Biology Reference
In-Depth Information
membrane-enclosed compartments as well. Although this process occurs to a
large extent in the cytosol, enzymatic hydrolysis of AM esters also can take
place within subcellular organelles. Therefore, some fraction of the indicator
molecules tend to be trapped in noncytosolic compartments. Second, some cell
types actively endocytose material from the incubation medium (
Malgaroli
et al.,1987
), including dispersed AM esters, which are then hydrolyzed to
release fluorescent indicator molecules within organelles of the endocytotic
pathway. Presumably, indicator molecules liberated in this way can end up in
a variety of organelles that are connected to the endocytotic pathway by
vesicular tra
c. Because most subcellular organelles [e.g., endoplasmic reticu-
lum (ER), lysosomes] tend to have high intraorganellar [Ca
2
þ
](
Y
>m
M), indica-
tors confined to these organelles would be saturated with Ca
2
þ
and would
contribute a high-[Ca
2
þ
] fluorescence signal that would not vary with changes
in cytosolic [Ca
2
þ
]. Therefore, the net e
ect of compartmentalized dye is
biasing measured cytosolic free Ca
2
þ
concentration toward higher values.
The first cause of dye compartmentalization just stated is a reflection of an
inherent imperfection of the AM ester loading technique and cannot be remedied
easily. The second cause is a cell biological process and can be attenuated.
Because endocytosis is a temperature-dependent process, cells loaded at lower
temperatures with AM ester tend to show less compartmentalization of indicator
(
Malgaroli et al., 1987
). The following results illustrate this point. In REF52
fibroblast cells, roughly 30% of total intracellular Fura-2 was in noncytosolic
compartments when loading via the AM ester was carried out at 37
C, whereas
only 10% was compartmentalized when loading was performed at 23
C. Although
endocytosis is known to be blocked at 10
C in mammalian cells and at 4
Cin
amphibian cells, loading at the lowest biologically permissible temperature does
not necessarily yield the best results, because processing of AM esters by esterases
in the cytosol is also temperature-dependent. At very low temperatures, the con-
centration of indicator accumulated in the cytosol can be quite low. Optimal
loading temperature is determined empirically to be the temperature at which
dye compartmentalization is minimized while good cytosolic loading is main-
tained. In practice, convenience often dictates loading at room temperature as a
reasonable compromise.
9
V
2. Assessing Extent of Compartmentalization
The extent of indicator compartmentalization can be estimated through a simple
semiquantitative procedure that is based on the observation that micromolar
concentrations of digitonin primarily permeabilizes the plasma membrane
9
When loading is done in air, the incubation medium should be bicarbonate-free (i.e., some other
bu
er such as HEPES should be used to maintain the pH of the medium). Otherwise, steady loss of CO
2
will shift the CO
2
-HCO
3
-CO
3
2
equilibrium and rapidly alkalinize the medium.
V
