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Electron microscope observations on the submicroscopic vesicular component of the subesophageal body and pericardial cells of the grasshopper, Melanoplus differentialis differentialis (Thomas)
Experimerlfal
108
Cell Research 22 108-119 (1961)
ELECTRON MICROSCOPE OBSERVATIONS ON THE SUBMICROSCOPIC VESICULAR COMPONENT OF THE SUBESOPHAGEAL BODY AND PERICARDIAL CELLS OF THE GRASSHOPPER, MELANOPLUS DIFFERENTIALIS DIFFERENTIALIS (THOMAS)l R. G. KESSEL” Department Bwwnan
of Zoology, Stote University of Iooloa and the Department of An.atomy, Gray School of Medicine, K’inston-Salem, North Carolina, U.S.A.
Received January 41, 1960
the electron microscope it has been shown in a variety of cells that the plasma membrane is infolded extensively [ 1, 2, 13, 11, 161. Moreover, in cells possessing such an elaboration of the plasma membrane, a vesicular component of the cytoplasm is often displayed as isolated units lying close to In cells without this surface or continuous with the plasma membrane. specialization, the vesicular component may be found concentrated adjacent to the surface and, indeed, may appear in contact with shallow inraginations (caueola intracellularis of Tamada [17]) of the plasma membrane [-I, 5, 10, 11, 121. Furthermore, a fusion of vesicles resulting in the formation of a larger vawole has been reported [15]. Because the small vesicular component of the cytoplasm has been observed aggregated in prorimity with the plasma membrane as well as close to or continuous with shallow inuaginations of the plasma membrane, various investigators hare suggested such spatial relationships indicate that the sub-
%‘ITH
1 This investigation was supported, in part, by a research grant (RG-6942) from the Division of General iUedica1 Sciences, Public Health Service, and Fluid Research Funds from the Bowman Gray School of Medicine. Bowman Gray School of Medicine, Winston% Present address: Department of Anatomy, Salem, North Carolina, U.S.A.
Fig. l.-Periphery of a pericardial cell (S clay adult raginations of the plasma membrane (ICM), small submicroscopic vesicles (PV) are shown. 52,500 x .
male). The plasma membrane (CAfj, inperipheral vacuoles (SV) and numerous
Fig. T.-Edge of a pericardial cell (one day adult male) containing numerous vesicles (PI’), small vacuoles (S V) and infoldings of the plasma membrane (IC31). One such infolding appears to extend the entire length of the section. 14,800 y. Experimenfnl
Cell Research 22
Submicroscopic
vesicles
ctnd cell Iranspori
ERrperimental
Cell Research 22
R. G. Kessel microscopic vesicles may be formed by a pinching oif process from the plasma membrane or, conversely, they may represent vesicles destined to coalesce with the plasma membrane [5, la]. Recently Bennett [3] advanced a hypothetical mechanism whereby membrane flow and membrane resiculation could play a role in active transport in the cell based, in part, on the above findings. A similar hypothesis was postulated by Odor [ 111 on the basis of experimental studies on the mesothelial cells of rats. During the course of cytological and physiological experiments on the subesophageal body cells in embryos and on pericardial cells in adult grasshoppers, a vesicular component of the cytoplasm was observed which, because of the morphological relationships between it and the specializations of the cell surface and vacuoles within the cells, suggested that this structure might play a role in transport of material into or out of the cell, thus substantiating Bennett’s theory of membrane flow and membrane \resiculation as mechanisms for active transport and ion pumping. Because of the early theories presented by physiologists with respect to membrane permeability and because of new techniques cleveloped in electron microscop? demonstrate a vesicular component suggesting that materials can move into or out of cells by a type of microvesiculation, it would appear of interest that the more widespread this type of vesiculation is found at the submicroscopic level the more support would be delivered to the hypothesis advanced by Bennett [3 j. It is with this thought in mind that the present observations are herein presented. MATERIALS
AND
METHODS
Pericardial
cells from adult, laboratory reared, grasshoppers, Melazzoplus dif(Thomas), and subesophageal body cells from pre-diapause embryos mere examined with the electron microscope. Pericardial cells were immediately removed from the animals by a ventral dissection and placed in cold 1 per cent osmium tetroxide solution buffered with acetate-Verona1 at a pH of 7.2,
ferentialis
differenticclis
