- Email: [email protected]
Closure of the plasma membrane around microneedle in Amoeba Proteus
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l I II-0105$02.00/0
Experimental
CLOSURE AROUND
Cell Research I1 1 (1978) 105-l 15
OF THE
PLASMA
MICRONEEDLE
IN AMOEBA
An Ultrastructural BARBARA
MEMBRANE PROTEUS
Study
SZUBINSKA
Department of Biological Structure, University of Washington, School of Medicine, Seattle, WA 98195, USA
SUMMARY Microneedle perforations of the plasma membrane of Amoeba proteus were studied on the ultrastructural level. In each individual cell one hole was produced, which subsequently was marked with an eyelash left in place. Cells were quickly fixed, and sections cut parallel to the longitudinal axis of the eyelash. It was clear that the eyelash penetrated the plasma membrane, and that its free tip was located in the interior of the cell. A gap remained between the plasma membrane and the eyelash which may correspond to the electrical leak sometimes found by microelectrode punctures. The edges of the broken plasma membrane curled back into the cytoplasm. Here, a great redundancy of the plasma membrane was observed. Within these membrane accumulations, a large quantity of dense droplets was apposed at the inner leaflet of the plasma membrane. Their involvement in the formation and expansion of the plasma membrane in Amoeba proteus and Xenopus faevis has been suggested previously [ 1, 181. Present studies offer more supportive evidence to that effect. Therefore, the interpretation seems to be plausible, that these membrane accumulations are the result of membrane expansion to minimize the hole produced during injury. This is in agreement with Holtfreter’s [8] and Bluemink’s [l] concept that the wound closure may occur by proliferation of the plasma membrane.
Electrophysiologists have been routinely impaling single cells with microelectrodes in order to measure transmembrane potentials. Thus, there is substantial information available on the reaction of cells to such injuries. With fine microelectrodes (0.5 pm tip diameter) the potentials are stable, but if the electrodes are larger (up to 9 pm) or the insertion is defective, low potentials result from a drop in membrane resistance [Z, 6, 12, 211, which may or may not recover [5]. There is also evidence of occasional leakage back along the microelectrode to the surface [3]. The most plausible explanation of these results is imperfect “sealing” of the electrode into the cell membrane with a persisting open channel. Yet from the morphological point of
view, very little is known about what exactly happens at the moment when a microelectrode is inserted into a cell, and what changes occur subsequently. It would be of interest to many scientists to know what the hole itself looks like, how the membrane accommodates around the microelectrode, and finally, how the perforation closes. Correlation of these morphological and physiological studies could yield useful information on cell function. An attempt has been made to follow these phenomena on the fine structure level and preliminary observations have been published [18, 191. From these studies [18], it appeared that holes inflicted with fine microneedles in individual cells of amoeba could be localized. Nevertheless, pictures Exp Cell
Res I I I (1978)
106
B. Szubinska
which showed the perforation and two edges of the broken plasma membrane in the same section, were obtained only fortuitously. It became obvious that in order to be able to trace the exact pathway of the microneedle, some sort of marker was needed. For this purpose an eyelash (5 pm tip diameter) has been employed [ 191, which was inserted into the cell along with the microneedle, and left there after the microneedle was withdrawn. This marker is not toxic, is easily seen in the light microscope, and is fully compatible with conventional EM procedures. It marks the hole in an unambiguous manner, and above all, it marks the exact pathway of the microneedle, offering at the same time the precise orientation of the hole. Injured cells marked in this way were fixed within 45 set, processed individually for electron microscopy, and sectioned along the longitudinal axis of the eyelash. Electron micrographs obtained from such cells reveal the interruption of the plasma membrane with its edges curled back into the cytoplasm, the tip of the eyelash in the cytoplasm free of the membrane, and the eyelash shaft closely approximated on both sides by remarkably smooth surfaces of the plasma membrane. In addition, a large number of electron dense droplets was found in close contact with the cytoplasmic surface of the plasma membrane near the hole. These have been previously associated with mechanical damage to cell membranes, both in Amoeba proteus [ 181 and Xenopus laevis [ 11.
MATERIALS
AND
METHODS
Amoebae were cultured and handled as described previously [B]. Cells were injured individually with tine glass microneedles (3-4 pm tip diameter), also as in previous study, but only one type of injury was employed in the present experiments, and the holes were Exp CeNRes
111 (1978)
labeled with an eyelash as the marker. Eyelashes were degreased by soaking in 75 % ethanol for 1 h, and then washed in several changes of distilled water prior to experiments. Each amoeba was speared only once, so that only one hole/cell was produced. The microneedle was inserted gently into the cell, from the side (at about a 45” angle) and held in that position while the eyelash (about 5 pm tip diameter) was introduced along its side. Then, the microneedle was withdrawn with the eyelash held exactly in the same position. It is important to note that although the description of injury and marking of the hole is rather simple, in fact, it is very difftcult to accomplish, considering the possibility of cell detachment from the bottom of the experimental dish, as well as introducing artifacts by unnecessary movements. Three cells marked in this manner were thoroughly examined in the present study. (An attempt has been made to mark holes in amoebae with other markers, such as Alcian blue as well as India ink, but both approaches were unsuccessful. Alcian blue dispersed in the cytoplasm of amoebae, and even India ink particles when dried on the microneedle and applied to the cell, did not stay in one particular spot on the cell, but instead moved quickly as observed with the light microscope.) Marked cells were fixed in situ within 35-45 set from the moment of injury. Fixation was carried out in 2.5 % acrolein buffered with 0.067 M cacodylate buffer at pH 7.2-7.4 for 15 min. Then the cells were post-fixed in 1.3 % 0~0, buffered as before, for 30 min. Dehydration was done in 35,70, 95, and 100% ethanol, respectively. Low viscosity epoxy resin based on diglycidyl ether [ 11, 201 was used as an embedding medium. For sectioning, each cell was oriented so that the sections would be cut parallel to the longitudinal axis of the eyelash. Sections were collected onto grids with a single hole of 500 pm diameter, coated with Pioloform [16] and carbon. Sections were double-stained with a saturated aqueous solution of uranyl acetate followed by Reynolds’ lead citrate [IS] for 5 and 10 min, respectively. More details on fixation, dehydration and embedding are given in the previous report [18]. No ruthenium dyes were used in this study.
