Biology Reference
In-Depth Information
mixture of bu
V
ers in the cytoplasm. Examples of such solutions of the di
V
usion
equation exist for spherical di
usion inward from the cell surface (
Nowycky and
Pinter, 1993; Sala and Hern
´
ndez-Cruz, 1990
), cylindrical di
V
usion inward from
membranes of nerve processes (
Stockbridge and Moore, 1984; Zucker and
Stockbridge, 1983
), di
V
V
usion from a point source (
Fryer and Zucker, 1993;
Stern, 1992
), and di
usion from arrays of point sources (
Fogelson and Zucker,
1985; Matveev et al., 2002, 2004, 2006, 2009; Pan and Zucker, 2009; Simon and
Llin´s, 1985; Tang et al., 2000; Yamada and Zucker, 1992
). For large cells, the
spatial nonuniformity of light intensity and photolysis rate also must be consid-
ered, taking into account the absorbances of all the species of diazo and the
changes in their concentration with photolysis. Like azid-1, the self-screening
imposed by diazo chelators is reduced with photolysis, so successive flashes (or
prolonged illumination) are progressively more e
V
V
ective.
V. Introduction into Cells
Photolabile chelators are introduced into cells by pressure injection from micro-
pipettes, perfusion from whole-cell patch pipettes, or permeabilization of the cell
membrane. Iontophoresis is also suitable for diazo compounds, since this proce-
dure inserts only the Ca
2
þ
-free form. For the caged Ca
2
þ
substances, this method
of introduction requires that the chelator load itself with Ca
2
þ
by absorbing it from
cytoplasm or intracellular stores. Filling cells from a patch pipette has the special
property that, if the photolysis light is confined to the cell and excludes all but the
tip of the pipette inside the cell, the pipette barrel acts as an infinite reservoir of
unphotolyzed chelator. Then the initial conditions of solutions in the pipette can be
restored within minutes after photolysis of the chelator in the cell. The nitr and
diazo compounds are soluble at concentrations over 100 mM and DM-nitrophen
is soluble at 75 mM, so levels in cytoplasm exceeding 10 mM can be achieved
relatively easily, even by microinjection, making the exogenous chelator com-
pound the dominant bu
er.
Nitr and diazo chelators also have been produced as membrane-permeant acet-
oxymethyl (AM) esters (
Kao et al., 1989
). Exposure of intact cells to medium
containing these esters (available from CalBiochem and Molecular Probes, respec-
tively) might result in the loading of cells with nearly millimolar concentrations, if
su
V
cient activity of intracellular esterase is present to liberate the membrane-
impermeant chelator. However, nitr-5 or nitr-7 introduced in this manner is not
bound to Ca
2
þ
, so it must sequester Ca
2
þ
from cytoplasm, from intracellular stores,
or after Ca
2
þ
influx is enhanced, for example, by depolarizing excitable cells. The
final concentration, level of Ca
2
þ
loading, and localization of the chelator are
uncertain, so this method of incorporation does not lend itself to quantification
of e
Y
ects of photolysis unless cells are coloaded with a Ca
2
þ
indicator.
During loading and other preparatory procedures, the photolabile chelators may
be protected fromphotolysis with low pass UV-blocking filters in the light path of the
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