Biology Reference
In-Depth Information
being isolated on a single atom,
the positive charge can reside on many
di
erent atoms in the structure—that is, the positive charge is delocalized over
the entire highlighted structure. A hydrophobic organic ion whose charge
is delocalized can pass through lipid membranes. Mitochondria maintain a re-
markably negative membrane potential—the mitochondrial lumen is typically at
V
200 mV relative to the cytosol. Therefore, Rhod-2 AM, which is a
hydrophobic organic cation whose positive charge is delocalized, can permeate
through the plasma membrane and the mitochondrial membranes and preferen-
tially partition into the negative lumen of mitochondria. In mitochondria, cleavage
of AM ester groups by esterases liberates the Ca
2
þ
-sensitive form of Rhod-2 -
(bearing multiple nondelocalized negative charges), which is not membrane-per-
meant and thus trapped in the mitochondrial lumen. Therefore, Rhod-2 can be
used to monitor intramitochondrial Ca
2
þ
signals (
Babcock et al., 1997; Tsien and
Bacskai, 1995
).
150 to
A. Estimating the Fraction of Intracellular Rhod-2 Indicator that Resides in Mitochondria
While Rhod-2 can be preferentially loaded into mitochondria, the discrimina-
tion against loading into other subcellular compartments is imperfect. Some
intracellular Rhod-2 is expected to reside in the cytosol and nonmitochondrial
organelles. A simple procedure based on di
erential permeabilization of cellular
membranes can be used to estimate the fraction of intracellular Rhod-2 that
actually resides in mitochondria. The procedure is a modification of the one
described in
Section III.B.2
. The procedure consists of monitoring total Rhod-
2 fluorescence from a cell or a group of cells bathed in low-Ca
2
þ
medium and
treating the cells sequentially with (1) a Ca
2
þ
ionophore (ionomycin or Br-
A23187), (2) the mild detergent digitonin to permeabilize the plasma membrane,
and (3) a strong detergent, for example, Triton X-100 or sodium dodecyl sulfate
(SDS), to permeabilize all membranes.
Figure 16
shows the procedure being
applied to a vagal sensory neuron that had been incubated with 1
m
M Rhod-
2 AM for 1 h at room temperature. Application of ionomycin abolishes significant
di
V
erences in [Ca
2
þ
] between di
erent subcellular compartments. This ensures that
Rhod-2 in all compartments is at comparable levels of Ca
2
þ
-binding, and thus
would contribute fluorescence intensity in proportion to their actual content in
each compartment. Once the fluorescence reaches a steady baseline after ionomy-
cin treatment, digitonin permeabilization of the plasma membrane allows cytosolic
Rhod-2 to escape,
27
giving a decrement in total fluorescence (labeled ''C'' in
Fig. 16
). Subsequent permeabilization of all cellular membranes by SDS allows
V
V
27
Digitonin treatment leads to release of Rhod-2 from the nucleus as well. The nuclear pores have a
size exclusion limit of 35-40 kDa; molecules with molecular mass less than the exclusion limit can freely
exchange between the nucleoplasm and cytosol. Therefore, with respect to low-molecular-mass solutes
such as simple ions (e.g., Ca
2
þ
,Na
þ
,Cl
) and small organic molecules (e.g., glucose, ATP, fluorescent
indicators), the nucleo-cytoplasm functions as a single ''cytosolic'' compartment.
