From: Howard Shapiro (hms@shapirolab.com)
Date: Wed Nov 19 2003 - 19:04:49 EST
Nick Holmes wrote:
>Howard Shapiro wrote:
> > Stephen Sullivan wrote:
> >
> > >I am studying nuclear reprogramming in cell hybrids. I want develop a
> FACS
> > >based system that will allow me to sort heterokaryons (fused cells where
> > >the two diploid nuclei from the fused cells are still distinct) from cell
> > >hybrids (where the nuclei have fused to form a single tetraploid
> nucleus).
> > >I am currently thinking about using FRET (fluorescence resonance energy
> > >transfer) to allow me so discriminate between heterokaryons and cell
> > >hybrids. .
>
> > >Does anyone have an
> > >alternative way to sort large numbers of heterokaryons from cell
> hybrids ?
> > >Thanks, Stephen
> >
> > I wouldn't FRET about this...but, all kidding aside, I think there may
> be a
> > somewhat simpler way to identify the hybrids. Michnick and his coworkers
> > detect interactions of proteins ("A" and "B" by introducing sequences
> > coding for complementary fragments of enzymes such as dihydrofolate
> > reductase (DHFR) and beta-lactamase to the sequences coding for the
> > proteins under study; when DHFR is used, the interaction is detected by
> > binding of fluorescently labeled methotrexate. The heterokaryons should
> not
> > bind the labeled compound, whereas the hybrids should.
>
>I cant see how your suggestion would work Howard (or Stephen's).
>
>The 2 complementary fragments are synthesized on ribosomes in the cytoplasm.
>Thus fuse the 2 cells and you can form hybrids. Even if the proteins were
>really localised tightly to the nucleus (and I dont think they generally
>are), there would be crossover of the newly synthesized proteins after
>cytoplasmic fusion, so that the nuclear protein makeup will become
>homogenised over time (rate depending on turn over etc.).
>
>I cant think of a reliable way to do what Stephen wants. To really
>discriminate between binucleate and mononucleate fused cells seems to me to
>require detection of a hybrid structure where the subunits cant mix between
>nuclei in the same cytoplasm.
You're almost certainly right; I was taking for granted Stephen's implied
assertion that the proteins were tightly localized to the nucleus, even
though made in the cytoplasm. If this were the case, either the
complementary fragment approach or FRET might provide a stronger signal
from hybrids than from heterokaryons, even in the presence of background
signal from cytoplasmic protein, mixing over time, etc.
One still could, however, use two-color cytoplasmic labeling plus vital DNA
staining to discriminate the heterokaryons and hybrids from diploid (and
G2/M tetraploid) cells of either strain, sort the heterokaryons and hybrids
into multiwell plates, and tell them apart by morphology, probably
utilizing visual observation. There are some flow cytometers with imaging
capability, but I don't think any of them can sort fast enough to do this job.
>
>
>I have a different query tho. Stephen's question implies that when 2
>cells fuse at some frequency less than 100% but more than 0%, the nuclei go
>on to fuse. Do the nuclei in fact fuse in heterokaryons or does the
>tetraploid (subsequently aneuploid usually) nucleus only form after mitosis?
>I am sure this question was answered by someone, probably about 40 years
>ago.
Now that you mention it, I knew enough (at least from reading) about cell
hybridization 25 or 30 years ago to answer that question; I suspect the
neurons devoted to that information have all apoptosed by now.
>Incidentally what species are you working in Stephen? I know tetraploids are
>stable in some species.
Including humans; tetraploid cells are found in liver, among other places,
if my dim memory serves me correctly.
On this one, I guess I violated my own "Know Thy Cells" rule; the only
excuse is that they weren't really *my* cells.
-Howard
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