Stephen Royle

We have a new preprint out &#8211; it is a cautionary tale about using GFP nanobodies in cells. This short post gives a bit of background to the work. Please read the paper if you are interested in using GFP nanobodies in cells, you can find it <a href="https://doi.org/10.1101/590984">here</a>.



Paper in a nutshell: Caution is needed when using GFP nanobodies because they can inhibit their target protein in cells.



People who did the work: Cansu Küey did most of the work for the paper. She discovered the inhibition side effect of the dongles. Gabrielle Larocque contributed a figure where she compared dongle-knocksideways with regular knocksideways. The project was initiated by Nick Clarke who made our first set of dongles and tested which fluorescent proteins the nanobody binds in cells. Lab people and their profiles can be found <a href="http://mechanochemistry.org/Royle/people/">here</a>. 


Background:&nbsp;Many other labs have shown that nanobodies can be functionalised so that you can stick new protein domains onto GFP-tagged proteins to do new things. This is useful because it means you can “retrofit” an existing GFP knock-in cell line or organism to do new things like <a href="https://doi.org/10.1016/j.devcel.2009.12.015">knocksideways</a> without making new lines. In fact there was a recent <a href="https://doi.org/10.1101/544700">preprint</a> which described a suite of functionalised nanobodies that can confer all kinds of functions to GFP.
Like many other labs we were working on this method. We thought functionalised GFP nanobodies resembled &#8220;dongles&#8221; &#8211; those adaptors that Apple makes so much money from &#8211; that convert one port to another.


<div class="wp-block-image"><figure class="aligncenter"><img loading="lazy" decoding="async" width="500" height="403" data-attachment-id="1799" data-permalink="https://quantixed.org/2019/03/29/not-what-you-want-our-new-paper-on-a-side-effect-on-gfp-nanobodies/5575298582_9d7bd341f2/" data-orig-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?fit=500%2C403&amp;ssl=1" data-orig-size="500,403" data-comments-opened="1" data-image-meta="{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}" data-image-title="5575298582_9d7bd341f2" data-image-description="" data-image-caption="" data-medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?fit=300%2C242&amp;ssl=1" data-large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?fit=500%2C403&amp;ssl=1" src="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?resize=500%2C403&#038;ssl=1" alt="" class="wp-image-1799" srcset="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?w=500&amp;ssl=1 500w, https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?resize=300%2C242&amp;ssl=1 300w" sizes="(max-width: 500px) 100vw, 500px" data-recalc-dims="1" /><figcaption>Dongles, dongles, dongles&#8230; (photo by Rex Hammock, licensed for reuse https://www.flickr.com/photos/rexblog/5575298582)</figcaption></figure></div>



A while back we made several different dongles. We were most interested in a GFP nanobodies with an additional FKBP domain that would allow us to do knocksideways (or <a href="https://doi.org/10.1038/s41467-018-04993-0">FerriTagging</a>) in GFP knock-in cells. For those that don&#8217;t know, knocksideways is not a knockout or a knockdown, but a way of putting a protein somewhere else in the cell to inactivate it. The most common place to send a protein is to the mitochondria.



<figure class="wp-block-image"><img loading="lazy" decoding="async" width="611" height="988" data-attachment-id="1803" data-permalink="https://quantixed.org/2019/03/29/not-what-you-want-our-new-paper-on-a-side-effect-on-gfp-nanobodies/dongleks/" data-orig-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?fit=611%2C988&amp;ssl=1" data-orig-size="611,988" data-comments-opened="1" data-image-meta="{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}" data-image-title="dongleKS" data-image-description="" data-image-caption="" data-medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?fit=186%2C300&amp;ssl=1" data-large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?fit=611%2C988&amp;ssl=1" src="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?resize=611%2C988&#038;ssl=1" alt="" class="wp-image-1803" srcset="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?w=611&amp;ssl=1 611w, https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?resize=186%2C300&amp;ssl=1 186w" sizes="(max-width: 611px) 100vw, 611px" data-recalc-dims="1" /><figcaption>Knocksideways works by joining FKBP and FRB (on the mitochondria) using rapamycin. Normally FKBP is fused to the protein of interest (top). If we just have a GFP tag, we can&#8217;t do knocksideways (middle). If we add a dongle (bottom) we can attach FKBP domains to allow knocksideways to happen.</figcaption></figure>



We found that dongle-knocksideways works really well and we were very excited about this method. Here we are removing GFP-clathrin from the mitotic spindle in seconds using dongle knocksideways.



