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membrane/cytoplasmatic ratio

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Valeria Berno-3

membrane/cytoplasmatic ratio

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Dear all,

One of the facility user is trying to quantify Inositol phosphates (PIP) in the plasma membrane of our cells. We manage to visualize it transfecting the cells with a recombinant GFP.

The idea is that the GFP-PIP should migrate from membrane to cytoplasm with specific treatment. We counterstained the cells with a cytoplasmatic marker.

We decided to quantify this with a line scan method (drawing a line through the cells) and measure the ratio between intensity in the membrane and intensity in the cytoplasm. This is a very common method in different paper but I couldn´t find more specifics.

My questions are:
-how can I determine where is my membrane a priori only looking at the cytoplasmatic staining?.
First of all the cytoplasmatic staining doesn´t drop in a specific postion but gradually decrease where I can clearly see the peak of the membrane.

Assuming the membrane should start where the peak of the cytoplasm staining start to decrease...then should I assume that the membrane is always a specific width or should I calculate the GFP intensity up to when I reach background level for the green?

I know that the best should be a membrane staining as a reference but could you suggest a good one for fixed cells?

Thanks in advance for your support

Valeria

Valeria Berno, PhD
Imaging Facility
______

Istituto Nazionale di Genetica Molecolare
Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi"
Via F.Sforza 35 - 20122 - Milano (MI) - Italy
Tel Off.: +39 02.00660 219
Tel Lab.:+39 02.00660 338
E-Mail: [hidden email]
Web site: http://www.ingm.org

SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la RICERCA SANITARIA. Grazie 1000...al 5 per mille!
Please consider the environment before printing this mail note

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George McNamara

Re: membrane/cytoplasmatic ratio

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Hi Valeria,

I hope you are choosing cells that are expressing the GFP-PIP biosensor
at a "moderate" or even "low" level, not overexpressing. If you are
choosing cells by eye by fluorescence, if your eye is drawn to the
bright cells, avoid using them.

You should also try to work with very thin cells and/or using an optical
sectioning method (confocal, multiphoton, quantitative deconvolution).
At high resolution, for example 1.2NA water immersion lens -- I am
assuming point scanning confocal microscopy, so you can zoom to optimal
pixel size -- the cytoplasm itself should not be uniform, because your
cytoplasmic marker should be excluded by mitochondria and other membrane
bound organelles. The organelles may not be high contrast, but you
should see them.

Line scan - a typical 'rule of thumb' is to use the halfway point
between the bright plateau (bright cytoplasm) and the dark background.
If you were imaging a point, this would be full width at half maximum
(FWHM), since you are imaging one edge, half width at half maximum
(HWHM). If some of your cells have filopodia, they could be imaged to
give you a measure of FWHM, for either marker, since filopodia contain
both cytoplasm and plasma membrane.

best wishes,

George

On 3/2/2015 5:30 AM, Valeria Berno 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.
> *****
>
> Dear all,
>
> One of the facility user is trying to quantify Inositol phosphates (PIP) in the plasma membrane of our cells. We manage to visualize it transfecting the cells with a recombinant GFP.
>
> The idea is that the GFP-PIP should migrate from membrane to cytoplasm with specific treatment. We counterstained the cells with a cytoplasmatic marker.
>
> We decided to quantify this with a line scan method (drawing a line through the cells) and measure the ratio between intensity in the membrane and intensity in the cytoplasm. This is a very common method in different paper but I couldn´t find more specifics.
>
>
> My questions are:
> -how can I determine where is my membrane a priori only looking at the cytoplasmatic staining?.
> First of all the cytoplasmatic staining doesn´t drop in a specific postion but gradually decrease where I can clearly see the peak of the membrane.
>
> Assuming the membrane should start where the peak of the cytoplasm staining start to decrease...then should I assume that the membrane is always a specific width or should I calculate the GFP intensity up to when I reach background level for the green?
>
> I know that the best should be a membrane staining as a reference but could you suggest a good one for fixed cells?
>
> Thanks in advance for your support
>
> Valeria
>
>
> Valeria Berno, PhD
> Imaging Facility
> ______
>
> Istituto Nazionale di Genetica Molecolare
> Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi"
> Via F.Sforza 35 - 20122 - Milano (MI) - Italy
> Tel Off.: +39 02.00660 219
> Tel Lab.:+39 02.00660 338
> E-Mail: [hidden email]
> Web site: http://www.ingm.org
>
> SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la RICERCA SANITARIA. Grazie 1000...al 5 per mille!
> Please consider the environment before printing this mail note
>
> DISCLAIMER :
> Questa e-mail potrebbe contenere informazioni privilegiate o confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è pregato di contattare il mittente rispondendo a questa stessa dopodichè è pregato di cancellarla immediatamente.
>
> This e-mail may contain confidential or privileged information. If you think you have received this e-mail in error, please advise the sender by reply e-mail and then delete this e-mail immediately.
>

--

George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

Mel Symeonides

Re: membrane/cytoplasmatic ratio

In reply to this post by Valeria Berno-3

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*****

Hi Valeria,

I think George covered line scan analysis pretty well. If I'm
understanding your question correctly, you are also looking for a
formaldehyde-fixable membrane stain so that you can localize your GFP
moiety in comparison to it rather than just looking for the outer edge
of the cytoplasmic dye.