Fig. 3.-Periphery of a pericardial cell (8 day adult male) showing -the plastna membrane (CdZ), an infolding of the plasma membrane containing electron dense material (I), a small vxuole (SV) and numerous resicles (PI’) some of which are aligned along the plasma membrane (CJZ). 16,200 x . Fig. l.-Edge of another pericardial cell from the satne animal as Fig. 3. In addition to the plasma metnbrane (CM), infoldings of the plastna membrane (Zj, small vacuoles t.SL’), and isolated vesicles (PI-), a region is indicated (arrows) in which expansions of the inuaginated plasma membrane occur suggesting vesicle formation. These expansions are litnited by a double membrane. Golgi apparatus (Cd). 31,600 ): . Experimentnl
Cell Reseurch 22
Submicroscopic vesicles and cell ~ra~,~~o~~
Experimental
111
Cell Research 22
R. G. Kessel
112
7.5 or 7.8 and allowed to fix for l-2 hours. In half of the cases, the buffered solution of osmium tetroxide was injected into the hemocoel before making the dissection. The pericardial cells mere then dehydrated, embedded in methacrylate and sectioned with a Porter-Blum microtome equipped with a glass knife and set to section at 0.025 microns. The electron microscopes used included an RCA4 EMU-2B and an EMU-SD. Entire embryos (18-19 days development at 25°C) were fixed for l-2 hours in 1 per cent buffered osmium tetroxide (pH 7.8). The embryos were washed quickly with distilled mater and dehydrated rapidly through a graded series of alcohols. In one of the lower alcohols, the entire subesophageal body was freed by removing the remaining embryonic tissue. The remainder of the procedure for electron microscopy was identical to that described for the pericardial cells. OBSERVATIONS
The prominent features of the ultramicroscopic strwture of the peripheral region of the c.ytoplasm of both the subesophageal body and pericardial cells include: (1) a system of smooth surfaced invaginations or infoldings of the plasma membrane (Figs. 1, 2, 6, ICM), (2) small peripheral vacuoles (Figs. l-8, SIT), and numerous submicroscopic vesicles (Figs. 1-7, PV). The diameter of the invaginated plasma membrane is variable but in many instances ranges from 370450 ,&. These peripheral smooth membranes seen in electron micrographs of both cell types appear to arise as a result of an inragination of the plasma membrane (Fig. 9, double arrows). In some electron micrographs, the cut edges of the tubular imagination ap0 7, arrow). This suggests that the most external, uninvaginapear double (Fi,. ted portion of the plasma membrane is composed of a double membrane separated by a narrow intermembrane space. Electron dense material is often observed within the tubular inraginated plasma membranes in section (Fi g. 2, 10, arrows). In addition, expansions of these walls can also be seen (Fig. 4, arrows). The structure seen in Fig. 1 suggests that a vesicular formation from the invaginated plasma membrane was underway at the time of fixation of this pericardial cell and is reminiscent of the caveolae intracellularis [lT]. Tubular evaginations (careolae) of the inraginated portion of the plasma membrane are also evident in electronmicrographs of the subesophageal body cell (Fig. 6, arrow). Careolae formaFig. 5.PElectron micrograph of subesophageal body cell showing a portion of a large central vacuole (L V), smaller, peripheral vacuoles (S I’) and numerous submicroscopic vesicles (P F’). Note the relationship of several vesicles (arrows) to the invaginated plasma membrane (IJ1C). In addition many of the vesicles are closely applied to the small vacuoles (arrows). In lower left of micrograph (arrow), several vesicles are arranged in a circular area with fine interresicular con33,000 x. nections. Arrows in upper right of micrograph su,,ooest areas of caveolae formation. Experimental
Cell Research 22
Submicroscopic vesicles and cell transport’
5 - 601732.58
Experimental
113
Cell Research 22
R. G. Kessel tion is further suggested from the uninvaginated portion of the plasma membrane of both types of cells (Fig. 3, along ChI and Fig. 5, arrows). Further evidence that submicroscopic vesic.les found in the cytoplasm may be formed by a pinching-off process of the plasma membrane is presented in Figs. 3, 5, and 8. Here the proximity of the vesicles to plasma membrane, or continuity in some instances, is such as to suggest that the vesicles may have formed from the latter (Figs. 5, S, arrows). If the submicroscopic vesicles are formed by a pinching-oil process from the double-membraned wall of the invaginated plasma membrane, then one would espec.t the vesicles to appear in certain cases to be surrounded by a double membrane separated by a very small intermembrane space. That certain of the vesicles do possess this structure is shown in Fig. 9 (single arrows). The variation in size of the submicroscopic vesicles suggests that a fusion of vesicles to produce a larger structure might occur. What may represent a stage in such a process in both the subesophageal body and pericaidial cell is shown in Fig. 5 (arrow) and 8 (double arrow). In these two figures, one of a pericardial cell and the other a subesophageal body cell, several vesicles appear aggregated in a circular fashion with fine intervesiwlar connections between them. This may represent a stage in the fusion of several submicroscopic vesicles. It is also possible to observe a close association or connection between the vesicles with peripheral vacuoles. In Fig. 8, for example, several vesicles are located adjacent to and connected with two vacuoles in the electron micrograph. These structural relationships suggest that the vesicles may fuse with vacuoles thus resulting in a variety of sizes of peripheral vacuoles. Additional examples of a c,lose association between the submicroscopic vesicles and the peripheral vacuoles are shown in Fig. 5 and 6. DISCUSSION
Palade [12] first suggested that the vesicular component in endothelial cells of blood capillaries, owurring in the form of spherical or oval vesicles Fig. 6.-Portion of subesophageal body cell showing inclusions ( ILV), small vacuoles (S I’), numerous submicroscopic vesicles (PI:), golgi apparatus (GA), endoplasmic reticulum (ER) and invaginations of the plasma membrane (I). In lower portion of the micrograph note the tubular vesicles (caveolae) associated with an hivagination of the plasma membrane (to right of arrolts). Small vesicles also appear to be within several of the vacuoles (S V), with small arrows. 15,960 :-:. Fig. ‘I.--Edge of a pericardial cell (one day adult male) showing small vacuoles (S 13, invaginations of the plasma membrane (ICM) and numerous vesicles (P V). The double nature of the wall of the invaginated plasma membrane is apparent (arrow). 22,400 x . Experimental
Cell Research 22
Submicroscopic vesicles and cell transport
115
R. G. Kessel about (iSO A%in diameter, might play a role in capillary permeability bg providing a system for transporting fluids across the capillary wall by a process analogous to that of pinocytosis described by Lewis [7]; difyering in that the functional units consist of submicroscopic vesicles on the one hand, and microscopic vesic,les on the other. In Lewis’s motion picture depicting the process of pinocytosis, cells in tissue culture were observed .to take in globules of fluid from the surrounding medium, the uptake of fluid being accompanied by rigorous cytoplasmic motion at the edge of the cell, as if globules were being surrounded and engulfed by clasping folds of cytoplasm. Globules so taken in at the periphery of the cell were then observed bg means of the light microscope to be transported as vesicles to other portions of the cell several microns away. De Robertis and Bennett [5 j confirmed Palade’s observations in capillary endothelial cells in mammals and also observed oral and elongated vesicles in the peripheral cytoplasm of both Schwann and nerve satellite cells of the frog as well as in the synapses of the frog and earthworm [6]. This specific c.ellular component has been observed in both vertebrate and invertebrate cells by others as well. DeRobertis and Bennett [5] also concurred in the interpretation of the possible function of these vesicles as suggested earlier by Palade [ 121. -4s a result of Odor’s [ll j observations on the uptake of colloidal particles of mercuric sulkle or thorium dioxide from the peritoneal cavity by the mesothelial cells, she postulated that in these cells transport across the plasma membrane occurred by the formation of a minute invagination of the plasma membrane followed by the formation of an intracellular vacuole as the invaginated plasma membrane was pinc.hed of’f. Finally, the isolated vacuole so formed could assume any position in the cytoplasm and coalesce with other vacuoles. L\ system of tubules (caveolae) and associated vesicles has also been described in the electric tissue of several electric fish by Luft [9] who suggested these structures play a role in ion transport in this tissue as well. Fig. S.-Periphery of pericardial cell (12 day adult male). The arrows point to areas in which the submicroscopic vesicles have a close association or connection with the inoaginated plasma membrane (I) and vacuoles (S I-). Several vesicles in the lower center of micrograph (two arrows) are arranged in a circular fashion with fine intervesicular connections. 52,800 x . Fig. S.-Most outer edge of a pericardial cell (8 day adult male). Note the invagination area of the plasma membrane (two arrows), electron dense material within the invaginated plasma membrane (three arrows at I) and the vesicles (PV) which in certain areas can be seen to be surromlded by a double membrane (single arrows). 92,000 x . Fig. lo.-Pericardial brane. Note electron Experimental
cell showing vesicules, vacuoles and portion of invaginated plasma memdense material within the invaginated plasma membrane (arrows). 9,600 x .
