membrane-targeted fluorophores

[sjh]

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I have used membrane-targeted fluorophores for visualizing neuronal morphology
off and on over the years. Some colleagues have warned me about their potential
side affects, and although I have always been cautious of this I have not
experienced this until just recently. I also have never seen a publication that
describes these effects.

In my setup I have been using a membrane-targeted form of citrine I made by
adding the Ras CAAX prenylation site to the C-term of citrine. Unless it is
expressed at very low concentrations it causes both obvious morphological
defects, and leads to early cell death. I have used a similarly made version of
Venus which has a very similar sequence, and not seen these effects, though in a
slightly different experiment.

Does anyone have more knowledge of what membrane-targeted fluorophores are
best. I am considering using a palmitoylation sequence since I have it on hand,
but am not sure if that will help. Not sure if it is the fluorophore, the linker or the
lipid modification that leads to the problem, and am just generally curious what
other people think.

[Ana Gil-Bernabe]

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Dear Steve,

I have used PKH26 (Sigma Aldrich) to label platelets for both in vitro and in vivo (injected in mouse) experiments. It works well, the platelets are still functional and the signal is really bright (565 nm excitation) and lasts long time. They also have the green version, which I have not tried yet.

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.
Michelson AD1, Barnard MR, Hechtman HB, MacGregor H, Connolly RJ, Loscalzo J, Valeri CR.

Best wishes,

Ana (Oxford University, Dpt. Oncology)
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Steve [[hidden email]]
Sent: 04 April 2014 19:57
To: [hidden email]
Subject: membrane-targeted fluorophores

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I have used membrane-targeted fluorophores for visualizing neuronal morphology
off and on over the years. Some colleagues have warned me about their potential
side affects, and although I have always been cautious of this I have not
experienced this until just recently. I also have never seen a publication that
describes these effects.

In my setup I have been using a membrane-targeted form of citrine I made by
adding the Ras CAAX prenylation site to the C-term of citrine. Unless it is
expressed at very low concentrations it causes both obvious morphological
defects, and leads to early cell death. I have used a similarly made version of
Venus which has a very similar sequence, and not seen these effects, though in a
slightly different experiment.

Does anyone have more knowledge of what membrane-targeted fluorophores are
best. I am considering using a palmitoylation sequence since I have it on hand,
but am not sure if that will help. Not sure if it is the fluorophore, the linker or the
lipid modification that leads to the problem, and am just generally curious what
other people think.

[Joachim Goedhart-2]

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Dear Steve,

There are a few details that are missing to fully understand the issue. You
mention that the lipidation motif is from Ras, but which isoform of Ras? I guess
that it is the C-terminus of KRas, which is widely used. This motif carries a lot of
basic residues and is highly positively charged. It therefore interacts with
negatively charged phospholipids at the plasma membrane (PI(4,5)P2, PI(4)P
and PA), which is good for plasma membrane localization. However, these lipids
are essential modulators of various processes, e.g. interacting directly or
indirectly with the cytoskeleton
(http://www.ncbi.nlm.nih.gov/pubmed/24534649).
Another issue is the probe. GFP variants, certainly YFP, can dimerize when it is
concentrated at the surface of the membrane
(http://www.ncbi.nlm.nih.gov/pubmed/11988576). So do you know whether you
are using monomeric YFP variants (e.g. carrying the A206K mutation)? If these
proteins are of the 206A type, dimerization may increase PM interaction (good
for localization), thereby competing more strongly with endogenous proteins
depend on interaction with the PM via negatively charged lipids.
We have some experience with the lipidation motif of Lck which gives very good
PM labeling, still it does not seem to interfere with cell morphology. The Lck
motif was successfully used in fusion proteins with mTurquoise2 and mVenus
(the aa sequence preceding the CDS of the FP we used is MGCVCSSNPELPVAT).
However, our expts are mainly performed in HeLa cells, so it might be different
in neuronal cells.

Best Regards,
Joachim Goedhart
Section Molecular Cytology
SILS - University of Amsterdam
The Netherlands

[lechristophe]

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Hi Steve,

We've been using membrane-associated fluorescent proteins in cultured
neurons for several years now, as they nicely delineate neuronal
morphology. We use the mainly use EGFP-F (Clontech), which is the 20-aa
farnesylation signal of c-Ha-Ras fused to the C-Terminus of EGFP. It does
not accumulate in endomembranes when expressing for more than 24 hours,
which happens in our system with other "membrane" targeting signals like
the neuromodulin aminoterminus. Using nucelofection at the time of plating,
we usually get expression for > 10 days with no aggregation and no
concentration in endomembranes.

