Stephen Royle

We have a new paper out! This post is to describe what our paper is
about. The paper is free to read, so please take a look if you are
interested.

Gabrielle Larocque, Daniel J. Moore, Méghane Sittewelle, Cansu Kuey,
Joseph H.R. Hetmanski, Penelope J. La-Borde, Beverley J. Wilson,
Nicholas I. Clarke, Patrick T. Caswell & Stephen J. Royle

**Intracellular nanovesicles mediate α5β1 integrin trafficking during
cell migration**

*J Cell Biol* 220 (10): e202009028. doi:
[10.1083/jcb.202009028](https://doi.org/10.1083/jcb.202009028)

## A quick guide to the paper {#a-quick-guide-to-the-paper .wp-block-heading}

**The paper in a nutshell:** We show that normal cell migration and
invasion in a cancer context depend on TPD54. The mechanism involves the
recycling of integrins, which we demonstrate occurs via intracellular
nanovesicles.

**Huh? What is cell migration?** Cells move a lot and this movement
(migration) is needed in the body for all kinds of normal, healthy
things, such as the immune response.

**That sounds good, so what about cancer?** There are times when you
don't want cells to move, such as in cancer. Tumour cells that move away
are said to "metastasise". This means they can make new tumours in
different places in the body. This can make the cancer more difficult to
treat and can mean a bad outcome for the patient. Previous work had
found that patients with certain cancers, e.g. breast cancer, had high
levels of Tumor Protein D52 (TPD52) and Tumor Protein D54 (TPD54). Not
only that, but these patients tended to do worse because their cancer
was more aggressive and migratory.

> Our lab had previously found that TPD54 is on tiny vesicles inside
> cells and we call them intracellular nanovesicles, or INVs for short.

To read more about that work, here's a
[post](https://quantixed.org/2019/10/31/tiny-universe-new-paper-on-intracellular-nanovesicles/){data-type="post"
data-id="1981"} and the [paper](http://doi.org/10.1083/jcb.201812044).

## TPD54 is the link between INVs and cell migration {#tpd54-is-the-link-between-invs-and-cell-migration .wp-block-heading}

We can watch cells move around (migrating) under the microscope. We
noticed that if we *decrease* the amount of TPD54 in cells the cells
migrate less. This is shown in the video below. Notice how the cells on
the right move slower and less far than the normal cells on the left.
Not only that but if we make the cells express *more* TPD54, like in
cancer, the cells migrate more. Their speed is higher.

<figure class="wp-block-video">

</figure>

This result is the main finding in the paper, but there is a lot of
other information in there and lots of experiments. A few other
highlights:

- We found that TPD52 and TPD53 (other members of the family) behave in
  the same way
- The changes in migration are due to altered integrin recycling

**Integrin recycling?** When cells move, they need to break and remake
their attachment with the surface they are moving on. These attachments
are made by integrins on the cell's surface, binding to "extracellular
matrix" proteins. One matrix protein is fibronectin and it is bound by
an integrin named alpha5/beta1. The integrin's connection with matrix is
broken by it being taken up into the cell and moved in vesicles and then
it is recycled to the cell surface to make a new connection. We found
that alpha5/beta1 integrins were recycled differently in cells with less
TPD54. This suggests that the recycling of this integrin type happens in
INVs.

As well as watching cells move on a flat surface -- like in the movie
above -- we imaged cells invading a 3D matrix. This is more similar to
the way that cancer cells would move in the body. We saw similar results
with these experiments suggesting that integrin recycling in INVs is
important here too. This part of the paper was contributed by Pat
Caswell's group at University of Manchester.

## **The people** {#the-people .wp-block-heading}

The paper is mainly the work of Gabrielle Larocque but there were a
bunch of other people who made important contributions, particularly
during the revision stage. The invasion experiments were in
collaboration with Joe Hetmanski, Bev Wilson, and Pat Caswell at
University of Manchester. Nick Clarke did the correlative light-electron
microscopy imaging of INVs. Penny La-Borde figured out how to test if
TPD54 binds direct to vesicles. Daniel Moore extended this analysis
during the revisions as well as doing proteomic analysis of INVs.
Méghane Sittewelle did many cell migration experiments and integrin
imaging for the revisions. Cansu Kuey imaged INVs wiggling around and
she quantified mitochondrial aggregation. You can meet the folks in our
lab [here](http://mechanochemistry.org/Royle/people/).

