Curr Biol 2013 PMID: 23394833 DOI: 10.1016/j.cub.2013.01.008

This paper defines the microtubule binding protein ensconsin as an obligate co-factor for kinesin-1 drive in motility in Drosophila. In a series of elegant experiments, the authors show that ensconsin is not required for the recruitment of kinesin to membranes but is in some way involved in activating the motor. Ensconsin does not seem to affect the amount of membrane-bound kinesin in cells so the authors sought to define its role in motor activation. Kinesin-1 normally exists in an auto-inhibited confirmation in cells. In a key experiment the authors showed that removal of this auto-inhibition within the kinesin-1 motor by mutation eliminates the requirement for ensoconsin in vivo. These data suggest a model in which ensconsin acts to relieve the auto-inhibition. An important point is that this function of ensconsin did not require its own microtubule binding activity. The authors postulate that spatial restriction of ensconsin to microtubules acts to refine the spatial activation of kinesin-1, adding to the diversity of mechanisms that control the spatial and temporal organization of microtubule motor activation.

Note: Ensconsin is also known as E-MAP-115 and MAP7.

These comments also appear on Faculty of 1000 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. 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.

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.

PhD studentship opportunity in our lab

We have a potential PhD studentship available to start in October 2013.

This studentship is part of the MRC Doctoral Training Programme at the University of Bristol. As such you apply to the Programme and the selection process is in competition with all other advertised projects. You can find full details on the programme, including the other available projects here:

MRC Doctoral Training Programme at the University of Bristol.

This is an exciting inter-disciplinary project bridging ongoing work in Biochemistry and Physics. The project would suit a graduate in biochemistry, cell biology, or biophysics. Full training will be provided and the training elements can be tailored to the interests of the successful candidate.

Please note the eligibility criteria: Only applicants from the UK/EU are eligible for this programme.

Title: Analysis Of Integration Between Membrane And Cytoskeleton Dynamics Using Advanced Light Microscopy

Supervisors: Professor David Stephens (Biochemistry) & Dr Henkjan Gersen (Physics)

To apply for this project please select ‘Faculty of Medical and Veterinary Sciences’ and ‘Biochemistry (PhD)(4-yr)’. Please also identify ‘MRCDTG’ as your fee payer in the Funding section of the online application.

The intricate relationship between endomembranes and cytoskeletal filaments governs the spatial organization, morphology, and function or organelles. Multiple cellular functions that coalesce around Golgi membranes are governed by small GTPases of the Rho family, Cdc42 being the most significant Rho GTPase at the Golgi (1). Recent years have seen the emergence of the septins as a critical component of this system; Cdc42 is known to dictate septin filament organization (2). Septin filaments act in concert with microtubules to direct trafficking around the Golgi (3). Septins also dictate the formation and function of primary cilia, a “cellular antenna” that integrates key signalling pathways essential to normal organism development and tissue function (4, 5, 6). Through selective disruption of Cdc42, Golgi, or septin function, we will define how the classical structure of the Golgi apparatus is defined by septin filaments and vice versa.

Septins adopt a highly conserved structural organization within filaments that can be detected by polarization fluorescence microscopy (7, 8), allowing the subunit architecture of septin filaments to be analysed in an intact cell context. This advanced bioimaging approach will form a core training aspect of the work and would suit a biomedical science graduate with a keen interest in imaging or a biophysics graduate with a strong interest in cell biology. The project bridges the Biochemistry and Physics departments at the University of Bristol. You would be based in the Stephens lab in the School of Biochemistry within newly refurbished cell biology laboratories and the project will involve considerable mammalian cell biology using gene silencing and advanced light microscopy. The Gersen lab, located a short distance away, will provide training in development and application of novel optical microscopy methods, notably fluorescence polarization. Successful PhD training is ensured through links to existing cell biology and nanoscience students in both labs as well as international collaboration.