Regarding expression levels, when transfecting high-level expression
vectors (such as lentiviral expression cassettes with enhancers), we see
some morphology changes in highly-expressing neurons, with more filopodias.
Regarding fluorescent proteins others than EGFP, we've had a hard time
finding FPs for other wavelength channels that behave as nicely as EGFP.
Most red FPs tested so far (DsRed2, mRFP, mCherry) tend to aggregate over
time, most likely due to their tendency to oligomerize. We recently made an
TdTomato-F construct, and it seems to be present in endomembrane more than
EGFP after several days of expression. I'd like to test the farnesylated
version of FusionRed FP (see
http://www.evrogen.com/products/FusionRed/FusionRed_Detailed_description.shtml)
which is advertised as equivalent to mCherry, with a lot less aggregation,
but I'd be happy to have some feedback form people using it before paying
for the plasmid. In the blue channel, we also tested an mTagBFP2-F (blue
FP), which also resulted in lots of intracellular labeling.

Hope this helps,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France



2014-04-04 20:57 GMT+02:00 Steve <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have used membrane-targeted fluorophores for visualizing neuronal
> morphology
> off and on over the years. Some colleagues have warned me about their
> potential
> side affects, and although I have always been cautious of this I have not
> experienced this until just recently. I also have never seen a publication
> that
> describes these effects.
>
> In my setup I have been using a membrane-targeted form of citrine I made by
> adding the Ras CAAX prenylation site to the C-term of citrine. Unless it is
> expressed at very low concentrations it causes both obvious morphological
> defects, and leads to early cell death. I have used a similarly made
> version of
> Venus which has a very similar sequence, and not seen these effects,
> though in a
> slightly different experiment.
>
> Does anyone have more knowledge of what membrane-targeted fluorophores are
> best. I am considering using a palmitoylation sequence since I have it on
> hand,
> but am not sure if that will help. Not sure if it is the fluorophore, the
> linker or the
> lipid modification that leads to the problem, and am just generally
> curious what
> other people think.
>

[lechristophe]

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I should add that the nucleofection/long term expression in hippocampal
neurons in a quite stringent test for the quality of an FP, as it is very
sensitive to toxicity, aggregation and mislocalization tendencies. Also, it
is simpler and faster than in-vivo tests using viruses. If you have a red
or blue, soluble or membrane-targeted FP that you think is good, I'd be
happy to try it, and share the results with you or the broader audience of
the list.

Thanks,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France



2014-04-07 9:33 GMT+02:00 Christophe Leterrier <
[hidden email]>:

> Hi Steve,
>
> We've been using membrane-associated fluorescent proteins in cultured
> neurons for several years now, as they nicely delineate neuronal
> morphology. We use the mainly use EGFP-F (Clontech), which is the 20-aa
> farnesylation signal of c-Ha-Ras fused to the C-Terminus of EGFP. It does
> not accumulate in endomembranes when expressing for more than 24 hours,
> which happens in our system with other "membrane" targeting signals like
> the neuromodulin aminoterminus. Using nucelofection at the time of plating,
> we usually get expression for > 10 days with no aggregation and no
> concentration in endomembranes.
>
> Regarding expression levels, when transfecting high-level expression
> vectors (such as lentiviral expression cassettes with enhancers), we see
> some morphology changes in highly-expressing neurons, with more filopodias.
> Regarding fluorescent proteins others than EGFP, we've had a hard time
> finding FPs for other wavelength channels that behave as nicely as EGFP.
> Most red FPs tested so far (DsRed2, mRFP, mCherry) tend to aggregate over
> time, most likely due to their tendency to oligomerize. We recently made an
> TdTomato-F construct, and it seems to be present in endomembrane more than
> EGFP after several days of expression. I'd like to test the farnesylated
> version of FusionRed FP (see
> http://www.evrogen.com/products/FusionRed/FusionRed_Detailed_description.shtml)
> which is advertised as equivalent to mCherry, with a lot less aggregation,
> but I'd be happy to have some feedback form people using it before paying
> for the plasmid. In the blue channel, we also tested an mTagBFP2-F (blue
> FP), which also resulted in lots of intracellular labeling.
>
> Hope this helps,
>
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>
>
>
> 2014-04-04 20:57 GMT+02:00 Steve <[hidden email]>:
>
> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> I have used membrane-targeted fluorophores for visualizing neuronal
>> morphology
>> off and on over the years. Some colleagues have warned me about their
>> potential
>> side affects, and although I have always been cautious of this I have not
>> experienced this until just recently. I also have never seen a
>> publication that
>> describes these effects.
>>
>> In my setup I have been using a membrane-targeted form of citrine I made
>> by
>> adding the Ras CAAX prenylation site to the C-term of citrine. Unless it
>> is
>> expressed at very low concentrations it causes both obvious morphological
>> defects, and leads to early cell death. I have used a similarly made
>> version of
>> Venus which has a very similar sequence, and not seen these effects,
>> though in a
>> slightly different experiment.
>>
>> Does anyone have more knowledge of what membrane-targeted fluorophores are
>> best. I am considering using a palmitoylation sequence since I have it on
>> hand,
>> but am not sure if that will help. Not sure if it is the fluorophore, the
>> linker or the
>> lipid modification that leads to the problem, and am just generally
>> curious what
>> other people think.
>>
>
>