## Path to publication {#path-to-publication .wp-block-heading}

This was a pandemic paper. Although some of this work dates back to
2014, Gabrielle wrote the first draft of the paper during the first UK
national lockdown in 2020. We posted it to bioRxiv in summer 2020 and
sent it to J Cell Biol for consideration. As always, the review process
was robust and a number of suggestions came back. Unfortunately they
required a lot of experimental work to answer at a time when we had only
limited access to the lab due to the pandemic. Not only that but many
authors, including Gabrielle, had moved on to other jobs. Other
scientists were drafted in to do the work here and in Manchester.
Eventually we sent the revised paper back. J Cell Biol waived their
normal time allowance for revisions which was great. It was a tough
situation and the contributors, particularly Daniel and Méghane put in
so much work to complete everything. The paper was accepted at the end
of June 2021.

The work was funded by UKRI MRC and the money for publishing the paper
Open Access came from the University's UKRI block grant. Did I mention
it's free to read? Here is the
[link](https://doi.org/10.1083/jcb.202009028).

---

The post title is taken from "Culture Move" by Asian Dub Foundation from
their album Rafi's Revenge. If you are having some déjà vu, this is an
updated version of an earlier
[post](https://quantixed.org/2020/08/21/culture-move-new-preprint-on-cell-migration/){data-type="post"
data-id="2226"}.


We have a new paper out! This post is to describe what our paper is about. The paper is free to read, so please take a look if you are interested. Gabrielle Larocque, Daniel J. Moore, Méghane Sittewelle, Cansu Kuey, Joseph H.R. Hetmanski, Penelope J. La-Borde, Beverley J. Wilson, Nicholas I. Clarke, Patrick T. Caswell &amp;

Culture Move: new paper on integrin recycling and cell migration

We have a [new paper](http://dx.doi.org/10.1038/s41467-018-04993-0) out.
It's not exactly news, because the paper has been up on bioRxiv since
December 2016 and hasn't changed too much. All of the work was done by
Nick Clarke when he was a PhD student in the lab. This post is to
explain our new paper to a general audience.

**The paper in a nutshell**

We have invented a new way to tag proteins in living cells so that you
can see them by light microscopy and by electron microscopy.

**Why would you want to do that?**

Proteins do almost all of the jobs in cells that scientists want to
study. We can learn a lot about how proteins work by simply watching
them down the microscope. We want to know their precise location. Light
microscopy means that the cells are alive and we can watch the proteins
move around. It's a great method but it has low resolution, so seeing a
protein's precise location is not possible. We can overcome this
limitation by using electron microscopy. This gives us higher
resolution, but the proteins are stuck in one location. When we
correlate images from one microscope to the other, we can watch proteins
move and then look at them with high resolution. All we need is a way to
see the proteins so that they can be seen in both types of microscope.
We do this with tagging.

Tagging proteins so that we can see them by light microscopy is easy. A
widely used method is to use a fluorescent protein such
as [GFP](https://en.wikipedia.org/wiki/Green_fluorescent_protein). We
can't see GFP in the electron microscope (EM) so we need another method.
Again, there are several tags available but they all have drawbacks.
They are not precise enough, or they don't work on single proteins. So
we came up with a new one and fused it with a fluorescent protein.

