Artificially inducing close apposition of endoplasmic reticulum and mitochondria induces mitochondrial fragmentation.

We have just released a new paper from our lab arising from Vicky Miller’s work.

Mitochondrial fragmentation

In this paper we show that artificially juxtaposing the ER and mitochondrial membranes is sufficient to drive fragmentation of the mitochondria. Vicky used the rapamycin-dependent knock-sideways system developed by Scottie Robinson in Cambridge to achieve this. Using an engineered ER-localized transmembrane domain tethered to FKBP and a mitochondrially targeted FRB domain she was able to induce close apposition of the two membranes in a rapamycin dependent manner. Two things were immediately evident on doing this – first that there is a rapid sequestration of the mito-YFP-FRB into puncta that presumably represent a sub-domain of the mitochondrial membrane and second, over the course of the next 5-30 minutes, fission of mitochondria was evident. This was shown using fluorescence recovery after photobleaching to show that these fragments were indeed independent of one another. Vicky also showed that these fragments retained their mitochondrial membrane potential.

Our work shows that this artificial “knocking” of the ER to the mitochondria is sufficient to drive fission. We have not explored this phenomenon in further detail as it lies considerably outside of our area of expertise. However, we do believe that this engineered system might have some use in dissecting the mechanisms that control ER-mitochondrial contacts and the consequences of this. Consequently, we have made this work available as a preprint on BiorXiv and have also submitted it for peer review. Constructs will be freely available and when we get the chance we will deposit them with Addgene.

The full PDF is available on BiorXiv here: http://www.biorxiv.org/content/early/2014/05/28/005645

Miller, V.J. and Stephens, D.J. (2014) Artificially inducing close apposition of endoplasmic reticulum and mitochondria induces mitochondrial fragmentation. BiorXiv dx.doi.org/10.1101/005645

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Our first BioRxiv preprint uploaded

I have just uploaded our first paper to the BioRxiv preprint server:

Figure from Brown-et-al

“OPPOSING MICROTUBULE MOTORS CONTROL MOTILITY, MORPHOLOGY, AND CARGO SEGREGATION DURING ER-TO-GOLGI TRANSPORT.”

Anna K Brown, Sylvie D Hunt, David J Stephens
doi: 10.1101/001743

The paper is a follow up to our Journal of Cell Science paper from 2013 in which we showed similar outcomes for motors in the endosomal system.

This paper has been long in the making and was in fact our starting point for the project. Despite us thinking that this would prove  a more tractable system (with a defined end-point for trafficking etc) the endosomal system provided a better model for this work. In this project we have also really been trying to find membrane anchors for dynein that direct ER-to-Golgi transport. I’ll admit that we held off on publishing early in the hope that we did and that we could incorporate these assays into that work. Unfortunately it didn’t work out that way. We also could have published this some time ago before Anna (first author) left for maternity leave. I retained the hope of extending the study through other work even while she was away. This didn’t happen (but we still have hopes). We left these data out of Sylvie’s paper for clarity. That paper remained entirely focussed on the endosomal network.So now, we have decided to publish as a follow-up to Sylvie’s paper on sorting nexins and motors from last year, we had to choose what to do. This in our view did not have the necessary detail (“mechanistic insight”??) for somewhere like Molecular Biology of the Cell or Journal of Cell Science. This choice coincided with the opening of BioRxiv as a preprint server for Biology.

[Declaration: I (David) am an “Affiliate” of BioRxiv]

This seemed like a very good option for this work to get it in the public domain ASAP. We will also submit this to a journal for full peer review as I believe that this remains an important part of the process. As such posting on BioRxiv is not the end point on this journey just an extra step for us that makes our data (and interpretation) freely available to all immediately. We don’t have to wait for peer review, revision, formatting and hosting. I cannot see that as anything but a good thing. The preprint has a DOI so can be cited and easily referred to. We can now decide where to submit this for full peer review. Many journals already allow posting (including EMBO and PLOS) and likely others will soon change their editorial policies to follow suit. Some won’t and that is their prerogative. Would we still send our future work to one of the journals that does not allow BioRxiv posting first? Yes, if we thought it was the most appropriate place, we certainly would. 

Our funder for this work (the UK Medical Research Council or MRC) mandates open access and I think BioRxiv is a good part of this process. I also chose the CC-BY license option as the most liberal in terms of re-use, data mining etc. This is also consistent with our funder policy.

For further info on BioRxiv I recommend you take a look. The common questions are readily answered on the BioRxiv website.

Alfonso Martinez-Arias has written on his blog on why he supports the preprint server system and I see no point in reiterating comments that I agree with! I strongly recommend that you read this if you are still wondering whether this is a good idea or not.

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.

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

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.