Informal enquires to David Stephens (david.stephens@bristol.ac.uk) or Henkjan Gersen (H.Gersen@bristol.ac.uk) are welcome.

For further details see:

http://www.bristol.ac.uk/biochemistry/stephens/index.html

http://www.bristol.ac.uk/physics/people/henkjan-gersen/index.html

References

• S. Etienne-Manneville, Cdc42–the centre of polarity. J. Cell Sci. 117, 1291 (Mar 15, 2004).
• G. Joberty et al., Borg proteins control septin organization and are negatively regulated by Cdc42. Nat. Cell Biol. 3, 861 (Oct, 2001).
• E. T. Spiliotis, Regulation of microtubule organization and functions by septin GTPases. Cytoskeleton 67, 339 (Jun, 2010).
• Q. Hu et al., A septin diffusion barrier at the base of the primary cilium maintains ciliary membrane protein distribution. Science 329, 436 (Jul 23, 2010).
• J. R. Bowen, D. Hwang, X. Bai, D. Roy, E. T. Spiliotis, Septin GTPases spatially guide microtubule organization and plus end dynamics in polarizing epithelia. J. Cell Biol. 194, 187 (Jul 25, 2011).
• E. T. Spiliotis, S. J. Hunt, Q. Hu, M. Kinoshita, W. J. Nelson, Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules. J. Cell Biol. 180, 295 (Jan 28, 2008).
• B. S. DeMay et al., Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals. The Journal of cell biology 193, 1065 (Jun 13, 2011).
• S. A. Rosenberg, M. E. Quinlan, J. N. Forkey, Y. E. Goldman, Rotational Motions of Macromolecules by Single-Molecule Fluorescence Microscopy, . Accounts of Biochemical Research 38, 583 (2005).

Potential applicants are encouraged to contact David when applying.

The deadline for applications is Wednesday 16th January 2013 and interviews are likely to be in the weeks of 11th February and 18th February 2013.

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.

Movies:

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”!

David

Evaluated: Boncompain et al (2012) Synchronization of secretory protein traffic in populations of cells.

I evaluated this for F1000 (yes, I’ve been busy!). Looks like a really useful system for many, especially given its adaptability.

G Boncompain, S Divoux, N Gareil, H de Forges, A Lescure, L Latreche, V Mercanti, F Jollivet, G Raposo and F Perez  (2012) Synchronization of secretory protein traffic in populations of cells. Nature Methods 2012 9 PMID: 22406856 DOI: 10.1038/nmeth.1928

This paper presents an exciting and widely modifiable system to study organelle dynamics and secretory protein traffic in a controlled manner. The retention using selective hooks (RUSH) system is based on controlled retention of proteins in the endoplasmic reticulum (ER) allowing one to exploit any number of targets through genetic engineering of relevant reporters. There are many advantages over other systems that have been highlighted by the other evaluations. The system presents some great options for both image-based and biochemical assays. I entirely agree with their comments and look forward to seeing this system in wide use (I note there are no restrictions on academic use).

PowerPoint with embedded movies on the iPad

Having recently bought and iPad but not being a Mac user I found some issues with getting the content I wanted to loaded.

The simple thing was Powerpoint presentations with embedded movies. On my PC these are all WMV files which I generated from original AVI files.

I am sure there are 1001 ways of doing this but I thought I would share mine as clearly there are 1001 websites with conflicting info on there. The easy solution I found was to buy Keynote from the app store. This runs all my PowerPoint presentations easily it seems. The only issue was then getting the embedded movies in.
Solution was to convert them to MP4 for which I use the (free) Leawo MP4 Converter.

This was a very quick easy and free way to convert and enables batch conversion. The whole folder took about 10 mins. A couple did not convert but I think this is down to old codecs used to make them in the first place.

I then uploaded these to my iPad using iTunes (Photos folder, manually selecting which folders to use).