**What is your EM tag?**

[![](https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ft_schema-300x140.png?resize=300%2C140){.alignright
.size-medium .wp-image-1568 loading="lazy" decoding="async"
attachment-id="1568"
permalink="https://quantixed.org/2018/07/04/ferrous-new-paper-on-ferritagging-proteins-in-cells/ft_schema/"
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image-title="ft_schema" image-description="" image-caption=""
medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ft_schema.png?fit=300%2C140&ssl=1"
large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ft_schema.png?fit=640%2C299&ssl=1"
width="300" height="140"
srcset="https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ft_schema.png?resize=300%2C140&ssl=1 300w, https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ft_schema.png?resize=768%2C359&ssl=1 768w, https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ft_schema.png?resize=1024%2C479&ssl=1 1024w, https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ft_schema.png?w=1307&ssl=1 1307w"
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recalc-dims="1"}](http://quantixed.org/2018/07/04/ferrous-new-paper-on-ferritagging-proteins-in-cells/ft_schema/){rel="attachment wp-att-1568"}We
call it FerriTag. It is based on Ferritin which is a large protein shell
that cells use to store iron. Because iron scatters electrons, this
protein shell can be seen by EM as a particle. There was a problem
though. If Ferritin is fused to a protein, we end up with a mush. So, we
changed Ferritin so that it could be attached to the protein of interest
by using a drug. This meant that we could put the FerriTag onto the
protein we want to image in a few seconds. In the picture on the right
you can see how this works to FerriTag clathrin, a component of vesicles
in cells.

[![](https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ferritag.gif?resize=110%2C71){.alignright
.size-full .wp-image-1569 loading="lazy" decoding="async"
attachment-id="1569"
permalink="https://quantixed.org/2018/07/04/ferrous-new-paper-on-ferritagging-proteins-in-cells/ferritag/"
orig-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ferritag.gif?fit=110%2C71&ssl=1"
orig-size="110,71" comments-opened="1"
image-meta="{\"aperture\":\"0\",\"credit\":\"\",\"camera\":\"\",\"caption\":\"\",\"created_timestamp\":\"0\",\"copyright\":\"\",\"focal_length\":\"0\",\"iso\":\"0\",\"shutter_speed\":\"0\",\"title\":\"\",\"orientation\":\"0\"}"
image-title="ferritag" image-description="" image-caption=""
medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ferritag.gif?fit=110%2C71&ssl=1"
large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/ferritag.gif?fit=110%2C71&ssl=1"
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recalc-dims="1"}](http://quantixed.org/2018/07/04/ferrous-new-paper-on-ferritagging-proteins-in-cells/ferritag/){rel="attachment wp-att-1569"}We
can watch the tagging process happening in cells before looking by EM.
The movie on the right shows green spots (clathrin-coated pits in a
living cell) turning orange/yellow when we do FerriTagging. The cool
thing about FerriTag is that it is genetically encoded. That means that
we get the cell to make the tag itself and we don't have to put it in
from outside which would damage the cell.

**What can you use FerriTag for?**

Well, it can be used to tag many proteins in cells. We wanted to
precisely localise a protein called HIP1R which links clathrin-coated
pits to the cytoskeleton. We FerriTagged HIP1R and carried out what we
call "contextual nanoscale mapping". This is just a fancy way of saying
that we could find the FerriTagged HIP1R and map where it is relative to
the clathrin-coated pit. This allowed us to see that HIP1R is found at
the pit and surrounding membrane. We could even see small changes in the
shape of HIP1R in the different locations.

[![](https://i0.wp.com/quantixed.org/wp-content/uploads/2018/06/hip1r_ferritag-1024x432.png?resize=640%2C270){.aligncenter
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image-title="hip1r_ferritag" image-description="" image-caption=""
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We're using FerriTag for lots of projects. Our motivation to make
FerriTag was so that we could look at proteins that are important for
cell division and this is what we are doing now.

**Is the work freely available?**

Yes! The paper is available
[here](http://dx.doi.org/10.1038/s41467-018-04993-0) under CC-BY
licence. All of the code we wrote to analyse the data and run computer
simulations is available [here](https://github.com/quantixed/FerriTag).
All of the plasmids needed to do FerriTagging are available from
[Addgene](http://www.addgene.org/browse/article/28190094/) (a non-profit
company, there is a small fee) so that anyone can use them in the lab to
FerriTag their favourite protein.

**How long did it take to do this project?**

Nick worked for four years on this project. Our first attempt at using
ribosomes to tag proteins failed, but Nick then managed to get Ferritin
working as a tag. This paper has broken our lab record for longest
publication delay from first submission to final publication. The
diagram below tells the whole saga.