<div class="wp-block-image"><figure class="aligncenter"><img loading="lazy" decoding="async" width="600" height="600" data-attachment-id="1800" data-permalink="https://quantixed.org/2019/03/29/not-what-you-want-our-new-paper-on-a-side-effect-on-gfp-nanobodies/composite-2/" data-orig-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?fit=600%2C600&amp;ssl=1" data-orig-size="600,600" data-comments-opened="1" data-image-meta="{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}" data-image-title="Composite-2" data-image-description="" data-image-caption="" data-medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?fit=300%2C300&amp;ssl=1" data-large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?fit=600%2C600&amp;ssl=1" src="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?resize=600%2C600&#038;ssl=1" alt="" class="wp-image-1800" data-recalc-dims="1"/><figcaption>GFP-clathrin, shown here in blue is sent to the mitochondria (yellow) using rapamycin. This effect is only possible because of the dongle which adds FKBP to GFP via a GFP nanobody.</figcaption></figure></div>



Since there are no specific inhibitors of endocytosis, we thought dongle knocksideways would be cool to try in cells with dynamin-2 tagged with GFP at both alleles. There is a line from David Drubin&#8217;s lab which is widely used. This would mean we could put the dongle plasmids on Addgene and everyone could inhibit endocytosis on-demand!



Initial results were encouraging. We could put dynamin onto mitochondria alright.



<figure class="wp-block-video aligncenter"><video controls src="https://quantixed.org/wp-content/uploads/2019/03/SV1.mov"></video><figcaption>Dynamin-2-GFP undergoing dongle-knocksideways. The Mitotrap is shown in red and dynamin is green.</figcaption></figure>



But we hit a problem. It turns out that dongle binding to dynamin inhibits endocytosis. So we have unintended inhibition of the target protein. This is a big problem because the power of knocksideways comes from being able to observe normal function and then rapidly switch it off. So if there is inhibition before knocksideways, the method is useless.



Now, this problem could be specific to dynamin or it might be a general problem with all targets of dongles. Either way, we’ve switched from this method and wrote this manuscript to alert others to the side effects of dongles. We discuss possible ways forward for this method and also point out some applications of the nanobody technology that are unaffected by our observations.



&#8212;



The post title comes from &#8220;Not What You Want&#8221; by Sleater-Kinney from their wonderful Dig Me Out record.

We have a new preprint out -- it is a cautionary tale about using GFP
nanobodies in cells. This short post gives a bit of background to the
work. Please read the paper if you are interested in using GFP
nanobodies in cells, you can find it
[here](https://doi.org/10.1101/590984).

**Paper in a nutshell:** Caution is needed when using GFP nanobodies
because they can inhibit their target protein in cells.

**People who did the work:** Cansu Küey did most of the work for the
paper. She discovered the inhibition side effect of the dongles.
Gabrielle Larocque contributed a figure where she compared
dongle-knocksideways with regular knocksideways. The project was
initiated by Nick Clarke who made our first set of dongles and tested
which fluorescent proteins the nanobody binds in cells. Lab people and
their profiles can be found
[here](http://mechanochemistry.org/Royle/people/).

**Background: **Many other labs have shown that nanobodies can be
functionalised so that you can stick new protein domains onto GFP-tagged
proteins to do new things. This is useful because it means you can
"retrofit" an existing GFP knock-in cell line or organism to do new
things like
[knocksideways](https://doi.org/10.1016/j.devcel.2009.12.015) without
making new lines. In fact there was a recent
[preprint](https://doi.org/10.1101/544700) which described a suite of
functionalised nanobodies that can confer all kinds of functions to GFP.

Like many other labs we were working on this method. We thought
functionalised GFP nanobodies resembled "**dongles**" -- those adaptors
that Apple makes so much money from -- that convert one port to another.

::: {.wp-block-image}
<figure class="aligncenter">
<img
src="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?resize=500%2C403&amp;ssl=1"
class="wp-image-1799" loading="lazy" decoding="async"
data-attachment-id="1799"
data-permalink="https://quantixed.org/2019/03/29/not-what-you-want-our-new-paper-on-a-side-effect-on-gfp-nanobodies/5575298582_9d7bd341f2/"
data-orig-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?fit=500%2C403&amp;ssl=1"
data-orig-size="500,403" data-comments-opened="1"
data-image-meta="{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}"
data-image-title="5575298582_9d7bd341f2" data-image-description=""
data-image-caption=""
data-medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?fit=300%2C242&amp;ssl=1"
data-large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?fit=500%2C403&amp;ssl=1"
srcset="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?w=500&amp;ssl=1 500w, https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/5575298582_9d7bd341f2.jpg?resize=300%2C242&amp;ssl=1 300w"
sizes="(max-width: 500px) 100vw, 500px" data-recalc-dims="1" width="500"
height="403" />
<figcaption>Dongles, dongles, dongles… (photo by Rex Hammock, licensed
for reuse https://www.flickr.com/photos/rexblog/5575298582)</figcaption>
</figure>
:::

A while back we made several different dongles. We were most interested
in a GFP nanobodies with an additional FKBP domain that would allow us
to do knocksideways (or
[FerriTagging](https://doi.org/10.1038/s41467-018-04993-0)) in GFP
knock-in cells. For those that don't know, knocksideways is not a
knockout or a knockdown, but a way of putting a protein somewhere else
in the cell to inactivate it. The most common place to send a protein is
to the mitochondria.