Cell Research 22
118
R. G. Kessel
A distinction has been made by Moore and Ruska [lo] between the fluid incorporation by the cell for its own supply (pinocytosis) and fluids being transmitted through the cytoplasm rather than utilized by the cell (cytoempsis). \Vhether cytoempsis is occurring in the pericardial cell and subesophageal body is unknown. However, since the cells previously mentioned occur as loosely packed or clustered units, it is probable that such a process does not operate here; rather this process is better characterized by the term pinocytosis. The ultramicroscopic morphology of various subesophageal body and pericardial cells, presumably in different phases of physiological activity, suggest pinocytosis operating as a mechanism of cell transport. This proposed mechanism of cell transport involves a process of microresic.ulation. The vesicle formation appears to occur from the plasma membrane; this membrane being highly infolded or inraginated in both cell types and apparently serves, in part at least, to increase tremendously the surface from which vesicles may form or coalesce. The method of vesicle formation appears similar to that described by others [+I, 5, S, 9, 10, 121. Namely, a shallow depression (caveola intracellularis) forms which finally detaches completely from the plasma membrane resulting in an intracellular 17esicle. Apparently, the vesicles map fuse resulting in the formation of racuoles (droplets) which can be observed at certain times in the peripheral cytoplasm of the pericardial cells with the light microscope. The vacuolar contents then presumably undergo various metabolic changes. The submicroscopic anatomy presented in the electron micrographs, however, does not preclude the possibility of a reverse mechanism occurring in the cells. Thus, it is possible that the peripheral cytoplasmic vacuoles may undergo a vesiculation (in some cases vesicles appear to lie within vacuoles (Figs. 4 and 8). After further resiculation, the smallest resicles could fuse with the plasma membrane releasing their contents to the outside. Thus, as has been suggested by others [3, 5, 121 such a membrane resiculation mechanism can provide a means whereby particles can more from one side of a membrane to the other without actually going through the membrane. In addition, one need not necessarily postulate the presence of pores in membranes in order to account for the passage of material from one side of the plasma membrane to the other.
Submicroscopic
119
vesicles and celZ tmnsport SUMMARY
Electron microscope obserrations on the subesophageal body and pericardial cells of the grasshopper reveal a small vesicular component of the c$oplasm which is believed to provide a mechanism of transport of material into or out of these cells. The relationships of these T:esicles to each other and to other structures in the cell, including the plasma membrane and its specialization as well as peripheral vacuoles, are described. These results thus compliment several other studies leading to an increasing evidence that microresiculation is a prominent mechanism of active transport and ion exchange by many kinds of cells. REFERENCES 1. BEANS, 2. 3. 4. 5. 6. i. S. 9. 10. 11. 12. 13. 14. 15. 16.
17.
H. IV., TAHI\IISI.\X. T. N. and DEVINE, R. L., J. Biopfys. Biochem. (1955). BEAMS, H. W.. ;\NDERSON, E. and PRESS, N.. Cyfologitr 21, 50 (1956). BEKNETT, I-1. S., J. Biophgs. Biochem. Cyfol. 2. 99 Suppl. (1956). BENKETT, H. S.. LUFT, J. H. and HAMPTON, J. C., J. Physiol. 196, 351 (1959). UEROBERTIS. E. D. P. and BENNETT, H. S., Expff. Ceff Reseurch 6, 543 (1954). -J. Biciphys. Biochem. Cyfol. 1. 1-i (1955). Ixw~s, W. H.. BuU. Johns Hopkins Hosp. 49, 1’7 (1931). Lu~r, 'J. ar~cl 0. HECHTER, J. ‘Biophys. &iochem. kyfol. 3: 615 (1957). LUFT. ,J. H.. Esufl. CeZZ Resectrch, Supnl. 5, 168 cl95S). RIoo&, D. H. a;ld RUSIU, H.. J. Biiphys.’ Biochem. byfof. 3, 45i (1957). ODOR. LOUISE D.. J. Biophys. Biochem. Cyfol. 2. 105 %qqd. il956j. P.\Z.kDE. G. E.. J. dppl. Phys. 24. 1424 (1953). .~ Anat. Record 118, 335 (abstract) (1954). __ J. Biophys. Biochem. CgfoZ. 1, 257 (1955). F;PP.~. G. -D.-and BRANDT, k. \I’., J. Biophys. Biochem. Cyful. 4. 485 (19%). PEASE. D. C., dnut. Record 121, iZ3 (1955). YAM.&L E., J. Biophys. Biochim. Cyfol. i, 455 (1955).