RESULT Individual cells of amoebae when injured, marked with an eyelash and embedded for electron microscopy yield a typical picture as seen in fig. 1. The cytoplasm throughout Fig. I. Light micrograph of a whole amoeba marked with the eyelash. The picture was taken in polymerized epoxy resin before sectioning. x 173. Fig. 2. Low power electron micrograph of the entire thin section of the same amoeba depicted in fig. 1. In this montage the eyelash is seen as the dark object. Plasma membrane is shown outlined in black and its continuity is traceable except for two regions. x2000.
Plasma
membrane
around microneedle
Exp Cdl
107
RPS 111 (1978)
108
B. Szubinska
the cell is distributed somewhat unevenly, giving rise to darker and lighter regions. The nucleus can be recognized approximately in the center of the cell. The site of the injury is marked with the eyelash, and is located to the right of the nucleus. Sections cut parallel to the longitudinal axis of the eyelash reveal the hole itself (fig. 2). In this low power electron micrograph of the entire section through a portion of an amoeba, the hole is clearly seen as a perforation in the plasma membrane, allowing the tip of the eyelash to penetrate into the interior of the cell. The plasma membrane of the cell has been deliberately outlined in order to facilitate the tracing of its continuity. Indeed, it appeared that despite its convolutions, continuity of the plasma membrane could be traced easily. There are several regions where continuity is disrupted. From studies done on injured amoebae with holes marked in this way, it is obvious that there are three discrete regions which deserve closer attention. First, there is the region through which the microneedle and later the eyelash enters the cell. This is the region in which the eyelash shaft is located, and it looks like a narrow tunnel or invagination. This leads into a second region which is characterized by abundant plasma membrane convolutions, and which finally opens up through the perforation in the plasma membrane into the interior of the cell, where the very tip of the eyelash is seen. Higher magnification pictures illustrate in more detail certain significant features of each of these regions obtained from the same cell. Fig. 4 shows the eyelash shaft being followed closely on both sides of the plasma membrane. The gap between the eyelash and plasma membrane on both sides is very small, about 1650 A on the left hand side and 7 300 A on the right. In fig. 3 it can be seen clearly that the tip of Exp Cd
Rev I 11 (1978)
the eyelash is located deep in the cytoplasm free of membrane. However, several outer cuticular cells of the eyelash have lifted slightly from the tip, giving an illusion of a membrane. In figs 3, 4 and 5, cytoplasm seems to be diluted and rather free from organelles, which is characteristic of the region in the immediate vicinity of the hole. Sections obtained from regions situated further away from the hole showed all organelles in good condition. The region most important to this study is the middle or central part of the hole Fig. 3. Higher magnification picture of the uppermost region in fia. 2, revealing the free tip of the eyelash located in the cytoplasm: Two dark bands across the eyelash represent an artifact which arises during the staining procedure. Eyelash does not adhere to the carbon film as tightly as does the rest of the section, and as a result, the staining solutions enter between the carbon film and plastic leaving a residue. x3 000. Fig. 4. Higher magnification picture of the lowermost region in fig. 2, showing the eyelash shaft located in the invagination of the cell. Note the small distance between the plasma membrane and the eyelash on both sides, but especially on the left hand side (1650 A), as well as surmisinalv smooth surfaces of the ulasma membranes: x 3 O@. Fin. 5. Higher magnification picture of the central region in fig-2 illust&ing the hole itself. Actual perforation in the plasma membrane is marked with arrows. A large accumulation of the plasma membrane is apparent around the hole. Plasma membrane of the periphery of the cell is at the upper right and lower left. x3ooo. Fig. 6. Higher magnification picture of the right upper region in fig. 5, showing the low density of the plasma membrane at the periphery of the cell and higher density of the plasma membrane in the vicinity of the eyelash. Lumen at L and arrow shows location of tig. 7. X identities the location of fig. 9. x7 950. Fig. 9. Higher magnification picture of the area in tig. 6 marked with the arrow showing the dark droplets apposed at the cytoplasmic leatlet of the plasma membrane. Extraneous coat is facing the lumen (L) of the invagination, and the cytoplasm (C) of the cell is on the right. x56400. Fig. 8. inset shows the close contact of the dense droplets with the cytoplasmic leaflet of the plasma membrane. Trilaminar membrane is clearlv seen as well as the extraneous coat. x 160000. Fig. 9. Depicts the edge of the curled plasma membrane identified at X in fig. 6. The tip contains five dense droplets, as well as a few tine filaments which are more obvious in the membrane loop itself (arrow). x55 300. These membrane-associated filaments are different from the usual cytoplasmic filaments.
