Re: How to differentiate plasma membrane vs. cytoplasm in Jurkat lymphocytes?

Posted by TSwayne on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-differentiate-plasma-membrane-vs-cytoplasm-in-Jurkat-lymphocytes-tp7586566p7586597.html

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Dear confocalists,

I am grateful for all of the excellent suggestions!

I don’t have easy access to a fluorescence polarization or FLIM system, though there are facilities nearby. The protein is not exposed to the extracellular milieu, so I wouldn’t be able to label it that way.

However, we could certainly try the ideas for TIRF, FRAP, “squeezing,” and simply maximizing resolution in confocal. But I also see that first I need to clarify the possibilities for localization, because if the protein is expected to accumulate near the PM it will be a very different problem to distinguish membrane-attached from non-attached.

To summarize the strategies that were recommended, roughly in reverse chronological order:

1) CellMask stays on membrane for up to ~40 min in macrophages, but is more internalized after fixation. (Darren Thomson)

2) If the protein is cytoplasmic but accumulated near the membrane, SIM or other techniques would be required to determine the localization. If it is distributed throughout the cytoplasm, you could use Brilliant Violet or similar antibody for protein of interest and controls; use a green cytoplasmic marker. Image cells with highest NA, small pinhole, and coverslip correction, at equator, and look for cells with > 400 nm width (at least 4 pixels) of visible cytoplasm. Compare the violet intensity in the half of cytoplasm closer to the membrane versus the half near the nucleus. (Sylvie Le Guyader)

3)   It’s too risky to assess external vs. internal via LM and EM should be used. (Mike Ignatius)

4) An advanced profile analysis could be used similar to that employed by the Ewa Paluch lab (http://dx.doi.org/10.1016/j.bpj.2013.05.057) who determined actin cortex localization and thickness with precision better than the diffraction limit. (Christian Eggeling)

5) FLIM based on refractive index differences in membrane vs. cytosol, as done by the Cees Otto lab (http://dx.doi.org/10.1529/biophysj.107.127837) (Artem Pliss)

6) For fixed cells, TIRF at a series of angles (cytosolic proteins would get brighter as you go deeper); for live cells, FRAP/FLIP, looking for greater mobility of soluble proteins. (Tim Feinstein)

7) Make resolving the cytoplasm easier by changing cell morphology: compression, activation, or expansion microscopy. (George McNamara)

8) Fluorescence polarization, looking for greater anisotropy in cytosolic proteins (Tim Feinstein)

9) Identify surface-exposed epitopes by labeling live cells on ice. (Renato Mortara)


Thank you again. I love this community!


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Swayne, Theresa C.
Sent: 06 March 2017 19:48
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Subject: How to differentiate plasma membrane vs. cytoplasm in Jurkat lymphocytes?

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Dear fellow confocalists,

One of my users would like to determine whether an epitope-tagged protein, expressed in Jurkat cells, is localized to the plasma membrane or the cytosol. These cells have only a thin layer of cytoplasm between the nucleus and PM.

It seems to me that at minimum we need a good plasma membrane marker, and control proteins known to be in cytosol and plasma membrane respectively.  Then I’m thinking we could do scanning confocal with high NA lens, and profile analysis or overlap analysis to measure the degree of coincidence between the membrane marker and the tagged protein.   Does that make sense as a strategy?

If so, do folks have a preferred membrane marker for this cell type? I’ve seen things like CellMask and PKH26, but the images I’ve seen online and in other cell types don’t show “pure” plasma membrane labeling.

Thanks in advance for any suggestions.

------------------------------------
Theresa Swayne, Ph.D.
Manager
Confocal and Specialized Microscopy Shared Resource<http://hiccc.columbia.edu/research/sharedresources/confocal>

Herbert Irving Comprehensive Cancer Center
Columbia University Medical Center
1130 St. Nicholas Ave., Room 222A
New York, NY 10032
Phone: [phone]
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