Esperimer~fal
Cyfof. 1. 197
Ce!f Resectrch 22
108
Cell Research 22 108-119 (1961)
ELECTRON MICROSCOPE OBSERVATIONS ON THE SUBMICROSCOPIC VESICULAR COMPONENT OF THE SUBESOPHAGEAL BODY AND PERICARDIAL CELLS OF THE GRASSHOPPER, MELANOPLUS DIFFERENTIALIS DIFFERENTIALIS (THOMAS)l R. G. KESSEL” Department Bwwnan
of Zoology, Stote University of Iooloa and the Department of An.atomy, Gray School of Medicine, K’inston-Salem, North Carolina, U.S.A.
Received January 41, 1960
the electron microscope it has been shown in a variety of cells that the plasma membrane is infolded extensively [ 1, 2, 13, 11, 161. Moreover, in cells possessing such an elaboration of the plasma membrane, a vesicular component of the cytoplasm is often displayed as isolated units lying close to In cells without this surface or continuous with the plasma membrane. specialization, the vesicular component may be found concentrated adjacent to the surface and, indeed, may appear in contact with shallow inraginations (caueola intracellularis of Tamada [17]) of the plasma membrane [-I, 5, 10, 11, 121. Furthermore, a fusion of vesicles resulting in the formation of a larger vawole has been reported [15]. Because the small vesicular component of the cytoplasm has been observed aggregated in prorimity with the plasma membrane as well as close to or continuous with shallow inuaginations of the plasma membrane, various investigators hare suggested such spatial relationships indicate that the sub-
%‘ITH
1 This investigation was supported, in part, by a research grant (RG-6942) from the Division of General iUedica1 Sciences, Public Health Service, and Fluid Research Funds from the Bowman Gray School of Medicine. Bowman Gray School of Medicine, Winston% Present address: Department of Anatomy, Salem, North Carolina, U.S.A.
Fig. l.-Periphery of a pericardial cell (S clay adult raginations of the plasma membrane (ICM), small submicroscopic vesicles (PV) are shown. 52,500 x .
male). The plasma membrane (CAfj, inperipheral vacuoles (SV) and numerous
Fig. T.-Edge of a pericardial cell (one day adult male) containing numerous vesicles (PI’), small vacuoles (S V) and infoldings of the plasma membrane (IC31). One such infolding appears to extend the entire length of the section. 14,800 y. Experimenfnl
Cell Research 22
Submicroscopic
vesicles
ctnd cell Iranspori
ERrperimental
Cell Research 22
R. G. Kessel microscopic vesicles may be formed by a pinching oif process from the plasma membrane or, conversely, they may represent vesicles destined to coalesce with the plasma membrane [5, la]. Recently Bennett [3] advanced a hypothetical mechanism whereby membrane flow and membrane resiculation could play a role in active transport in the cell based, in part, on the above findings. A similar hypothesis was postulated by Odor [ 111 on the basis of experimental studies on the mesothelial cells of rats. During the course of cytological and physiological experiments on the subesophageal body cells in embryos and on pericardial cells in adult grasshoppers, a vesicular component of the cytoplasm was observed which, because of the morphological relationships between it and the specializations of the cell surface and vacuoles within the cells, suggested that this structure might play a role in transport of material into or out of the cell, thus substantiating Bennett’s theory of membrane flow and membrane \resiculation as mechanisms for active transport and ion pumping. Because of the early theories presented by physiologists with respect to membrane permeability and because of new techniques cleveloped in electron microscop? demonstrate a vesicular component suggesting that materials can move into or out of cells by a type of microvesiculation, it would appear of interest that the more widespread this type of vesiculation is found at the submicroscopic level the more support would be delivered to the hypothesis advanced by Bennett [3 j. It is with this thought in mind that the present observations are herein presented. MATERIALS
AND
METHODS
Pericardial
cells from adult, laboratory reared, grasshoppers, Melazzoplus dif(Thomas), and subesophageal body cells from pre-diapause embryos mere examined with the electron microscope. Pericardial cells were immediately removed from the animals by a ventral dissection and placed in cold 1 per cent osmium tetroxide solution buffered with acetate-Verona1 at a pH of 7.2,
ferentialis
differenticclis