Plasma
membrane
around microneedle
Exp Cell
Res III
109
(1978)
110
Exp Cell
B. Szubinska
Rcs II I (1978)
Plasma
S-771807
membrane
around microneedle
111
112
B. Szubinska
where the actual perforation of the plasma membrane is present (fig. 5). Perforation is recognized where two edges of the plasma membrane on opposite sides of the eyelash are curled and found at the same level as indicated by the arrows. An abundance of convoluted plasma membrane is apparent around the hole and there is nearly complete continuity with at least the left edge of the curled plasma membrane. This has been checked carefully on higher magnification pictures which would be impractical to show here. The significance of this large membrane accumulation in this particular region will be discussed later. For the time being, however, it is important to look at a still higher magnification picture of portions of the plasma membrane itself. Fig. 6 illustrates the region obtained from the upper right side of the eyelash in fig. 5, which includes the curled edge of the plasma membrane. There is a distinct difference in density between the plasma membrane at the periphery of the cell and its portion in the vicinity of the eyelash. The peripheral membrane appears quite pale while the portion near eyelash shows discrete black droplets. Still higher magnification (fig. 7), taken from the area marked with the arrow in fig. 6, reveals dense droplets aligned on the plasma membrane at more or less regular intervals. The inset (fig. 8) illustrates that these dense droplets are apposed very closely at the inner leaflet of the plasma membrane. Trilaminar membrane is seen clearly and the amorphous and tilamentous layer of the extraneous coat of the cell points to the outside. Fig. 9 shows further details of the right edge of the curled plasma membrane. Dense droplets are again prominent throughout the picture, and are attached to the membrane including the very end of the curled plasma membrane. In addition, an aggregation of Exp Cell
Res 111 (1978)
fine filaments 5&100 A in diameter is apparent at the cytoplasmic surface in the membrane loop (arrow), and a few strands occur in the very end of the curled plasma membrane. They terminate in a dense knob about twice their diameter and have been seen previously in wounded amoebae [18]. These membrane-associated filaments may be responsible for the curling properties of the plasma membrane. DISCUSSION Cell injuries raise many questions of biological importance, but first of all it is pertinent to consider the advantage of the approach used in this study with respect to the already existing methods. For a long time electrophysiologists have been interested in the location of microelectrode tips in cells of various tissues; therefore, the literature records many attempts to accomplish that task. Several dyes were used for this purpose such as methylene blue, fast green, Alcian blue and crystal violet. Usually, the dyes were driven into the cell by iontophoresis. In recent years Stretton & Kravitz [17] suggested the use of the fluorescent dye, Procion yellow, as a marking agent. Also, iontophoresis has been employed to introduce various ions, such as silver, into the tissue for localization. A more recent method is that described by Nickels [13] ibr accurate localization of a recording glass microelectrode in a muscle cell. The method involves precipitation of ferrocyanide and phosphate from the microelectrode with lead added externally together with glutaraldehyde, and followed with ammonium sulfide. The end product is in the form of a dark sediment which is easily seen both in the light and electron microscope. Also, intracellular injection of cobaltous chloride by Pitman & Tweedle
Plasma [ 141 was used for labeling central neurons for later identification in the EM. This metal is likewise precipitated as a sulfide, and is also electron opaque. In addition, there have been some recent improvements in dye application as a marker. Christensen [4] described the usage of Procion brown as an intracellular dye for light and electron microscopy. The advantage of this dye over Procion yellow and cobalt is that in addition to remaining within the cell after injection, Procion brown does not require further reaction to make it visible in the EM. In spite of the number of these methods available for marking the site of injury, none was adequate for the studies intended here. None of the dye markers could have been employed successfully since all dyes disperse rapidly in the interior of the cells, marking the entire cell. This is especially true for the cytoplasm of amoebae which is in constant movement at all times. Even the histochemical method proposed by Nickels [13] was insufficient. Although it permits localization of the microelectrode tip, it does not allow for precise axial orientation as desired. Besides, all these dyes and chemicals involved in the methods mentioned above, are toxic to the cell and therefore may interfere with natural wound closure. With regard to the biological findings, the experiments conducted in this study reveal several unusual findings. First of all, when looking at the light micrograph of an amoeba marked with the eyelash (fig. l), it is not certain whether the eyelash is in the interior of the cell or whether it lies only in an invagination of the plasma membrane produced during cell injury. Ultrastructural studies done on these amoebae offer an undisputable answer. It is obvious from the pictures that the eyelash did penetrate the
membrane
around microneedle
113
