Vakuola Kontraktil PDF
Vakuola Kontraktil PDF
Vakuola Kontraktil PDF
Abstract. Amoebae of the eukaryotic microorganism connected tubules and cisternae comprise the contrac-
Dictyostelium discoideum were found to contain an in- tile vacuole system of Dictyostelium. Earlier studies
terconnected array of tubules and cisternae whose had demonstrated that contractile vacuole membranes
membranes were studded with 15-nm-diameter "pegs" in Dictyostelium are extremely rich in calmodulin
Comparison of the ultrastructure and freeze-fracture (Zhu, Q., and M. Clarke. 1992. J. Cell Biol. 118:
behavior of these pegs with similar structures found in 347-358). Light microscopic immunofluorescence
other cells and tissues indicated that they were the confirmed that antibodies against the vacuolar proton
head domains of vacuolar-type proton pumps. Sup- pump colocalized with anti-calmodulin antibodies on
porting this identification, the pegs were observed to these organelles. Time-lapse video recording of living
decorate and clump when broken amoebae were ex- amoebae imaged by interference-reflection microscopy,
posed to an antiserum against the B subunit of mam- or by fluorescence microscopy after staining contrac-
malian vacuolar H+-ATPase. The appearance of the tile vacuole membranes with potential-sensitive styryl
peg-rich cisternae in quick-frozen amoebae depended dyes, revealed the extent and dynamic interrelationship
on their osmotic environment: under hyperosmotic of the cisternal and tubular elements in Dictyostelium's
conditions, the cisternae were fiat with many narrow contractile vacuole system. The high density of proton
tubular extensions, while under hypo-osmotic condi- pumps throughout its membranes suggests that the
tions the cisternae ranged from bulbous to spherical. generation of a proton gradient is likely to be an im-
In all cases, however, their contents deep etched like portant factor in the mechanism of fluid accumulation
pure water. These properties indicated that the inter- by contractile vacuoles.
HE contractile vacuoles in protozoa have long re- 1984; Walter and Gutkneck, 1986). Contractile vacuole
Figure 2. Time-lapse video microscopy of an AX2 Dictyostelium amoeba imaged by IRM. Recording starts in the upper left panel and
proceeds uninterrupted at 4-s intervals to the lower fight. Contractile vacuoles and their associated membrane tubules appear dark against
the lighter grey of the cell bottom, due to their close approach to the plasma membrane. Arrows denote the onsets of three discharge events,
which in subsequent frames can be seen to involve disappearance of the contractile vacuole and its replacement by a faintly visible and
dispersed reticulum of dark dots and threads. These fine elements then gradually enlarge and appear to coaelesce into a new vacuole at
or near the site of the former one. A particularly clear example o f a tubulo-cisternal intermediate in this process is indicated at the asterisk.
Bar, 10 t~m.
8 a), whereas they were more widely and uniformly dis- unequivocally distinguished elements of the contractile vac-
persed on the rounder cisternae with puffy or varicose arms uole system from other organelles such as food vacuoles and
(Figs. 8, b and c). Thus, the freeze-dry images suggested that endosomes, which were invariably filled with a dense matrix
in Dictyostelium, proton pumps cannot fit into the most of non-etchable material (Fig. 11 b). It also ruled out an ear-
highly convoluted regions of contractile vacuole membrane, lier hypothesis that contractile vacuoles might be filled with
but can readily redistribute within it as it fills and changes an expandable hydrocolloid that accumulated and retained
shape. water (Heywood, 1978).
The second distinctive feature of contractile vacuoles was
their unique fracturing behavior. In unfixed preparations,
Freeze Etch Views of Intact, Quick-Frozen
contractile vacuole membranes displayed an unusually high
Dictyostelium Cells
concentration of large intramembrane particles on the E
The quick-freeze, deep-etch technique revealed two impor- fracture-face (i.e., the luminal leaflet of their bilayer), corre-
tant features of the contractile vacuole system (Figs. 10 and sponding in density to the 15-nm pegs on their surfaces
11). First, regardless of the degree of distention of the cister- (Figs. 10 and 11 d, asterisks). In contrast, few IMPs were
nae, they appeared utterly empty inside; that is, the etching present on the E fracture-faces of ER, lysosomal, or en-
behavior of their contents was equivalent to that of pure wa- dosomal membranes (not shown). Interestingly, glutaralde-
ter or a dilute electrolyte (Fig. 11 a). This empty appearance hyde fixation before freezing altered the fracturing properties
of contractile vacuole membranes, such that IMPs parti- ious interconnections of the system could be seen in different
tioned almost exclusively to the P fracture-face, leaving the focal planes (Fig. 12, lower panels).