Fig. 3.-Periphery of a pericardial cell (8 day adult male) showing -the plastna membrane (CdZ), an infolding of the plasma membrane containing electron dense material (I), a small vxuole (SV) and numerous resicles (PI’) some of which are aligned along the plasma membrane (CJZ). 16,200 x . Fig. l.-Edge of another pericardial cell from the satne animal as Fig. 3. In addition to the plasma metnbrane (CM), infoldings of the plastna membrane (Zj, small vacuoles t.SL’), and isolated vesicles (PI-), a region is indicated (arrows) in which expansions of the inuaginated plasma membrane occur suggesting vesicle formation. These expansions are litnited by a double membrane. Golgi apparatus (Cd). 31,600 ): . Experimentnl
Cell Reseurch 22
Submicroscopic vesicles and cell ~ra~,~~o~~
Experimental
111
Cell Research 22
R. G. Kessel
112
7.5 or 7.8 and allowed to fix for l-2 hours. In half of the cases, the buffered solution of osmium tetroxide was injected into the hemocoel before making the dissection. The pericardial cells mere then dehydrated, embedded in methacrylate and sectioned with a Porter-Blum microtome equipped with a glass knife and set to section at 0.025 microns. The electron microscopes used included an RCA4 EMU-2B and an EMU-SD. Entire embryos (18-19 days development at 25°C) were fixed for l-2 hours in 1 per cent buffered osmium tetroxide (pH 7.8). The embryos were washed quickly with distilled mater and dehydrated rapidly through a graded series of alcohols. In one of the lower alcohols, the entire subesophageal body was freed by removing the remaining embryonic tissue. The remainder of the procedure for electron microscopy was identical to that described for the pericardial cells. OBSERVATIONS
The prominent features of the ultramicroscopic strwture of the peripheral region of the c.ytoplasm of both the subesophageal body and pericardial cells include: (1) a system of smooth surfaced invaginations or infoldings of the plasma membrane (Figs. 1, 2, 6, ICM), (2) small peripheral vacuoles (Figs. l-8, SIT), and numerous submicroscopic vesicles (Figs. 1-7, PV). The diameter of the invaginated plasma membrane is variable but in many instances ranges from 370450 ,&. These peripheral smooth membranes seen in electron micrographs of both cell types appear to arise as a result of an inragination of the plasma membrane (Fig. 9, double arrows). In some electron micrographs, the cut edges of the tubular imagination ap0 7, arrow). This suggests that the most external, uninvaginapear double (Fi,. ted portion of the plasma membrane is composed of a double membrane separated by a narrow intermembrane space. Electron dense material is often observed within the tubular inraginated plasma membranes in section (Fi g. 2, 10, arrows). In addition, expansions of these walls can also be seen (Fig. 4, arrows). The structure seen in Fig. 1 suggests that a vesicular formation from the invaginated plasma membrane was underway at the time of fixation of this pericardial cell and is reminiscent of the caveolae intracellularis [lT]. Tubular evaginations (careolae) of the inraginated portion of the plasma membrane are also evident in electronmicrographs of the subesophageal body cell (Fig. 6, arrow). Careolae formaFig. 5.PElectron micrograph of subesophageal body cell showing a portion of a large central vacuole (L V), smaller, peripheral vacuoles (S I’) and numerous submicroscopic vesicles (P F’). Note the relationship of several vesicles (arrows) to the invaginated plasma membrane (IJ1C). In addition many of the vesicles are closely applied to the small vacuoles (arrows). In lower left of micrograph (arrow), several vesicles are arranged in a circular area with fine interresicular con33,000 x. nections. Arrows in upper right of micrograph su,,ooest areas of caveolae formation. Experimental
Cell Research 22
Submicroscopic vesicles and cell transport’
5 - 601732.58
Experimental
113
Cell Research 22
R. G. Kessel tion is further suggested from the uninvaginated portion of the plasma membrane of both types of cells (Fig. 3, along ChI and Fig. 5, arrows). Further evidence that submicroscopic vesic.les found in the cytoplasm may be formed by a pinching-off process of the plasma membrane is presented in Figs. 3, 5, and 8. Here the proximity of the vesicles to plasma membrane, or continuity in some instances, is such as to suggest that the vesicles may have formed from the latter (Figs. 5, S, arrows). If the submicroscopic vesicles are formed by a pinching-oil process from the double-membraned wall of the invaginated plasma membrane, then one would espec.t the vesicles to appear in certain cases to be surrounded by a double membrane separated by a very small intermembrane space. That certain of the vesicles do possess this structure is shown in Fig. 9 (single arrows). The variation in size of the submicroscopic vesicles suggests that a fusion of vesicles to produce a larger structure might occur. What may represent a stage in such a process in both the subesophageal body and pericaidial cell is shown in Fig. 5 (arrow) and 8 (double arrow). In these two figures, one of a pericardial cell and the