plasma membrane, and that its free tip is located in the interior of the cell. The plasma membrane is broken, and its two edges are curled back on both sides of the eyelash. In other words, there is an opening which leads to the interior of the cell obstructed only by the presence of the eyelash. The perforation itself is large (8-10 pm) when measured from one end of the plasma membrane to another; nevertheless, the smallest open spaces on both sides of the eyelash at the level of the curled edges correspond only to 1200 and 340 A each. Assuming that both the eyelash and the perforation are circular, and the open space to be uniform around the eyelash, this is equivalent to about 1.8 pm2. From these data it appears that the area through which leakage from the cell can occur is relatively small. The great length of the pathway from cytoplasm to the exterior also must impede leakage. It is important to remember, however, that the measurements given in this paper are taken from fixed and embedded cells which include shrinkage and distortion of uncertain magnitude as compared to the living cell. Nevertheless, the small channel remaining open between the plasma membrane and eyelash may be the structural counterpart of the electrical leak sometimes found with microelectrodes in other cells [3]. Another interesting feature, which emerges from these experiments is the fact that the hole is not yet completely closed. This seems to be in contradiction with the information existing already in the literature, that wound closure in amoebae occurs very rapidly. Two explanations can be offered to that effect. (1) The object itselfthe eyelash-represents quite a large foreign body. It must take some time for the cell to accommodate itself to such a dramatic disturbance. On the other hand, the Exp Cd
RES I I I (1978)
114
B. Szubinska
eyelash may also act as a cork in a bottle which slows the leakage of the cytoplasm (or ions) to the outside, and therefore does not force the cell to rapid closure of the hole. (2) The speed with which the fixative was delivered (35-45 set) from the moment of injury certainly must interrupt the process of closure. Another aspect that should be discussed here is the presence and involvement of cytoplasmic filaments in wound closure. Much of the information that is present in the literature suggests the involvement of contractile filaments in cellular wound healing [7, lo]. Recent ultrastructural studies done on Amoeba proteus by Jeon & Jeon [9] are supportive of this idea. However, studies done onXenopus eggs by Bluemink [l], although showing the presence of cytoplasmic filaments in the vicinity of the wound, indicate also that they may not be an absolute requirement for wound closure. In view of Bluemink’s findings, it is easier to explain the lack of these cytoplasmic tilaments around the wound in my studies. Although they are present in other portions of the same cell, they certainly are not present around the hole itself. There is a possibility that there are two mechanisms operating independently in wound closure, depending on the type of the injury, size of the wound and requirement of the cell at the moment. Expulsion of the nucleus [9], for example, which must require rapid contraction, may necessitate mobilization of the filaments, whereas the closure of the holes, which does not require rapid contraction, may be not entirely filament dependent. A different concept with regard to wound closure was long ago expressed by Holtfreter [8] and, more recently, reexamined by Bluemink [l]. According to these studies, wound closure may also occur by Exp Cell Res 111 (1978)
means of growth of new membrane surface. Since supportive evidence for this concept comes from my studies, it seems to be worthwhile to concentrate for a moment on this aspect. Electron micrographs presented in this work show around the hole substantial accumulation of the plasma membrane in at least partial continuity with its broken and curled edges (fig. 5). The question arises at this point why there is such accumulation of the plasma membrane at this particular location? There is no reason to think that these packages of membranes are non-specific artifacts produced during cell injury, since such artifacts were recognized early in other cells [18]. These consist of highly disorganized strands of broken membranes, pulled in all possible directions. A more plausible explanation is that this is, in fact, greatly expanded plasma membrane (composed of both old and new elements) made by the cell to minimize the hole. It would be too much of a coincidence to find such an aggregation of the plasma membrane only near the vicinity of the wound and nowhere else. At this point, it is important to consider how the plasma membrane expansion can occur so fast. In previous studies on amoebae [18] I have shown and discussed the mechanism of plasma membrane expansion by injection into the already existing membrane of membrane precursors present in the cytoplasm in the form of dense or foamy droplets. Here again, the same dense droplets are present in abundance within these membrane accumulations at the site of the hole, but only occasionally at the periphery of the cell (fig. 6). They are apposed very closely to the cytoplasmic leaflet of the plasma membrane, including the regions of two curled edges of the membrane. Similar material has been demonstrated by Bluemink [l, p. 1091 in the eggs
Plasma
ofxenopus with a different technique and is also found to be associated with cell injury. The results presented in this paper further reinforce the possibility of wound closure by proliferation of the plasma membrane.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Bluemink, J G, J ultrastruct res 41 (1972) 95. de1 Castillo, J & Katz, B, J physiol 125 (1954) 546. -Ibid 128 (1955) 157. Christensen, B N, Science 182 (1973) 1255. De Mello, W C, Proc natl acad sci US 70 (1973) 982. Fatt, P, Methods med res 9 (l%l) 381. Gingell, D, J embryo1 exp morph01 23 (1970) 583. Holtfreter, J, J exp zoo1 93 (1943) 251. Jeon, K W 62 Jeon, M S, J cell bio167 (1975) 243.
membrane
around microneedle
115
:;. Luckenbill, L M, Exp cell res 66 (1971) 263. Luft, J H, Advanced techniques in biological . electron microscopy (ed J K Koehler) p. 1. ,2, Springer-Verlag, Berlin (1973).
