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Hi Julia,
Vesicle aging and incorporation into plasma membrane:
See Robert H Chow's papers, PubMed 12634788 (commentary at 12888291) and
12887320 ... now could use a "Fast (or medium or slow) Timer FP" - see
20121102 for entry point to Fast-FT etc - also check out superfast GFP
PubMed 18545653, whose topic looks close to your interests.
For exocytosis of vesicles to contribute to plasma membrane, see
21903928 and 21903923 as an entry point to FM1-43 dye.
Richard Pagano routinely added fluorescent lipids to the plasma membrane
by adding fluorescent lipid-BSA to cells (and could exchange slipids off
with BSA as well). I suggest doing a pubmed search for:
pagano re membrane fluorescence
as an entry point.
You could do FlAsH/ReAsH tetracysteine pulse-chase kinds of experiments,
usually works best in the redox conditions of the cytoplasm, so would
want to put the tetracys motify on the cytoplasmic side of a protein or
possibly in the membrane spanning - see PubMed 21500332.
Diffusion happens - can measure it:
Xiaowei Zhuang recently published a STORM (and 2 color) STORM paper
studying single organelle marking (standard, straight out of Mol Probes
catalog) fluorophores - including single molecule diffusion:
Super-resolution fluorescence imaging of organelles in live cells
with photoswitchable membrane probes. </pubmed/22891300> Shim SH,
Xia C, Zhong G, Babcock HP, Vaughan JC, Huang B, Wang X, Xu C, Bi
GQ, *Zhuang X*. Proc Natl Acad Sci U S A. 2012 Aug
28;109(35):13978-83. PMID: 22891300
Imaging membranes in live cells with nanometer-scale resolution
promises to reveal ultrastructural dynamics of organelles that are
essential for cellular functions. In this work, we identified
photoswitchable membrane probes and obtained super-resolution
fluorescence images of cellular membranes. We demonstrated the
photoswitching capabilities of eight commonly used membrane probes,
each specific to the plasma membrane, mitochondria, the endoplasmic
recticulum (ER) or lysosomes. These small-molecule probes readily
label live cells with high probe densities. Using these probes, we
achieved dynamic imaging of specific membrane structures in living
cells with 30-60 nm spatial resolution at temporal resolutions down
to 1-2 s. Moreover, by using spectrally distinguishable probes, we
obtained two-color super-resolution images of mitochondria and the
ER. We observed previously obscured details of morphological
dynamics of mitochondrial fusion/fission and ER remodeling, as well
as heterogeneous membrane diffusivity on neuronal processes.
Generally FM1-43 gets endocytosed (or is thought to get retrieved) - I
now think that under the right environmental conditions (see O2
scavenging reagents of Shim et al and dSTORM papers) that any
fluorophore will be single molecule imagable - so FM1-43 molecules that
escape endocytosis could be tracked on the plasma membrane (i.e.
TIRF-STORM).
George
On 9/9/2012 8:43 AM, Julia Edgar wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Dear All
> Is there a way of labelling newly generated cell membrane with a fluorescent tag so that we can visualise where 'new' membrane is being added to the cell (in vitro)?
>
> I assume that if we 'switch on' (e.g. using an inducible system) a membrane associated fluorescent tag it will very quickly diffuse throughout the entire cell membrane and prevent us from determining what is 'new' membrane.
>
> Thank you in advance for any advice.
>
> Julia Edgar
> University of Glasgow
>
>
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