other a subesophageal body cell, several vesicles appear aggregated in a circular fashion with fine intervesiwlar connections between them. This may represent a stage in the fusion of several submicroscopic vesicles. It is also possible to observe a close association or connection between the vesicles with peripheral vacuoles. In Fig. 8, for example, several vesicles are located adjacent to and connected with two vacuoles in the electron micrograph. These structural relationships suggest that the vesicles may fuse with vacuoles thus resulting in a variety of sizes of peripheral vacuoles. Additional examples of a c,lose association between the submicroscopic vesicles and the peripheral vacuoles are shown in Fig. 5 and 6. DISCUSSION
Palade [12] first suggested that the vesicular component in endothelial cells of blood capillaries, owurring in the form of spherical or oval vesicles Fig. 6.-Portion of subesophageal body cell showing inclusions ( ILV), small vacuoles (S I’), numerous submicroscopic vesicles (PI:), golgi apparatus (GA), endoplasmic reticulum (ER) and invaginations of the plasma membrane (I). In lower portion of the micrograph note the tubular vesicles (caveolae) associated with an hivagination of the plasma membrane (to right of arrolts). Small vesicles also appear to be within several of the vacuoles (S V), with small arrows. 15,960 :-:. Fig. ‘I.--Edge of a pericardial cell (one day adult male) showing small vacuoles (S 13, invaginations of the plasma membrane (ICM) and numerous vesicles (P V). The double nature of the wall of the invaginated plasma membrane is apparent (arrow). 22,400 x . Experimental
Cell Research 22
Submicroscopic vesicles and cell transport
115
R. G. Kessel about (iSO A%in diameter, might play a role in capillary permeability bg providing a system for transporting fluids across the capillary wall by a process analogous to that of pinocytosis described by Lewis [7]; difyering in that the functional units consist of submicroscopic vesicles on the one hand, and microscopic vesic,les on the other. In Lewis’s motion picture depicting the process of pinocytosis, cells in tissue culture were observed .to take in globules of fluid from the surrounding medium, the uptake of fluid being accompanied by rigorous cytoplasmic motion at the edge of the cell, as if globules were being surrounded and engulfed by clasping folds of cytoplasm. Globules so taken in at the periphery of the cell were then observed bg means of the light microscope to be transported as vesicles to other portions of the cell several microns away. De Robertis and Bennett [5 j confirmed Palade’s observations in capillary endothelial cells in mammals and also observed oral and elongated vesicles in the peripheral cytoplasm of both Schwann and nerve satellite cells of the frog as well as in the synapses of the frog and earthworm [6]. This specific c.ellular component has been observed in both vertebrate and invertebrate cells by others as well. DeRobertis and Bennett [5] also concurred in the interpretation of the possible function of these vesicles as suggested earlier by Palade [ 121. -4s a result of Odor’s [ll j observations on the uptake of colloidal particles of mercuric sulkle or thorium dioxide from the peritoneal cavity by the mesothelial cells, she postulated that in these cells transport across the plasma membrane occurred by the formation of a minute invagination of the plasma membrane followed by the formation of an intracellular vacuole as the invaginated plasma membrane was pinc.hed of’f. Finally, the isolated vacuole so formed could assume any position in the cytoplasm and coalesce with other vacuoles. L\ system of tubules (caveolae) and associated vesicles has also been described in the electric tissue of several electric fish by Luft [9] who suggested these structures play a role in ion transport in this tissue as well. Fig. S.-Periphery of pericardial cell (12 day adult male). The arrows point to areas in which the submicroscopic vesicles have a close association or connection with the inoaginated plasma membrane (I) and vacuoles (S I-). Several vesicles in the lower center of micrograph (two arrows) are arranged in a circular fashion with fine intervesicular connections. 52,800 x . Fig. S.-Most outer edge of a pericardial cell (8 day adult male). Note the invagination area of the plasma membrane (two arrows), electron dense material within the invaginated plasma membrane (three arrows at I) and the vesicles (PV) which in certain areas can be seen to be surromlded by a double membrane (single arrows). 92,000 x . Fig. lo.-Pericardial brane. Note electron Experimental
cell showing vesicules, vacuoles and portion of invaginated plasma memdense material within the invaginated plasma membrane (arrows). 9,600 x .