Nastuk, W L & Hodgkin, A L, J cell camp physiol 35 (1950) 39. 13. Nickels, J, Acta physiol Stand 80 (1970) 360. 14. Pitman, R M, Tweedle, C D & Cohen, M J, Science 176 (1971) 412. 15. Reynolds, E S, J cell biol 17 (1%3) 208. 16. Stockem, W, Mikroskopie 26 (1970) 185. 17. Stretton, A 0 W & Kravitz, E A, Science 162 -‘. (1968) 132. 18. Szubinska, B, J cell bio149 (1971) 747. - Ibid 55 (1972) 256 a. ::: Szubinska, B & Luft, J H. Unpublished dam. 21. Woodbury, L A, Hecht, H H & Christopherson, A R, Am j physiol 164 (1951) 307. Received March 17, 1977 Revised version received August 10, 1977 Accepted August 11, 1977
Exp Cell
Res I I I (1978)
Experimental
CLOSURE AROUND
Cell Research I1 1 (1978) 105-l 15
OF THE
PLASMA
MICRONEEDLE
IN AMOEBA
An Ultrastructural BARBARA
MEMBRANE PROTEUS
Study
SZUBINSKA
Department of Biological Structure, University of Washington, School of Medicine, Seattle, WA 98195, USA
SUMMARY Microneedle perforations of the plasma membrane of Amoeba proteus were studied on the ultrastructural level. In each individual cell one hole was produced, which subsequently was marked with an eyelash left in place. Cells were quickly fixed, and sections cut parallel to the longitudinal axis of the eyelash. It was clear that the eyelash penetrated the plasma membrane, and that its free tip was located in the interior of the cell. A gap remained between the plasma membrane and the eyelash which may correspond to the electrical leak sometimes found by microelectrode punctures. The edges of the broken plasma membrane curled back into the cytoplasm. Here, a great redundancy of the plasma membrane was observed. Within these membrane accumulations, a large quantity of dense droplets was apposed at the inner leaflet of the plasma membrane. Their involvement in the formation and expansion of the plasma membrane in Amoeba proteus and Xenopus faevis has been suggested previously [ 1, 181. Present studies offer more supportive evidence to that effect. Therefore, the interpretation seems to be plausible, that these membrane accumulations are the result of membrane expansion to minimize the hole produced during injury. This is in agreement with Holtfreter’s [8] and Bluemink’s [l] concept that the wound closure may occur by proliferation of the plasma membrane.
Electrophysiologists have been routinely impaling single cells with microelectrodes in order to measure transmembrane potentials. Thus, there is substantial information available on the reaction of cells to such injuries. With fine microelectrodes (0.5 pm tip diameter) the potentials are stable, but if the electrodes are larger (up to 9 pm) or the insertion is defective, low potentials result from a drop in membrane resistance [Z, 6, 12, 211, which may or may not recover [5]. There is also evidence of occasional leakage back along the microelectrode to the surface [3]. The most plausible explanation of these results is imperfect “sealing” of the electrode into the cell membrane with a persisting open channel. Yet from the morphological point of
view, very little is known about what exactly happens at the moment when a microelectrode is inserted into a cell, and what changes occur subsequently. It would be of interest to many scientists to know what the hole itself looks like, how the membrane accommodates around the microelectrode, and finally, how the perforation closes. Correlation of these morphological and physiological studies could yield useful information on cell function. An attempt has been made to follow these phenomena on the fine structure level and preliminary observations have been published [18, 191. From these studies [18], it appeared that holes inflicted with fine microneedles in individual cells of amoeba could be localized. Nevertheless, pictures Exp Cell
Res I I I (1978)
106
B. Szubinska
which showed the perforation and two edges of the broken plasma membrane in the same section, were obtained only fortuitously. It became obvious that in order to be able to trace the exact pathway of the microneedle, some sort of marker was needed. For this purpose an eyelash (5 pm tip diameter) has been employed [ 191, which was inserted into the cell along with the microneedle, and left there after the microneedle was withdrawn. This marker is not toxic, is easily seen in the light microscope, and is fully compatible with conventional EM procedures. It marks the hole in an unambiguous manner, and above all, it marks the exact pathway of the microneedle, offering at the same time the precise orientation of the hole. Injured cells marked in this way were fixed within 45 set, processed individually for electron microscopy, and sectioned along the longitudinal axis of the eyelash. Electron micrographs obtained from such cells reveal the interruption of the plasma membrane with its edges curled back into the cytoplasm, the tip of the eyelash in the cytoplasm free of the membrane, and the eyelash shaft closely approximated on both sides by remarkably smooth surfaces of the plasma membrane. In addition, a large number of electron dense droplets was found in close contact with the cytoplasmic surface of the plasma membrane near the hole. These have been previously associated with mechanical damage to cell membranes, both in Amoeba proteus [ 181 and Xenopus laevis [ 11.
MATERIALS
AND
METHODS
Amoebae were cultured and handled as described previously [B]. Cells were injured individually with tine glass microneedles (3-4 pm tip diameter), also as in previous study, but only one type of injury was employed in the present experiments, and the holes were Exp CeNRes
111 (1978)
labeled with an eyelash as the marker. Eyelashes were degreased by soaking in 75 % ethanol for 1 h, and then washed in several changes of distilled water prior to experiments. Each amoeba was speared only once, so that only one hole/cell was produced. The microneedle was inserted gently into the cell, from the side (at about a 45” angle) and held in that position while the eyelash (about 5 pm tip diameter) was introduced along its side. Then, the microneedle was withdrawn with the eyelash held exactly in the same position. It is important to note that although the description of injury and marking of the hole is rather simple, in fact, it is very difftcult to accomplish, considering the possibility of cell detachment from the bottom of the experimental dish, as well as introducing artifacts by unnecessary movements. Three cells marked in this manner were thoroughly examined in the present study. (An attempt has been made to mark holes in amoebae with other markers, such as Alcian blue as well as India ink, but both approaches were unsuccessful. Alcian blue dispersed in the cytoplasm of amoebae, and even India ink particles when dried on the microneedle and applied to the cell, did not stay in one particular spot on the cell, but instead moved quickly as observed with the light microscope.) Marked cells were fixed in situ within 35-45 set from the moment of injury. Fixation was carried out in 2.5 % acrolein buffered with 0.067 M cacodylate buffer at pH 7.2-7.4 for 15 min. Then the cells were post-fixed in 1.3 % 0~0, buffered as before, for 30 min. Dehydration was done in 35,70, 95, and 100% ethanol, respectively. Low viscosity epoxy resin based on diglycidyl ether [ 11, 201 was used as an embedding medium. For sectioning, each cell was oriented so that the sections would be cut parallel to the longitudinal axis of the eyelash. Sections were collected onto grids with a single hole of 500 pm diameter, coated with Pioloform [16] and carbon. Sections were double-stained with a saturated aqueous solution of uranyl acetate followed by Reynolds’ lead citrate [IS] for 5 and 10 min, respectively. More details on fixation, dehydration and embedding are given in the previous report [18]. No ruthenium dyes were used in this study.