Depending on the degree of relocalization you are looking for, you
should keep in mind that most fixable surface stains will not stain the
membrane itself, but structures on the membrane. So, given that your GFP
moiety, when localized to the membrane, faces into the cytoplasm, there
might be a considerable offset from your surface stain, even above the
diffraction limit (assuming that you are performing diffraction-limited
imaging). The closest you will be able to get is using a fixable
lipophilic dye, such as CM-DiI, though that will also stain
intracellular membranes as it is internalized. If you don't mind the
offset factor, your best bet for surface staining is either using an
antibody for an abundant surface protein (depending on your cell type,
e.g. CD81), or a fluorophore-conjugated lectin like wheat germ
agglutinin. You can also perform these stains after fixation (but before
permeabilization), but keep in mind that fixation does lightly
permeabilize the membrane, whereas a live cell stain on ice will be
strictly on the surface. I guess you could theoretically also perform
the CM-DiI stain immediately before fixation, though I have not tried
that and don't know if it would still diffuse through the cell membrane
in living cells.

I would also recommend a brief permeabilization step immediately after
fixation (e.g. PBS/0.2% Triton X-100 for 10 min) to prevent
post-fixation blebbing, which can happen to varying degrees depending on
the cell type and even cellular PIP2 levels (see
doi:10.1016/j.fob.2014.02.003), and which would affect your ability to
accurately localize cell subtructures. I have battled post-fixation
blebbing for a couple of years and now swear by the post-fixation perm
step. If you are using CM-DiI (or any lipophilic dye), do not
permeabilize with Triton as it will wash out, use digitonin instead.

Best regards,
Mel

On 3/2/2015 6:30 AM, Valeria Berno wrote:

--
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: 802-656-1161

Winfree, Seth

Re: membrane/cytoplasmatic ratio

In reply to this post by Valeria Berno-3

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Valeria,

I worked on a similar problem/assay with GFP-PIP probes and other membrane associated markers a few years back that had a couple iterations. Firstly, in terms of membrane markers, I never found one that was wholly satisfying, survived fixation reasonably and had fidelity, etc-admitededly it was nearly 10 years ago that we were working out the assay. In our first iterations we relied on wheat germ agglutinin(WGA) staining but found it to a be a mediocre marker with density that didn’t always correlate with plasma membrane location nor density (not surprising really). With that being said we used WGA as it was the best we could find. We imaged, as George recommends, cells expressing low to moderate levels of the GFP-PIP probes on a confocal microscope. To assess an enrichment as we called it, we measured GFP in the regions defined by the WGA and normalized to the cytosolic levels of GFP. This turned out to work fairly well for our system and the data was supported by commiserate increases in PIPs by mass spec [1]. We also used this approach to follow phosphorylation of Akt at the membrane with correlative western blot data[2]. Our second approach was to reframe the question-and consider local changes relative to a structure of interest-in our case a bacterium. This is not suitable for all systems of course[3].

We considered line scans but found that based on the plasma-membrane structure we were interested in this was a tenuous and too subjective an approach-where to draw/scan etc the line. We would’ve still used WGA though. Of course our first approach was not perfect either and required choices regarding where the cytsol was and what part of the plasma membrane to consider. We tried to counter any bias with systematic and blind sampling and blind analysis. In fact, this is what led us to recast the question and develop the second assay.

Hopefully this helps, and wasn’t too confusing or convoluted-feel free to contact me with any questions.

Happy hunting!

Seth

Winfree, Seth
[hidden email]<mailto:[hidden email]>

Indiana Center for Biological Microscopy
Indiana University Medical Center
950 W Walnut St, R2-202
Indianapolis, IN 46202-5116

1.Activation of Akt by the bacterial inositol phosphatase, SopB, is wortmannin insensitive.<http://www.ncbi.nlm.nih.gov/pubmed/21779406>
Cooper KG, Winfree S, Malik-Kale P, Jolly C, Ireland R, Knodler LA, Steele-Mortimer O.
PLoS One. 2011;6(7):e22260. doi: 10.1371/journal.pone.0022260. Epub 2011 Jul 14.
2.Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.<http://www.ncbi.nlm.nih.gov/pubmed/19614667>
Knodler LA, Winfree S, Drecktrah D, Ireland R, Steele-Mortimer O.
Cell Microbiol. 2009 Nov;11(11):1652-70. doi: 10.1111/j.1462-5822.2009.01356.x. Epub 2009 Jul 13.
3.The Annexin A2/p11 complex is required for efficient invasion of Salmonella Typhimurium in epithelial cells.<http://www.ncbi.nlm.nih.gov/pubmed/23931152>
Jolly C, Winfree S, Hansen B, Steele-Mortimer O.
Cell Microbiol. 2014 Jan;16(1):64-77. doi: 10.1111/cmi.12180. Epub 2013 Aug 28.