Cell Research 22
118
R. G. Kessel
A distinction has been made by Moore and Ruska [lo] between the fluid incorporation by the cell for its own supply (pinocytosis) and fluids being transmitted through the cytoplasm rather than utilized by the cell (cytoempsis). \Vhether cytoempsis is occurring in the pericardial cell and subesophageal body is unknown. However, since the cells previously mentioned occur as loosely packed or clustered units, it is probable that such a process does not operate here; rather this process is better characterized by the term pinocytosis. The ultramicroscopic morphology of various subesophageal body and pericardial cells, presumably in different phases of physiological activity, suggest pinocytosis operating as a mechanism of cell transport. This proposed mechanism of cell transport involves a process of microresic.ulation. The vesicle formation appears to occur from the plasma membrane; this membrane being highly infolded or inraginated in both cell types and apparently serves, in part at least, to increase tremendously the surface from which vesicles may form or coalesce. The method of vesicle formation appears similar to that described by others [+I, 5, S, 9, 10, 121. Namely, a shallow depression (caveola intracellularis) forms which finally detaches completely from the plasma membrane resulting in an intracellular 17esicle. Apparently, the vesicles map fuse resulting in the formation of racuoles (droplets) which can be observed at certain times in the peripheral cytoplasm of the pericardial cells with the light microscope. The vacuolar contents then presumably undergo various metabolic changes. The submicroscopic anatomy presented in the electron micrographs, however, does not preclude the possibility of a reverse mechanism occurring in the cells. Thus, it is possible that the peripheral cytoplasmic vacuoles may undergo a vesiculation (in some cases vesicles appear to lie within vacuoles (Figs. 4 and 8). After further resiculation, the smallest resicles could fuse with the plasma membrane releasing their contents to the outside. Thus, as has been suggested by others [3, 5, 121 such a membrane resiculation mechanism can provide a means whereby particles can more from one side of a membrane to the other without actually going through the membrane. In addition, one need not necessarily postulate the presence of pores in membranes in order to account for the passage of material from one side of the plasma membrane to the other.
Submicroscopic
119
vesicles and celZ tmnsport SUMMARY
Electron microscope obserrations on the subesophageal body and pericardial cells of the grasshopper reveal a small vesicular component of the c$oplasm which is believed to provide a mechanism of transport of material into or out of these cells. The relationships of these T:esicles to each other and to other structures in the cell, including the plasma membrane and its specialization as well as peripheral vacuoles, are described. These results thus compliment several other studies leading to an increasing evidence that microresiculation is a prominent mechanism of active transport and ion exchange by many kinds of cells. REFERENCES 1. BEANS, 2. 3. 4. 5. 6. i. S. 9. 10. 11. 12. 13. 14. 15. 16.
17.
H. IV., TAHI\IISI.\X. T. N. and DEVINE, R. L., J. Biopfys. Biochem. (1955). BEAMS, H. W.. ;\NDERSON, E. and PRESS, N.. Cyfologitr 21, 50 (1956). BEKNETT, I-1. S., J. Biophgs. Biochem. Cyfol. 2. 99 Suppl. (1956). BENKETT, H. S.. LUFT, J. H. and HAMPTON, J. C., J. Physiol. 196, 351 (1959). UEROBERTIS. E. D. P. and BENNETT, H. S., Expff. Ceff Reseurch 6, 543 (1954). -J. Biciphys. Biochem. Cyfol. 1. 1-i (1955). Ixw~s, W. H.. BuU. Johns Hopkins Hosp. 49, 1’7 (1931). Lu~r, 'J. ar~cl 0. HECHTER, J. ‘Biophys. &iochem. kyfol. 3: 615 (1957). LUFT. ,J. H.. Esufl. CeZZ Resectrch, Supnl. 5, 168 cl95S). RIoo&, D. H. a;ld RUSIU, H.. J. Biiphys.’ Biochem. byfof. 3, 45i (1957). ODOR. LOUISE D.. J. Biophys. Biochem. Cyfol. 2. 105 %qqd. il956j. P.\Z.kDE. G. E.. J. dppl. Phys. 24. 1424 (1953). .~ Anat. Record 118, 335 (abstract) (1954). __ J. Biophys. Biochem. CgfoZ. 1, 257 (1955). F;PP.~. G. -D.-and BRANDT, k. \I’., J. Biophys. Biochem. Cyful. 4. 485 (19%). PEASE. D. C., dnut. Record 121, iZ3 (1955). YAM.&L E., J. Biophys. Biochim. Cyfol. i, 455 (1955).
Esperimer~fal
Cyfof. 1. 197
Ce!f Resectrch 22