RESULT Individual cells of amoebae when injured, marked with an eyelash and embedded for electron microscopy yield a typical picture as seen in fig. 1. The cytoplasm throughout Fig. I. Light micrograph of a whole amoeba marked with the eyelash. The picture was taken in polymerized epoxy resin before sectioning. x 173. Fig. 2. Low power electron micrograph of the entire thin section of the same amoeba depicted in fig. 1. In this montage the eyelash is seen as the dark object. Plasma membrane is shown outlined in black and its continuity is traceable except for two regions. x2000.
Plasma
membrane
around microneedle
Exp Cdl
107
RPS 111 (1978)
108
B. Szubinska
the cell is distributed somewhat unevenly, giving rise to darker and lighter regions. The nucleus can be recognized approximately in the center of the cell. The site of the injury is marked with the eyelash, and is located to the right of the nucleus. Sections cut parallel to the longitudinal axis of the eyelash reveal the hole itself (fig. 2). In this low power electron micrograph of the entire section through a portion of an amoeba, the hole is clearly seen as a perforation in the plasma membrane, allowing the tip of the eyelash to penetrate into the interior of the cell. The plasma membrane of the cell has been deliberately outlined in order to facilitate the tracing of its continuity. Indeed, it appeared that despite its convolutions, continuity of the plasma membrane could be traced easily. There are several regions where continuity is disrupted. From studies done on injured amoebae with holes marked in this way, it is obvious that there are three discrete regions which deserve closer attention. First, there is the region through which the microneedle and later the eyelash enters the cell. This is the region in which the eyelash shaft is located, and it looks like a narrow tunnel or invagination. This leads into a second region which is characterized by abundant plasma membrane convolutions, and which finally opens up through the perforation in the plasma membrane into the interior of the cell, where the very tip of the eyelash is seen. Higher magnification pictures illustrate in more detail certain significant features of each of these regions obtained from the same cell. Fig. 4 shows the eyelash shaft being followed closely on both sides of the plasma membrane. The gap between the eyelash and plasma membrane on both sides is very small, about 1650 A on the left hand side and 7 300 A on the right. In fig. 3 it can be seen clearly that the tip of Exp Cd
Rev I 11 (1978)
the eyelash is located deep in the cytoplasm free of membrane. However, several outer cuticular cells of the eyelash have lifted slightly from the tip, giving an illusion of a membrane. In figs 3, 4 and 5, cytoplasm seems to be diluted and rather free from organelles, which is characteristic of the region in the immediate vicinity of the hole. Sections obtained from regions situated further away from the hole showed all organelles in good condition. The region most important to this study is the middle or central part of the hole Fig. 3. Higher magnification picture of the uppermost region in fia. 2, revealing the free tip of the eyelash located in the cytoplasm: Two dark bands across the eyelash represent an artifact which arises during the staining procedure. Eyelash does not adhere to the carbon film as tightly as does the rest of the section, and as a result, the staining solutions enter between the carbon film and plastic leaving a residue. x3 000. Fig. 4. Higher magnification picture of the lowermost region in fig. 2, showing the eyelash shaft located in the invagination of the cell. Note the small distance between the plasma membrane and the eyelash on both sides, but especially on the left hand side (1650 A), as well as surmisinalv smooth surfaces of the ulasma membranes: x 3 O@. Fin. 5. Higher magnification picture of the central region in fig-2 illust&ing the hole itself. Actual perforation in the plasma membrane is marked with arrows. A large accumulation of the plasma membrane is apparent around the hole. Plasma membrane of the periphery of the cell is at the upper right and lower left. x3ooo. Fig. 6. Higher magnification picture of the right upper region in fig. 5, showing the low density of the plasma membrane at the periphery of the cell and higher density of the plasma membrane in the vicinity of the eyelash. Lumen at L and arrow shows location of tig. 7. X identities the location of fig. 9. x7 950. Fig. 9. Higher magnification picture of the area in tig. 6 marked with the arrow showing the dark droplets apposed at the cytoplasmic leatlet of the plasma membrane. Extraneous coat is facing the lumen (L) of the invagination, and the cytoplasm (C) of the cell is on the right. x56400. Fig. 8. inset shows the close contact of the dense droplets with the cytoplasmic leaflet of the plasma membrane. Trilaminar membrane is clearlv seen as well as the extraneous coat. x 160000. Fig. 9. Depicts the edge of the curled plasma membrane identified at X in fig. 6. The tip contains five dense droplets, as well as a few tine filaments which are more obvious in the membrane loop itself (arrow). x55 300. These membrane-associated filaments are different from the usual cytoplasmic filaments.
