Tiny Universe: new paper on intracellular nanovesicles

We have a new paper out! You can access it here.

The paper in a nutshell

We have discovered a new class of trafficking vesicle inside cells.

These vesicles are very small (30 nm across) and we’ve called them intracellular nanovesicles, or INVs for short.

What is a trafficking vesicle?

Humans are built from lots of cells. Rather than being boring building blocks, each cell interior is a busy world where proteins are made and go to work, constantly moving around. All this traffic allows each cell to eat, drink, reproduce, and much more. We are interested in the cell’s membrane trafficking system: a transport network of different types of membrane vesicle that important proteins can travel in. Membrane trafficking ensures that cargo proteins go to the right place at the right time.

Several classes of trafficking vesicle have been described over the last sixty years or so. Vesicles are typically classified by their size, their role, the compartments they travel between, and the proteins associated with them. For example, COPII vesicles travel from the endoplasmic reticulum (ER) to the Golgi apparatus, carrying cargo proteins for export. These vesicles are 70-90 nm in diameter and have a COPII coat.

We made the unexpected discovery of a novel vesicle type that was much smaller than all the others that had been described.

How to catch a vesicle

We unexpectedly found INVs by catching them inside cells! We did this by moving a protein called TPD54 to the mitochondria. This is a trick we use a lot in the lab. We make the protein sticky so that it will attach specifically to mitochondria in a few seconds. Mostly this trick means that the protein attaches by itself or with a few close friends. This time, we found that TPD54 brought with it a whole vesicle! Not just one vesicle, but hundreds of them, becoming trapped at the mitochondria. Because TPD54 is all over these surface of the vesicles, and we’d made TPD54 sticky, this made the mitochondria fold over onto themselves. We could see this by EM.

EM view of lots of INVs trapped on the mitochondria

Are they really vesicles?

This was the main challenge: we needed to test whether the vesicles were functional transport vesicles. A real vesicle must be carrying cargo (otherwise, it’s not transporting anything!), and it must have other proteins called Rabs and SNAREs so that the vesicle can move, fuse and deliver the cargo. We were able to show that these trapped vesicles had cargo, Rabs and SNAREs. As far as we can tell, INVs are real functional vesicles.

The final thing to do was to try and see them in cells without trapping them. This is hard to do because the vesicles are so small (below the resolution limit of the light microscope). We were able to use a super resolution method called STORM to see TPD54 and, sure enough it was on small spots that were just over 30 nm in diameter.

Extreme close-up! INVs imaged by STORM

If INVs are really tiny, doesn’t that limit what they can carry?

Well it depends on how efficiently vesicles are loaded with cargo. Intuitively we think of large vesicles carrying a lot of cargo and small vesicles carrying less. However, even a 30 nm vesicle has a surprising capacity. The number of vesicles is important. There seem to be thousands of INVs in cells which mean that lots of cargo can be transported even if the vesicle capacity is limited. TPD54 is one of the most abundant proteins in cells, and the reason may be because there are lots of INVs in cells.

As analogies go this isn’t too bad. The Imperial Tie Fighters are the INVs and the Star Destroyer is the larger vesicle or carrier.

The emptiness of space as seen in Star Wars is not representative of life inside a cell. It’s a really crowded place with lots of other proteins and structures to negotiate.

The cytoplasm is a crowded place (source, Cell Signaling Technology)

In the paper we use the analogy that in the cell’s transport system, INVs could be like bike messengers, while other vesicle types are the cars, buses and trucks. Messengers can manoeuvre through slow moving traffic to quickly deliver their cargo.

Crazy bike messenger nipping through traffic (source, Pinterest)

What next?

We have many questions about the INVs themselves. How do they form? How do they move? Another direction is the TPD54 protein. It belongs to a family of proteins that were identified because their expression is altered in cancer, particularly breast cancer. We’re investigating the role of INVs in cancer.

The people behind the paper

Most importantly – I need to thank the people who actually did the work. Almost everything in the paper was done by Gabrielle Larocque. She really took this project and ran with it. She was helped by: Penny La-Borde who made a crucial gene-edited cell line, Nick Clarke who did correlative light-electron microscopy experiments, and Nick Carter who helped Gabrielle do the STORM imaging during the revisions. I did a bit of programming and writing (the code is released here).

Publication

We posted a bioRxiv preprint (with a very different title) in late 2018. This manuscript was focussed on the Rabs that are on the INVs. Some of these Rabs bind to TPD54 and we thought that TPD54 might be a Rab effector. The feedback from the community was that the INVs likely represent a new vesicle class and that we should rewrite the paper to highlight that. During peer review of the paper at J Cell Biol we got some incredibly helpful input that took almost nine months to address. During this time our evidence for TPD54 behaving as a Rab effector (a very specific type of binding protein) did not get any stronger, so we removed this part of the paper and rewrote the manuscript to focus on the INVs. Anyone who has the time (and energy) can read what happened since all the preprint versions are available and the review process file is online.

The publication process took a long time and was hard on Gabrielle and Penny who worked hard on the revisions, but it actually restored my faith in pre-publication peer review. I’ve published quite a few papers and peer review did not materially change any of them. Sure, we’ve had some good suggestions and our papers have improved a bit (we’ve had plenty of negative experiences too), but I don’t recall ever needing to change the main message of a paper. This is the first time that peer review really altered the course of one of our papers. Maybe this is because we engaged totally with the process rather than trying to push through our original vision for the paper – who knows? In this case, the combination of community feedback from the preprint and from giving talks, together with pre-publication peer review where the paper is scrutinised in detail, worked well together. Thanks to the Editors and Reviewers at J Cell Biol for their input.

The post title is from “Tiny Universe” by Jesu from the Compilation album “Pale Sketches” released in 2007.

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