E fracture-face so depleted of mass that it sometimes fell Double-staining experiments verified that the proton pump-
completely apart during deep etching (not shown). Similar bearing network of tubules and vacuoles seen by deep-etch
fracturing properties have been reported for proton pump- EM is indeed the contractile vacuole system of Dic-
bearing membranes of other aldehyde-fixed tissues (Allen tyostelium. The antiserum specific for the vacuolar H +-
and Staehelen, 1981; Orci et al., 1981; Allen, 1984; Stetson ATPase (Moriyama and Nelson, 1989; see Figs. 1, 6, and 7),
and Steinmetz, 1985; Brown, 1989). when used together with anti-calmodulin antibodies, yielded
the results shown in Fig. 13. This figure shows three typical
Light Microscopic Immunocytochemistry examples of interphase amoebae as well as one anaphase cell
of Contractile Vacuoles in Dictyostelium Amoebae displaying the contractile vacuole dispersion that occurs in
It was previously demonstrated that antibodies against Dic- mitotic Dictyostelium cells (Zhu et al., 1993). In each case
tyostelium calmodulin strongly and selectively label contrac- there was a close correspondence between the distribution of
tile vacuole membranes (Zhu and Clarke, 1992). In the ear- the proton pump antigen and of calmodulin, confirming that
lier study, cells were simultaneously fixed and extracted with contractile vacuole membranes are the principal locus of
cold formaldehyde-methanol, typically after exposure of the vacuolar proton pumps in Dictyostelium. On the other hand,
cells to hypo-osmotic buffers that caused their contractile anti-proton pump staining of endosomes and phagosomes in
vacuoles to distend; under those conditions, staining was re- Dictyostelium was generally below threshold, even though a
stficted to the large vacuoles. With the present IRM and low concentration of ~15-nm pegs was observed on these
TEM images in mind, we tested several fixation conditions elements by TEM (data not shown).
to see whether we could preserve and immunolabel the tubu- Further confirmation of this proton pump distribution was
lar elements of the system as well as the vacuoles. Best obtained by visualizing the contractile vacuole system in
results were obtained using the two-step fixation procedure newly germinated wild-type (NC4) amoebae. Newly germi-
described in Methods and Materials and using cells that had nated cells typically contain a single prominent, very active
been exposed to hypo-osmotic medium only briefly (<5 min) contractile vacuole (Cotter and Raper, 1966; Cotter et al.,
or not at all (i.e., fixation directly in HL5, the axenic growth 1969; Zhu and Clarke, 1992). When such cells were labeled
medium). These conditions often revealed narrow extensions with the anti-proton pump antiserum, they displayed clear
radiating from the vacuoles (Fig. 12, upper panels), and var- staining of this large contractile vacuole, and also diffuse
Discussion
tozoa and suggested that these were involved in water ac- tebrate proton pumps, both in the TEM (Figs. 6 c and 7 i)
cumulation (McKanna, 1974, 1976). Here, the identification and in the light microscope (Figs. 12-14). Second, they oc-
of these pegs as being vacuolar-type proton pumps was made cur in great abundance in Dictyostelium membrane fractions
in three ways. First, the pegs decorate with antibodies to ver- that are enriched in proton pumps (unpublished observations
with T. Steck's laboratory, Department of Biochemistry,
University of Chicago; see Nolta et al., 1991). Finally,
they look identical to vacuolar proton pumps seen in other
tissues and phyla (Fig. 7). In particular, they look exactly like
the peg-like "portasomes" seen in the plasma membrane of
/ \ thin-sectioned insect gut epithelial cells (Harvey et al., 1981,
1983). These structures have recently been recognized to be
vacuolar-type proton pumps (Klein et al., 1991; Wieczorek
et al., 1991), and indeed, look exactly like the pegs seen in
Dictyostelium and other protozoal contractile vacuoles (Fig.
7 h).
IRM studies of Gingell et al. (1982) and the work presented and central vacuoles (storage reservoirs), has long encour-
here have shown that Dictyostelium's contractile vacuole sys- aged the idea that water is accumulated in a two-step reaction
tem actually consists of a network of relatively continuous as well (recently reviewed by Zeuthen, 1992). The first step
membrane channels that are always present but simply is thought to be ion transport into the peripheral collecting
change shape and organization during water accumulation. ducts, resulting in a passive or osmotic influx of water. The
This is also true of several other types of amoebae that we second step, presumably occurring in the central storage res-
have examined (unpublished observations.) The narrower ervoir, involves pumping the same ions back into the
tubular and cisternal elements of these systems would have cytoplasm, thereby preserving the cells' intracellular ions and
been invisible to light microscopists working without IRM producing a dilute contractile vacuole discharge (Schmidt-
and without special stains or antibodies, and would probably Nielsen and Schrauger, 1963; Mayer and Iverson, 1967;
have broken down into vesicles under earlier TEM fixation Riddick, 1968). For amoebae, this view was amended to
protocols (Doggenweiler and Heuser, 1964). Thus, the con- state that specific "satellite" vesicles pumped ions into them-
tractile vacuole system of Dictyostelium resembles that of selves and accumulated water osmotically, then fused to
other protozoa, except that its proton pumps are not confined form the contractile vacuole, whereupon the ions were pumped
to tubular spongiomes (and in fact are excluded from its nar- back into the cytoplasm (Schmidt-Nielsen and Schrauger,
rowest tubular elements, as shown in Fig. 8 a). Instead, pro- 1963).