Winfree, Seth
[hidden email]<mailto:[hidden email]>

Indiana Center for Biological Microscopy
Indiana University Medical Center
950 W Walnut St, R2-202
Indianapolis, IN 46202-5116

On Mar 2, 2015, at 6:30 AM, Valeria Berno <[hidden email]<mailto:[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.
*****

Dear all,

One of the facility user is trying to quantify Inositol phosphates (PIP) in the plasma membrane of our cells. We manage to visualize it transfecting the cells with a recombinant GFP.

The idea is that the GFP-PIP should migrate from membrane to cytoplasm with specific treatment. We counterstained the cells with a cytoplasmatic marker.

We decided to quantify this with a line scan method (drawing a line through the cells) and measure the ratio between intensity in the membrane and intensity in the cytoplasm. This is a very common method in different paper but I couldn´t find more specifics.

My questions are:
-how can I determine where is my membrane a priori only looking at the cytoplasmatic staining?.
First of all the cytoplasmatic staining doesn´t drop in a specific postion but gradually decrease where I can clearly see the peak of the membrane.

Assuming the membrane should start where the peak of the cytoplasm staining start to decrease...then should I assume that the membrane is always a specific width or should I calculate the GFP intensity up to when I reach background level for the green?

I know that the best should be a membrane staining as a reference but could you suggest a good one for fixed cells?

Thanks in advance for your support

Valeria

Valeria Berno, PhD
Imaging Facility
______

Istituto Nazionale di Genetica Molecolare
Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi"
Via F.Sforza 35 - 20122 - Milano (MI) - Italy
Tel Off.: +39 02.00660 219
Tel Lab.:+39 02.00660 338
E-Mail: [hidden email]
Web site: http://www.ingm.org

SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la RICERCA SANITARIA. Grazie 1000...al 5 per mille!
Please consider the environment before printing this mail note

DISCLAIMER :
Questa e-mail potrebbe contenere informazioni privilegiate o confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è pregato di contattare il mittente rispondendo a questa stessa dopodichè è pregato di cancellarla immediatamente.

This e-mail may contain confidential or privileged information. If you think you have received this e-mail in error, please advise the sender by reply e-mail and then delete this e-mail immediately.

Michael Model

Re: membrane/cytoplasmatic ratio

In reply to this post by Valeria Berno-3

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

That may not work with GFP but, generally speaking, quenchable membrane
markers can be used with live cells. For example, fluorescein is quenched
by acidification, red fluorophores by acid blue 9, pretty much everything
by trypan blue (not sure about the last one). Other than that, I would try
TIRF if you have it.

Mike Model

On Mon, Mar 2, 2015 at 6:30 AM, Valeria Berno <[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.
> *****
>
> Dear all,
>
> One of the facility user is trying to quantify Inositol phosphates (PIP)
> in the plasma membrane of our cells. We manage to visualize it transfecting
> the cells with a recombinant GFP.
>
> The idea is that the GFP-PIP should migrate from membrane to cytoplasm
> with specific treatment. We counterstained the cells with a cytoplasmatic
> marker.
>
> We decided to quantify this with a line scan method (drawing a line
> through the cells) and measure the ratio between intensity in the membrane
> and intensity in the cytoplasm. This is a very common method in different
> paper but I couldn´t find more specifics.
>
>
> My questions are:
> -how can I determine where is my membrane a priori only looking at the
> cytoplasmatic staining?.
> First of all the cytoplasmatic staining doesn´t drop in a specific postion
> but gradually decrease where I can clearly see the peak of the membrane.
>
> Assuming the membrane should start where the peak of the cytoplasm
> staining start to decrease...then should I assume that the membrane is
> always a specific width or should I calculate the GFP intensity up to when
> I reach background level for the green?
>
> I know that the best should be a membrane staining as a reference but
> could you suggest a good one for fixed cells?
>
> Thanks in advance for your support
>
> Valeria
>
>
> Valeria Berno, PhD
> Imaging Facility
> ______
>
> Istituto Nazionale di Genetica Molecolare
> Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi"
> Via F.Sforza 35 - 20122 - Milano (MI) - Italy
> Tel Off.: +39 02.00660 219
> Tel Lab.:+39 02.00660 338
> E-Mail: [hidden email]
> Web site: http://www.ingm.org
>
> SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE
> INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire
> il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD,
> 730 e UNICO nella sezione relativa al finanziamento per la RICERCA
> SANITARIA. Grazie 1000...al 5 per mille!
> Please consider the environment before printing this mail note
>
> DISCLAIMER :
> Questa e-mail potrebbe contenere informazioni privilegiate o
> confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è
> pregato di contattare il mittente rispondendo a questa stessa dopodichè è
> pregato di cancellarla immediatamente.
>
> This e-mail may contain confidential or privileged information. If you
> think you have received this e-mail in error, please advise the sender by
> reply e-mail and then delete this e-mail immediately.