[sjh]

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Hi Christophe,

Thanks for the information and suggestions. I had assumed that Citrine
would behave similarly to EGFP since there isn't that much difference in
their sequence, but perhaps that is not true. Unfortunately in my case it
would be difficult to see EGFP to label the membrane since I need that
color for labeling other structures. I also had a similar issue with
membrane targeted red proteins. They were all either too dim or cause side
affects. In particular the form of tdtomato we made got stuck in the ER,
and disrupted morphology. I had still hoped to try mOrange2 and TagRFP-T,
but had heard negative things about both of those have cytotoxic effects.
For that reason I decided to switch to doing cyan and yellow proteins since
I thought they were generally more amenable to being used as fusion tags.
Citrine and mTurquoise2 were the brightest and that is quite important for
my work. I am going to try membrane mTurquoise2 since I already have the
constructs, hopefully it will work out better than mTagBFP2-F did for you.

In regards to expression levels, I am electroporating into the retina
constructs expressing fluorescent proteins under the control of a CAG
promoter that are only expressed after being processed by Cre. I would
expect expression to be quite a bit lower than lentivirus, but have no
experience using it so I am not sure. In the retina I start to see obvious
deffects in morphology within 48 hours with the Citrine-CAAX, but do not
see anything unusual with cytoplasmic tdTomato. I should note that at this
point tdTomato is at least 5 times brighter, but since it is a brighter
fluorophore it is difficult to compare protein levels.

Thank you for the offer to test constructs in hippocampal cultures. First,
I am going to try a few different constructs I currently have in my system
to see if I can narrow down the issue a little more, but may contact you in
the future.

thanks,

Steve


On Mon, Apr 7, 2014 at 3:33 AM, Christophe Leterrier <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Steve,
>
> We've been using membrane-associated fluorescent proteins in cultured
> neurons for several years now, as they nicely delineate neuronal
> morphology. We use the mainly use EGFP-F (Clontech), which is the 20-aa
> farnesylation signal of c-Ha-Ras fused to the C-Terminus of EGFP. It does
> not accumulate in endomembranes when expressing for more than 24 hours,
> which happens in our system with other "membrane" targeting signals like
> the neuromodulin aminoterminus. Using nucelofection at the time of plating,
> we usually get expression for > 10 days with no aggregation and no
> concentration in endomembranes.
>
> Regarding expression levels, when transfecting high-level expression
> vectors (such as lentiviral expression cassettes with enhancers), we see
> some morphology changes in highly-expressing neurons, with more filopodias.
> Regarding fluorescent proteins others than EGFP, we've had a hard time
> finding FPs for other wavelength channels that behave as nicely as EGFP.
> Most red FPs tested so far (DsRed2, mRFP, mCherry) tend to aggregate over
> time, most likely due to their tendency to oligomerize. We recently made an
> TdTomato-F construct, and it seems to be present in endomembrane more than
> EGFP after several days of expression. I'd like to test the farnesylated
> version of FusionRed FP (see
>
> http://www.evrogen.com/products/FusionRed/FusionRed_Detailed_description.shtml
> )
> which is advertised as equivalent to mCherry, with a lot less aggregation,
> but I'd be happy to have some feedback form people using it before paying
> for the plasmid. In the blue channel, we also tested an mTagBFP2-F (blue
> FP), which also resulted in lots of intracellular labeling.
>
> Hope this helps,
>
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>
>
>
> 2014-04-04 20:57 GMT+02:00 Steve <[hidden email]>:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > I have used membrane-targeted fluorophores for visualizing neuronal
> > morphology
> > off and on over the years. Some colleagues have warned me about their
> > potential
> > side affects, and although I have always been cautious of this I have not
> > experienced this until just recently. I also have never seen a
> publication
> > that
> > describes these effects.
> >
> > In my setup I have been using a membrane-targeted form of citrine I made
> by
> > adding the Ras CAAX prenylation site to the C-term of citrine. Unless it
> is
> > expressed at very low concentrations it causes both obvious morphological
> > defects, and leads to early cell death. I have used a similarly made
> > version of
> > Venus which has a very similar sequence, and not seen these effects,
> > though in a
> > slightly different experiment.
> >
> > Does anyone have more knowledge of what membrane-targeted fluorophores
> are
> > best. I am considering using a palmitoylation sequence since I have it on
> > hand,
> > but am not sure if that will help. Not sure if it is the fluorophore, the
> > linker or the
> > lipid modification that leads to the problem, and am just generally
> > curious what
> > other people think.
> >
>