ton pumps populate nearly all parts of its contractile vacuole The basic problem with this classical view is that the final
membrane, including the larger vacuoles. This would be ex- storage reservoir would have to assume an exceptionally low
pected if the tubules merged with the vacuoles as they grew, water permeability to resist passive water egress as its ions
as suggested by our data (see Figs. 2, 8, and 15). were being reabsorbed and its contents were becoming hypo-
tonic relative to the cytoplasm. It is unclear how such a low
permeability could be maintained, since artificial bilayer
Implications for Contractile Vacuole Function studies suggest that even pure lipid membranes with no pro-
The clear differentiation of certain protozoal contractile tein insertions (pumps, channels, etc.) are relatively perme-
vacuole systems into two physically distinct and relatively able to water (Finkelstein, 1984; Walter and Gutkneck,
permanent phases, tubular spongiomes (collecting ducts) 1986). Moreover, isolated contractile vacuoles behave like
Another area for future study is the role of contractile tubules (Carasso and Favard, 1966). Spasmonemal tubules
vacuoles in cellular activities other than osmoregulation. For would thus be functionally equivalent to the sarcoplasmic
instance, the peritrich protozoa have in their attachment- reticulum of vertebrate muscle, and so it is interesting to note
stalks structures called "spasmonemes" composed of dense that there is now evidence that these tubules are physically
bundles of filaments riddled with membrane tubules. A continuous with the tubular spongiome that typifies peritrich
rapid, calcium-activated coiling of the filaments is thought contractile vacuoles (Patterson, 1980). Further indication of
to underlie these protozoa's escape reflex (Amos, 1972). The this interrelationship came from our finding in unpublished
source of calcium, and the pool to which it is returned as studies that peritrich spasmonemal tubules possess surface
coiling is relaxed, is thought to be the intervening membrane "pegs" similar in structure to the other proton pumps studied
here. Moreover, we find that the pegs on spasmonemal tu-
bules are arranged in the same beautiful spiral patterns char-
acteristic of the pegs on spongiome tubules in these and other
ciliate protozoa (Schneider, 1960; Carasso et al., 1962; Al-
len, 1973; Allen and Fok, 1988). By extension, it seems pos-
sible that contractile vacuoles in these and other protozoa
may also be calcium sequestering and eliminating or-
ganelles. Their rich endowment with proton pumps might,
for example, allow them to accomplish this by H/Ca2+ ex-
change, using a strong proton gradient established across
their membranes. In fact, it has been recently reported that
a proton pump-rich membrane fraction isolated from Dic-
tyostelium amoebae possesses ATP-driven Ca2/H antiport
activity (Rooney and Gross, 1992). This membrane fraction,
Figure 16. Stationary-phase AX3 amoebae stained with anti-proton
pump antibodies, examined 2 d after a culture had reached station- also described by Nolta et al. (1991) as containing proton
ary phase. Most amoebae were of normal size and still contained pump-rich "acidosomes," we believe to be fragmented con-
contractile vacuoles that became stained with the antibody. How- tractile vacuole membranes. In any case, the present demon-
ever, also present were many small refractile cells that appeared stration of the great abundance of proton pumps on bona fide
shrunken and contained no vacuoles. In these ceils, the anti-proton contractile vacuole membranes from Dictyostelium will, we
pump antibodies strongly labeled the plasma membrane, suggesting predict, be relevant to many aspects of protozoal cell physi-
that contractile vacuole membrane had merged with it. Bar, 10 #m. ology.
We thank Dr. Nathan Nelson (Roche Institute of Molecular Biology, Nutley, fracture evidence for differentiation and flow in Paramecium. J. Cell Biol.
NJ) for the gift of his antiserum against the vacuolar proton pump. Many 89:9-20.
Allen, R. D., and A. K. Fok. 1988. Membrane dynamics of the contractile
of the ideas generated in the course of this study resulted from helpful dis- vacuole complex of Paramecium. J. Protozool. 35:63-71.
cussions with Ted Steck (University of Chicago, Chicago, IL), Steve Gluck Amos, W. B. 1972. Structure and coiling of the stalk of the peritrich ciliates
(Washington University, St. Louis, MO), and Tom Maron (University of Vorticella and Carchesium. J. Cell Sci. 10:95-108.
Florida, Gainesville, FL); their valuable input was greatly appreciated. Betz, W. J., F. Mao, and G. S. Bewick. 1992. Activity-dependent fluorescent
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Helping with the immunofluorescence was Ms. Tongyau Liu; with the time- rosci. 12:363-375.
lapse video microscopy was J. Hiroshi Morisaki; with the deep elch EM Blair, H. C., S. L. Teitelbaum, R. Ghiselli, and S. Gluck. 1989. Osteoclastic
was Robyn Roth, Shailesh Patel, and Brenda Moore; and with the typing bone resorption by a polarized vacuolar proton pump. Science (Wash. DC).
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This work was supported by National Institutes of Health grants GM 323:630-632.
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