Cytosolic pH:

Margaret Clarke and I obtained pHi 7.55 using ratiometric pHlourin, a
modified GFP; see J Cell Science 115, 1907-1918 (2002).  More recent
measurments using an improved spectral unmixing algorithm now give values
closer to 7.45, similar to Rob's measurement.

A major uncertainty in all biosensor pH measurements concerns the location
of the biosensor, ie is it really all in the cytosol, or might some also
be in organelles?  Margaret's images of agar-overlayed,
pHlourin-expressing cells showed a clear "swiss-cheese" cytosolic pattern,
with holes where one expected acid vesicles and mitochondria.  Ratio
images showed pH uniform througout the cell.

Margaret, Jyoti Jaiswal and I also made ratio images (unpublished) using
the "esterase-loaded" small-molecule pH sensor BCECF.  These images showed
multiple round, low-pH objects surrounding the nucleus, presumably
endosomes; a higher-pH granular component near the cell surface,
presumably mitochondria, and wavy compartment of intermediate pH in
between, presumably ER.  BCECF appears to enter all of these compartments.
The cell edge was not sharp in these images, and filopodia were not
fluorescent, suggesting that BCECF is cleared from the cytosol.  Thus
BCECF measurements, and by implication measurements with other
esterase-loaded sensors, may simply reflect the relative amounts of acid
vesicles, ER and mitochondria in the individual cell under examination.

Using pHlourin it is easy to show that in phosphate buffers with pH
between 5 and 8.5, pHi in wildtype cells changes by at most a few tenths
of a point.  Acetate at low pHo, however, produces rapid, large changes in
pHi, from which wildtype cells recover in several hours even in the
continued presence of acetate.  Barry Coukell's patB- cells show enhanced
sensitivity to protonophores and do not recover.  An ER-targeted pHlourin
gives pH values lower than the cytosol, but Dicty mitochondria have
cytosolic pH.

We looked at pHi in the cell cycle and development using pHlourin, but
could see no modulation.  Slugs under cover slips often show an "acid
nose" but this disappears if the cover slip is left off, so it presumably
results from anoxia.  In ratio images, the prestalk-prespore boundary is
not apparent.  I also rechecked the pHi of rtoA nulls with pHlourin;
in my hands it was indistinguishable from the parental strain.

If anyone is interested in pHlourin pHi measurements, the vector is in the
stock center, and I can send cells.  If you have access to a
spectroflourimeter, recovery from acetate loading makes a nice student lab
experiment.

best wishes

harry

-------------

On Thu, 24 Jun 2010, Rob KayMRC wrote:

> Salts: Maeda years ago
> pH: about 7.4 from in vivo NMR Kay et al, 1986.
> Rob
>
> Sent from my iPhone
>
> On 24 Jun 2010, at 17:38, Richard Gomer <[log in to unmask]>
> wrote:
>
> > Dunno about specific ions, we measured osmolality of lysed cells (see
> > our 2004 JBC AlrA paper), its ~ 250 mOsm/ kg H2O for veg cells, and is
> > 400 at 6 hours.
> >
> > For cytosolic pH, see Aerts et al 1985 Cell.  We saw cytosolic pH's in
> > veg cells ranging from 6.4 to 7.4 depending on cell cycle phase (see
> > our
> > 2000 JBC RtoA paper)
> >
> > Also, be careful with 'Sorenson's phosphate buffer', about 15 years
> > ago
> > I read a protocol using this  buffer, and didn't have a recipe- I
> > emailed the Dicty listserver and got ~6 replies.  Each recipe was
> > different.....
> >
> > cheers
> > Richard Gomer
> >
> >
> > Richard Gomer
> > Professor
> > TAMU Biology
> > ILSB    MS 3474
> > 301 Old Main Drive
> > College Station, TX  77843-3474
> > 979 458 5745
> >
> >
> >
> >
> >>>> Ralph Gräf <[log in to unmask]> 06/24/10 6:54 AM >>>
> > Dear Dicty Researchers,
> >
> > I was trying to find a publication where somebody has measured or
> > estimated the cytosolic salt concentrations of prominent cations and
> > anions but I was not successful. I doubt that the commonly used low
> > ionic strength Soerenson phosphate buffer reflects a physiological
> > situation for cytosolic proteins.  We would like to use an optimal
> > physiological buffer for cytosolic proteins with regard to pH, ion
> > concentration and ion composition. Any helpful hints are welcome.
> >
> > All the best,
> >
> > Ralph
> >
> >
> > ---------------------------------------------------------
> >
> > Prof. Dr. Ralph Gräf
> >
> > University of Potsdam
> > Institute of Biochemistry and Biology
> > Department of CELL BIOLOGY
> >
> > Karl-Liebknecht-Strasse 24-25, Haus 26
> > 14476 Potsdam
> > Germany
> >
> > Tel.: +49-(0)331 9775520
> > FAX: +49-(0)331 9775522
> >
> > http://www.bio.uni-potsdam.de/professuren/zellbiologie/aktivitaten
>

----------------------------

Prof. Harry K. MacWilliams
Biologiezentrum der Ludwig-Maximilians-Universität
Grosshadenerstrasse 2
82152 Planegg-Martinsried
GERMANY

tel (office) +89 2180 74 288
tel (lab)    +89 2180 74 289
fax.         +89 2180 74 219

callers from North America please dial:
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