- Email: [email protected]
Observations on microextensions from the surface of isolated vertebrate cells
DEVELOPMENTAL
BIOLOGY,
Observations
7, 660-673
(1963)
on Microextensions of Isolated
from
Vertebrate
the Surface
Cells1
A. CECIL TAYLOR AND ELLIOTT ROBBINS The Rockefeller
Institute,
New York, and the Albert Einstein New York, New York Accepted
November
College
of Medicine,
30, 1962
INTRODUCTION
With the advent of the phase contrast microscope a new tool for visualization and photographic recording of living cells became available. Although certain minute projections from the surface of living cells had often been seen in ordinary light microscopy or with dark field illumination (Lewis and Lewis, 1924), their form and particularly their activities were first observed and described in detail by Gey (review, 1956) with phase contrast. In an early electron microscope study Porter and his collaborators (1945) observed similar projections from unsectioned, thinly spread cells which were mounted whole on Formvar-coated grids and examined with the benefit of the increased resolution afforded by this instrument. It was pointed out that in some instances the electron microscope revealed surface processes not visible in ordinary light. The microprocesses described were subsequently reported on different cells by other investigators (DeRobertis and Sotelo, 1952; Gey, 1956). Because of their possible bearing on problems of cell contact and selective adhesion, the characteristics and behavior of these microirregularities, which seem to appear on the surface of most cells, deserve more attention than they have received. To this end the ’ These investigations form Dr. Paul Weiss as principal American Cancer Society and of Health of the U. S. Public Supported in part also by
part of a program of study under the direction of investigator, aided in part by grants from the the National Cancer Institute (National Institutes Health Service). NIH grant no. GM07348-03. 660
OBSERVATIONS
ON
MICROEXTENSIONS
661
present preliminary report adds some light- and electron-microscopic observations made upon these cell extensions. METHODS
Light microscopy. Observations were made on two commercially obtained cell strains of human origin (liver and conjunctiva) and on freshly dissociated chick embryo liver and kidney. The cells were dispersed with trypsin or Versene and allowed to spread on a glass surface in liquid medium consisting of Eagle’s basal medium + 10% beef embryo serum. Spreading occurred during incubation for periods of from 6 to 24 hours, after which the cells were examined directly and photographed with 40~ and 100x oil immersion, phase contrast objectives or with dark field optics. Time-lapse motion pictures were also made at the same magnifications and at lo-second intervals. Electron microscopy. Glass slides were coated with 0.1% collodion or Formvar. One-half milliliter of a monodisperse cell suspension (human conjunctiva and HeP strain or various 7- to g-day chick embryo organs in Eagle’s basal medium plus 15% fetal calf serum) was carefully placed on one end of the coated slide so as to form a large drop. The cells were incubated in an atmosphere of 5% CO, + 95% air and 100% humidity until they were spread. Fixation was carried out on the glass slide with either 2% 0~0, in isotonic Tyrode’s solution at pH 7 or in a hypertonic solution similar to that already described (Robbins, 1961) except that 1% formalin was added. In some instances cells were postfixed with 0.1% phosphotungstic acid. After rinsing with distilled water, the collodion was floated off the slide and mounted on 200-mesh grids. The cells were allowed to dry slowly. Some were then shadowcast with either chromium or palladium at an angle of 20 degrees and carbon coated. Examination was carried out in an RCA EMU 3G at an accelerating voltage of XL 106 kv. OBSERVATIONS
In this report the term microextension will be employed as a general name for cell processes near or below the limits of light microscope resolution. Two distinct types of microextensions were identified on the basis of their characteristics as seen both in the living cell and under the electron microscope. One of these types will here be referred to as microspikes, a term first suggested by Weiss (1961) to designate
662
TAYLOR
AND
ROBBINS
microextensions with a rigid core; the other type will be called retraction fibrils, which seems an appropriate name for the strands of cytoplasm spun out when a cell border retreats from its attachment to the substratum. Light Microscopy
of Living
Cells
In general, our observations of microextensions made with the light microscope are in agreement with earlier descriptions. Certain features, however, require emphasis here because of their bearing upon electron microscope studies to be discussed later. A brief summary of observations made by light microscopy, including the study of time-lapse motion pictures, can be organized under the following headings: Form and dimensions. While significant measurements of the thickness of these microprocesses seen with the light microscope were difficult to make, they appeared to range in diameter from about 0.5 ,LLdownward to filaments barely visible with a 100 x oil phase objective (about 0.1 p). The length of most microspikes did not exceed 15 ,L whereas retraction fibrils were frequently longer, particularly when associated with extreme cell retraction. Most microspikes, particularly the finer ones, were of uniform diameter throughout their length (cf. a in Figs. 1 and 3). Some, however, appeared thicker near the base (Fig. 3, b) or were forked at the distal end (Fig. 3, c). Unlike microspikes, the retraction fibrils often did not have a uniform diameter and frequently did not extend along a straight line (Fig. 1, b). This may be due to irregular attachments to the substratum
along
their
length.