 

[![](https://i0.wp.com/quantixed.org/wp-content/uploads/2018/07/Ferritag-saga-1024x568.png?resize=640%2C355){.aligncenter
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The publication process was frustratingly slow. It took a few months to
write the paper and then we submitted to the first journal after
Christmas 2016. We got a rapid desk rejection and sent the paper to
another journal and it went out for review. We had two positive referees
and one negative one, but we felt we could address the comments and
checked with the journal who said that they would consider a revised
paper as an appeal. We did some work and resubmitted the paper. Almost
six months after first submission the paper was rejected, but with the
offer of a rapid (ha!) publication at Nature Communications using the
peer review file from the other journal.

Hindsight is a wonderful thing but I now regret agreeing to transfer the
paper to Nature Communications. It was far from rapid. They drafted in a
new reviewer who came with a list of new questions, as well as being
slow to respond. Sure, a huge chunk of the delay was caused by us doing
revision experiments (the revisions took longer than they should because
Nick defended his PhD, was working on other projects and also became a
parent). However, the journal was really slow. The Editor assigned to
our paper left the journal which didn't help and the reviewer they
drafted in was slow to respond each time (6 and 7 weeks, respectively).
Particularly at the end, after the paper was 'accepted in principle' it
took them three weeks to actually accept the paper (seemingly a week to
figure out what a bib file is and another to ask us something about
chi-squared tests). Then a further three weeks to send us the proofs,
and then another three weeks until publication. You can see from the
graphic that we sent back the paper in the third week of February and
only incurred a 9-day delay ourselves, yet the paper was not published
until July.

Did the paper improve as a result of this process? Yes and no. We
actually added some things in the first revision cycle (for Journal \#2)
that got removed in subsequent peer review cycles! And the message in
the final paper is exactly the same as the version on bioRxiv, posted 18
months previously. So in that sense, no it didn't. It wasn't all a total
waste of time though, the extra reviewer convinced us to add some new
analysis which made the paper more convincing in the end. Was this worth
an 18-month delay? You can download our
[paper](http://dx.doi.org/10.1038/s41467-018-04993-0) and the
[preprint](https://www.biorxiv.org/content/early/2016/12/18/095208) and
judge for yourself.

[![](https://i0.wp.com/quantixed.org/wp-content/uploads/2018/06/yearPlot-1-300x300.png?resize=300%2C300){.size-medium
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Were we unlucky with this slow experience? Maybe, but I know other
authors who've had similar (and worse) experiences at this journal. As
described in a previous
[post](http://quantixed.org/2018/06/23/ten-years-vs-the-spread-calculating-publication-lag-times-in-r/),
the publication lag times are getting longer at Nature Communications.
This suggests that our lengthy wait is not unique.

There's lots to like about this journal:

- It is open access.
- It has the Nature branding (which, like it or not, impresses many
  people).
- Peer review file is available
- The papers look great (in print and online).

But there are downsides too.

- The APC for each paper is £3300 (\$5200). Obviously open access must
  cost something, but there a cheaper OA journals available (albeit
  without the Nature branding).
- Ironically, paying a premium for this reputation is complicated since
  the journal covers a wide range of science and its kudos varies
  depending on subfield.
- It's also slow, and especially so when you consider that papers have
  often transferred here from somewhere else.
- It's essentially a mega journal, so your paper doesn't get the same
  exposure as it would in a community-focused journal.
- There's the whole ReadCube/SpringerNature thing...

Overall it was a negative publication experience with this paper.
Transferring a paper along with the peer review file to another journal
has worked out well for us recently and has been rapid, but not this
time. Please leave a comment particularly if you've had a positive
experience and redress the balance.

---

The post title comes from "Ferrous" by Circle from their album Meronia.


We have a new paper out. It’s not exactly news, because the paper has been up on bioRxiv since December 2016 and hasn’t changed too much. All of the work was done by Nick Clarke when he was a PhD student in the lab. This post is to explain our new paper to a general audience.

Messaggi di Rogue Scholar

Culture Move: new paper on integrin recycling and cell migration

Ferrous: new paper on FerriTagging proteins in cells