Plasma
membrane
around microneedle
Exp Cell
Res III
109
(1978)
110
Exp Cell
B. Szubinska
Rcs II I (1978)
Plasma
S-771807
membrane
around microneedle
111
112
B. Szubinska
where the actual perforation of the plasma membrane is present (fig. 5). Perforation is recognized where two edges of the plasma membrane on opposite sides of the eyelash are curled and found at the same level as indicated by the arrows. An abundance of convoluted plasma membrane is apparent around the hole and there is nearly complete continuity with at least the left edge of the curled plasma membrane. This has been checked carefully on higher magnification pictures which would be impractical to show here. The significance of this large membrane accumulation in this particular region will be discussed later. For the time being, however, it is important to look at a still higher magnification picture of portions of the plasma membrane itself. Fig. 6 illustrates the region obtained from the upper right side of the eyelash in fig. 5, which includes the curled edge of the plasma membrane. There is a distinct difference in density between the plasma membrane at the periphery of the cell and its portion in the vicinity of the eyelash. The peripheral membrane appears quite pale while the portion near eyelash shows discrete black droplets. Still higher magnification (fig. 7), taken from the area marked with the arrow in fig. 6, reveals dense droplets aligned on the plasma membrane at more or less regular intervals. The inset (fig. 8) illustrates that these dense droplets are apposed very closely at the inner leaflet of the plasma membrane. Trilaminar membrane is seen clearly and the amorphous and tilamentous layer of the extraneous coat of the cell points to the outside. Fig. 9 shows further details of the right edge of the curled plasma membrane. Dense droplets are again prominent throughout the picture, and are attached to the membrane including the very end of the curled plasma membrane. In addition, an aggregation of Exp Cell
Res 111 (1978)
fine filaments 5&100 A in diameter is apparent at the cytoplasmic surface in the membrane loop (arrow), and a few strands occur in the very end of the curled plasma membrane. They terminate in a dense knob about twice their diameter and have been seen previously in wounded amoebae [18]. These membrane-associated filaments may be responsible for the curling properties of the plasma membrane. DISCUSSION Cell injuries raise many questions of biological importance, but first of all it is pertinent to consider the advantage of the approach used in this study with respect to the already existing methods. For a long time electrophysiologists have been interested in the location of microelectrode tips in cells of various tissues; therefore, the literature records many attempts to accomplish that task. Several dyes were used for this purpose such as methylene blue, fast green, Alcian blue and crystal violet. Usually, the dyes were driven into the cell by iontophoresis. In recent years Stretton & Kravitz [17] suggested the use of the fluorescent dye, Procion yellow, as a marking agent. Also, iontophoresis has been employed to introduce various ions, such as silver, into the tissue for localization. A more recent method is that described by Nickels [13] ibr accurate localization of a recording glass microelectrode in a muscle cell. The method involves precipitation of ferrocyanide and phosphate from the microelectrode with lead added externally together with glutaraldehyde, and followed with ammonium sulfide. The end product is in the form of a dark sediment which is easily seen both in the light and electron microscope. Also, intracellular injection of cobaltous chloride by Pitman & Tweedle
Plasma [ 141 was used for labeling central neurons for later identification in the EM. This metal is likewise precipitated as a sulfide, and is also electron opaque. In addition, there have been some recent improvements in dye application as a marker. Christensen [4] described the usage of Procion brown as an intracellular dye for light and electron microscopy. The advantage of this dye over Procion yellow and cobalt is that in addition to remaining within the cell after injection, Procion brown does not require further reaction to make it visible in the EM. In spite of the number of these methods available for marking the site of injury, none was adequate for the studies intended here. None of the dye markers could have been employed successfully since all dyes disperse rapidly in the interior of the cells, marking the entire cell. This is especially true for the cytoplasm of amoebae which is in constant movement at all times. Even the histochemical method proposed by Nickels [13] was insufficient. Although it permits localization of the microelectrode tip, it does not allow for precise axial orientation as desired. Besides, all these dyes and chemicals involved in the methods mentioned above, are toxic to the cell and therefore may interfere with natural wound closure. With regard to the biological findings, the experiments conducted in this study reveal several unusual findings. First of all, when looking at the light micrograph of an amoeba marked with the eyelash (fig. l), it is not certain whether the eyelash is in the interior of the cell or whether it lies only in an invagination of the plasma membrane produced during cell injury. Ultrastructural studies done on these amoebae offer an undisputable answer. It is obvious from the pictures that the eyelash did penetrate the
membrane
around microneedle
113