When
they
were
anchored
only
at their
tips and were under tension, they appeared as straight strands. FIG. 1. A spread HeLa cell showing an active border above bearing microspikes (a), and a retreating border below with retraction fibrils attached to the substratum ( b ). Phase contrast, oil immersion. Magnification: x 1000. FIG. 2. HeLa cells spread on glass and photographed in dark field illumination. Processes extend between the cells which presumably originated as microspikes (a). Magnification: x 1000. FIG. 3. Frames from a motion picture film of a human conjunctiva cell showing the rapid movements of microspikes. Long microspikes with uniform diameter (a); tapered microspike (b ) ; forked microspike (c). Oil immersion; Ten-second interval between frames. Magnification: X 660.
OBSERVATIONS
ON
MICROEXTENSIONS
663
664
TAYLOR
AND
ROBBINS
Movements. The most striking distinction between the two types of microextensions, as seen on the living cell, was their difference in mobility. When first formed, the microspikes project from the cell surface into the liquid medium and, being unattached peripherally, are free to wave about in continuous activity. In these movements they remain rigid throughout their length, bending back and forth like rodlets hinged to the cell surface. Their action thus resembles that of the spines of a sea urchin rather than the tentacles of a coelenterate. These movements may be quite rapid, as illustrated by the changes visible in the reproduced frames from a motion picture strip (Fig. 3). The life of a single spike may last only a few minutes, for spikes are constantly disappearing and new ones are being formed nearby to take their place. In time-lapse films the formation of new microspikes was difficult to observe since they seemed at first appearance to be full-length structures. The regression of a spike, however, frequently was observed. This might occur by the folding back of a spike against the plasmalemma, with which it then appeared to fuse, or it might simply collapse, as if some rigid support had dissolved. Only rarely were microspikes seen in the process of forming an attachment to the substratum by their distal ends. When this was observed in two instances on a motion picture record, the anchored microspikes served as guides along which ectoplasm flowed out to form new cell processes. Figure 2 is a photograph of spreading HeLa cells viewed in dark field illumination which shows what appear to be microspikes that have formed connections between adjacent cells. Some of these have already become enlarged by the outflow of cytoplasm, When the same preparation was examined an hour later two of these contact points had become greatly broadened. Retraction fibrils, unlike the microspikes, were, from their beginning, anchored at the tips and showed no motion when stretched taut. When slack, or if detached, they were seen to flutter passively as they were buffeted by particles in Brownian motion. Detached retraction fibrils eventually coiled up and were slowly resorbed. FIG. 4. Electron micrograph of portions of a human conjunctiva cell showing microspikes and their cores. Composite microspikes ((I and c); unit microspikes ( b ) . Chromium shadowed. Magnification: X 7200. FIG. 5. Electron micrograph of part of a human conjunctiva cell with microspikes. The deformation of the cores at their tips (a and b) indicate a protrusive thrust of the core. Chromium shadowed. Magnification: x7200.