In Keynote on the iPad I then found I simply needed to deletee the embedded movie which wouldn’t run and insert (as a new item) the relevant MP4.
Bingo. Cost £6.99 for Keynote app, time taken ~ 2 hours to work it out and then achieve. Result, nice looking presentation on iPad. The display on the 2012 version is great for this.
The method which didn’t work was to use “replace” not delete and insert from scratch. Having looked at a variety of websites about this issue, this seems to be where some go wrong.

All good now – question remains when will I be brave enough to present using this alone and not take my trusty laptop with me? For the time being I can now load any presentation I have to at least show and discuss whenever I like.

Link

Following on from my earlier post….

While I decide whether we need anything further I have developed a Word form for the lab to use for each imaging experiment. It has a built in date stamp,drop boxes for commonly used antibodies and cell labels and customized content for our most commonly used imaging systems. I had no idea how to make forms in Word but it seems pretty easy; this also means it will be easy to adapt over time. We might reject this idea or use it for the next 10 years – time will tell.

I’ve attached this finalized draft: Experiment Log 2012a in case it is of any use to anyone else. It is read only such that the user gets to change the filename. The date in the top right should auto update as well. the idea is that the “user” saves this along with the image. Frighteningly simple but hopefully so simple it will be effective. Also something that I hope will be usable from undergrad to Professor.

Should you wish to adapt this for your self you will probably need to use this editable version: Experiment Log 2012 template unprotected.

How do I manage our microscopy data?

So today I solved one major lab issue (I think).

I have always used “personal” file servers to archive our data: essentially simple hard drive arrays in RAID5 configuration. These have been accessible to all in the lab from undergraduate project students to postdocs. These are great but they do fail .

Recently two of ours failed.

Had these two drives been in the same array, we would have lost a lot of our imaging data. So I felt the need to do something about this. My University has fortunately just implemented 5Tb (yes, Tb not Gb) of network storage for each “data steward” (essentially each academic). This is automatically backed up and can (in future) be archived to tape. This seems to solve the problem for 5Tb at least but also for more (if we pay). Local IT have also provided a possible solution for local file storage (i.e. within the building) which could be a very useful solution for our core research facilities such as Bioimaging and Proteomics that generate large data volumes.

The large network data store required me to write a data management plan which I did using this online Data Management Planning Tool by the UK Digital Curation Centre through a JISC funded project. This was surprisingly useful in the way it prompted me to think about the way that I organize lab data and consider its future use, access etc.

So, now to the question – HOW do I organize these data sets.

We take a lot of microscopy data from a lot of different imaging systems. Thus formats are complex and not always future-proofed. one plan here is to export as OME-TIFF using the Open Microscopy format that includes the META data. This is simple and fine. In fact we acquire most of our data using Volocity from Improvision/Perkin Elmer – this includes good metadata and (at least for now) the core software is free from Perkin-Elmer on registration.The OME-TIFF option would allow us to take everything including the meta data into ImageJ or equivalent very easily.

Its more the folder structure that requires better organization. Currently I list everything in date order (yy-mm-dd) followed by a brief experiment name. This date format means I can sort by date acquired (which to a large extent is how my brain remembers experiments). I am wondering whether to then include a Word/Text doc alongside detailing methods, labelling etc is enough. We can usually get this annotated directly on the microscope. The other thing I plan to include is a short description of the experimental goal and outcomes where possible. This might in fact be within a higher level directory where we organize by project/sub-project. I need to get the lab to properly commit to this. Progressive creep away from such rigid systems is also a concern but that is another issue…

So….does anyone have a better way? I wish to have something that is simple but highly effective. I want to look through our data archives and know exactly what everything is without referring to the person who acquired the data. I should already be there but I am not.

Before someone suggests OME, we don’t have the wherewithal to move to OMERO – the server setup is beyond me and not something that we can implement easily through our IT support. This is one for the future…

The good news is that I am taking data management more seriously as required by Research Councils (in the UK at least but I guess also internationally by all funders). The bad news is that I clearly have a long way to go with this.