plasma membrane, and that its free tip is located in the interior of the cell. The plasma membrane is broken, and its two edges are curled back on both sides of the eyelash. In other words, there is an opening which leads to the interior of the cell obstructed only by the presence of the eyelash. The perforation itself is large (8-10 pm) when measured from one end of the plasma membrane to another; nevertheless, the smallest open spaces on both sides of the eyelash at the level of the curled edges correspond only to 1200 and 340 A each. Assuming that both the eyelash and the perforation are circular, and the open space to be uniform around the eyelash, this is equivalent to about 1.8 pm2. From these data it appears that the area through which leakage from the cell can occur is relatively small. The great length of the pathway from cytoplasm to the exterior also must impede leakage. It is important to remember, however, that the measurements given in this paper are taken from fixed and embedded cells which include shrinkage and distortion of uncertain magnitude as compared to the living cell. Nevertheless, the small channel remaining open between the plasma membrane and eyelash may be the structural counterpart of the electrical leak sometimes found with microelectrodes in other cells [3]. Another interesting feature, which emerges from these experiments is the fact that the hole is not yet completely closed. This seems to be in contradiction with the information existing already in the literature, that wound closure in amoebae occurs very rapidly. Two explanations can be offered to that effect. (1) The object itselfthe eyelash-represents quite a large foreign body. It must take some time for the cell to accommodate itself to such a dramatic disturbance. On the other hand, the Exp Cd
RES I I I (1978)
114
B. Szubinska
eyelash may also act as a cork in a bottle which slows the leakage of the cytoplasm (or ions) to the outside, and therefore does not force the cell to rapid closure of the hole. (2) The speed with which the fixative was delivered (35-45 set) from the moment of injury certainly must interrupt the process of closure. Another aspect that should be discussed here is the presence and involvement of cytoplasmic filaments in wound closure. Much of the information that is present in the literature suggests the involvement of contractile filaments in cellular wound healing [7, lo]. Recent ultrastructural studies done on Amoeba proteus by Jeon & Jeon [9] are supportive of this idea. However, studies done onXenopus eggs by Bluemink [l], although showing the presence of cytoplasmic filaments in the vicinity of the wound, indicate also that they may not be an absolute requirement for wound closure. In view of Bluemink’s findings, it is easier to explain the lack of these cytoplasmic tilaments around the wound in my studies. Although they are present in other portions of the same cell, they certainly are not present around the hole itself. There is a possibility that there are two mechanisms operating independently in wound closure, depending on the type of the injury, size of the wound and requirement of the cell at the moment. Expulsion of the nucleus [9], for example, which must require rapid contraction, may necessitate mobilization of the filaments, whereas the closure of the holes, which does not require rapid contraction, may be not entirely filament dependent. A different concept with regard to wound closure was long ago expressed by Holtfreter [8] and, more recently, reexamined by Bluemink [l]. According to these studies, wound closure may also occur by Exp Cell Res 111 (1978)
means of growth of new membrane surface. Since supportive evidence for this concept comes from my studies, it seems to be worthwhile to concentrate for a moment on this aspect. Electron micrographs presented in this work show around the hole substantial accumulation of the plasma membrane in at least partial continuity with its broken and curled edges (fig. 5). The question arises at this point why there is such accumulation of the plasma membrane at this particular location? There is no reason to think that these packages of membranes are non-specific artifacts produced during cell injury, since such artifacts were recognized early in other cells [18]. These consist of highly disorganized strands of broken membranes, pulled in all possible directions. A more plausible explanation is that this is, in fact, greatly expanded plasma membrane (composed of both old and new elements) made by the cell to minimize the hole. It would be too much of a coincidence to find such an aggregation of the plasma membrane only near the vicinity of the wound and nowhere else. At this point, it is important to consider how the plasma membrane expansion can occur so fast. In previous studies on amoebae [18] I have shown and discussed the mechanism of plasma membrane expansion by injection into the already existing membrane of membrane precursors present in the cytoplasm in the form of dense or foamy droplets. Here again, the same dense droplets are present in abundance within these membrane accumulations at the site of the hole, but only occasionally at the periphery of the cell (fig. 6). They are apposed very closely to the cytoplasmic leaflet of the plasma membrane, including the regions of two curled edges of the membrane. Similar material has been demonstrated by Bluemink [l, p. 1091 in the eggs
Plasma
ofxenopus with a different technique and is also found to be associated with cell injury. The results presented in this paper further reinforce the possibility of wound closure by proliferation of the plasma membrane.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Bluemink, J G, J ultrastruct res 41 (1972) 95. de1 Castillo, J & Katz, B, J physiol 125 (1954) 546. -Ibid 128 (1955) 157. Christensen, B N, Science 182 (1973) 1255. De Mello, W C, Proc natl acad sci US 70 (1973) 982. Fatt, P, Methods med res 9 (l%l) 381. Gingell, D, J embryo1 exp morph01 23 (1970) 583. Holtfreter, J, J exp zoo1 93 (1943) 251. Jeon, K W 62 Jeon, M S, J cell bio167 (1975) 243.
membrane
around microneedle
115
:;. Luckenbill, L M, Exp cell res 66 (1971) 263. Luft, J H, Advanced techniques in biological . electron microscopy (ed J K Koehler) p. 1. ,2, Springer-Verlag, Berlin (1973).
Nastuk, W L & Hodgkin, A L, J cell camp physiol 35 (1950) 39. 13. Nickels, J, Acta physiol Stand 80 (1970) 360. 14. Pitman, R M, Tweedle, C D & Cohen, M J, Science 176 (1971) 412. 15. Reynolds, E S, J cell biol 17 (1%3) 208. 16. Stockem, W, Mikroskopie 26 (1970) 185. 17. Stretton, A 0 W & Kravitz, E A, Science 162 -‘. (1968) 132. 18. Szubinska, B, J cell bio149 (1971) 747. - Ibid 55 (1972) 256 a. ::: Szubinska, B & Luft, J H. Unpublished dam. 21. Woodbury, L A, Hecht, H H & Christopherson, A R, Am j physiol 164 (1951) 307. Received March 17, 1977 Revised version received August 10, 1977 Accepted August 11, 1977
Exp Cell
Res I I I (1978)