<figure class="wp-block-image">
<img
src="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?resize=611%2C988&amp;ssl=1"
class="wp-image-1803" loading="lazy" decoding="async"
data-attachment-id="1803"
data-permalink="https://quantixed.org/2019/03/29/not-what-you-want-our-new-paper-on-a-side-effect-on-gfp-nanobodies/dongleks/"
data-orig-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?fit=611%2C988&amp;ssl=1"
data-orig-size="611,988" data-comments-opened="1"
data-image-meta="{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}"
data-image-title="dongleKS" data-image-description=""
data-image-caption=""
data-medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?fit=186%2C300&amp;ssl=1"
data-large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?fit=611%2C988&amp;ssl=1"
srcset="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?w=611&amp;ssl=1 611w, https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/dongleKS.png?resize=186%2C300&amp;ssl=1 186w"
sizes="(max-width: 611px) 100vw, 611px" data-recalc-dims="1" width="611"
height="988" />
<figcaption>Knocksideways works by joining FKBP and FRB (on the
mitochondria) using rapamycin. Normally FKBP is fused to the protein of
interest (top). If we just have a GFP tag, we can’t do knocksideways
(middle). If we add a dongle (bottom) we can attach FKBP domains to
allow knocksideways to happen.</figcaption>
</figure>

We found that dongle-knocksideways works really well and we were very
excited about this method. Here we are removing GFP-clathrin from the
mitotic spindle in seconds using dongle knocksideways.

::: {.wp-block-image}
<figure class="aligncenter">
<img
src="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?resize=600%2C600&amp;ssl=1"
class="wp-image-1800" loading="lazy" decoding="async"
data-attachment-id="1800"
data-permalink="https://quantixed.org/2019/03/29/not-what-you-want-our-new-paper-on-a-side-effect-on-gfp-nanobodies/composite-2/"
data-orig-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?fit=600%2C600&amp;ssl=1"
data-orig-size="600,600" data-comments-opened="1"
data-image-meta="{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}"
data-image-title="Composite-2" data-image-description=""
data-image-caption=""
data-medium-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?fit=300%2C300&amp;ssl=1"
data-large-file="https://i0.wp.com/quantixed.org/wp-content/uploads/2019/03/Composite-2.gif?fit=600%2C600&amp;ssl=1"
data-recalc-dims="1" width="600" height="600" />
<figcaption>GFP-clathrin, shown here in blue is sent to the mitochondria
(yellow) using rapamycin. This effect is only possible because of the
dongle which adds FKBP to GFP via a GFP nanobody.</figcaption>
</figure>
:::

Since there are no specific inhibitors of endocytosis, we thought dongle
knocksideways would be cool to try in cells with dynamin-2 tagged with
GFP at both alleles. There is a line from David Drubin's lab which is
widely used. This would mean we could put the dongle plasmids on Addgene
and everyone could inhibit endocytosis on-demand!

Initial results were encouraging. We could put dynamin onto mitochondria
alright.

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

<figcaption>Dynamin-2-GFP undergoing dongle-knocksideways. The Mitotrap
is shown in red and dynamin is green.</figcaption>
</figure>

**But we hit a problem.** It turns out that dongle binding to dynamin
inhibits endocytosis. So we have *unintended inhibition* of the target
protein. This is a big problem because the power of knocksideways comes
from being able to observe normal function and then rapidly switch it
off. So if there is inhibition before knocksideways, the method is
useless.

Now, this problem could be specific to dynamin or it might be a general
problem with all targets of dongles. Either way, we've switched from
this method and wrote this manuscript to alert others to the side
effects of dongles. We discuss possible ways forward for this method and
also point out some applications of the nanobody technology that are
unaffected by our observations.

---

The post title comes from "Not What You Want" by Sleater-Kinney from
their wonderful Dig Me Out record.

We have a new preprint out – it is a cautionary tale about using GFP nanobodies in cells. This short post gives a bit of background to the work. Please read the paper if you are interested in using GFP nanobodies in cells, you can find it here.

 Paper in a nutshell:

Caution is needed when using GFP nanobodies because they can inhibit their target protein in cells.

Rogue Scholar Beiträge

Not What You Want: our new paper on a side effect of GFP nanobodies