OBSERVATIONS
ON
MICROEXTENSIONS
665
666
TAYLOR
AND
ROBBINS
After the retreat of a cell process and the formation of retraction fibrils, the cell border was frequently observed to advance again, flowing out along the anchored retraction fibrils it had previously formed, apparently reincorporating them into its substance. Distribution. Microspikes appeared larger, longer, and more conspicuously motile on the advancing borders of a cell than they did on more inactive portions of its surface. Although they were more obvious when projecting radially from the cell borders, and hence appearing in profile, their presence elsewhere over the surface of the flattened process could be verified by focusing directly upon them. Nearly as active, although somewhat smaller, microspikes were often observed on surfaces with no undulating membrane or microfrills. They apparently can arise from an otherwise inactive and consolidated surface membrane. Even completely contracted and rounded cells were sometimes covered with a fur of mixed microspikes and retraction fibrils. When cells were spread on glass, retraction fibrils were observed only at their margins or between two cells. These fibrils could be readily produced by any treatment, such as cold or high pH, which would initiate contraction of the flattened cell borders. Electron Microscopic
Observations
When cells of the same types as those studied in the living state were fixed and dried for electron microscope examination some distortion of the freely projecting microextensions was to be expected. However, examination at this stage by phase contrast showed them to be essentially the same in appearance as cells fixed and mounted for light microscopy. In accord with observations by Porter et al. (1945), structural differences revealed by the higher resolving power of the electron microscope substantiate the presence of the two types of microextensions as assumed from observing their behavior in vitro. Electron micrographs showed that one type of microextension is characterized by the presence of one or more cylindrical cores of uniform diameter extending from within the marginal cytoplasm to the tip of the process (Figs. 47). That this core is a rigid structure is evidenced by the straightness of the microextension itself, by the tentlike configuration of the plasmalemma where it is carried outward, by protruding core structures (Fig. 4, a), and by the bending or spiraling deformation of the distal ends of some cores where their protrusive
$
I
FIG. 6. Electron micrograph of 4 unit microspike at the margin of a human conjunctiva cell showing clearly the coarsely granular structure of the cores and their lack of periodicity. Chromium shadowed. Magnification: x 32,000. FIG. 7. Electron micrograph of a portion of a chick embryo kidney cell showing many unit microspikes (a). Magnification: X 3200. 667
668
TAYLOR
AND
ROBBINS
thrust has encountered an excessive resistance of the enveloping plasmalemma sheath (Fig. 5, a and b). These and other considerations strongly suggest that such processes are actively projected from within the cytoplasm rather than being protracted from without. The unit microextension of this type appears to be a single axial core surrounded by a thin sleeve of plasmalemma and a small amount of ectoplasm (Figs. 4 b, 6, and 7 a). The core itself measures between 500 and 800 A in diameter, and appears to have a rough granular structure with no evidence of a regular periodicity (Fig. 6). The unit process measures approximately 800-1006 A in diameter. Frequently, however, a single microextension contains more than one core. Whether such composite extensions are the result of lateral fusions of two or more unit extensions or the simultaneous protrusion of several cores, as may be occurring at a in Fig. 4, cannot be determined. Whereas the dimensions of a unit microextension would place it at the lower limit of light microscope visibility, composite structures, such as that at c in Fig. 4, are easily visible. This type of axiate microextension fits well into the category of the microspike as described from observations on the living cells. The rigid axial core would account for the observed uniform diameters and characteristic motions like those of a hinged rod. The fusion of unit spikes of unequal length or the extension into the same sleeve of several cores at different rates (Fig. 5, a) could explain the appearance of tapered composite microspikes, and forked extensions could result from an incomplete fusion (Fig. 4, c). A microspike would disappear rapidly after the collapse of its core by a solation process, and the formation of new spikes might occur through development of a core by rapid cytoplasmic gelation, or polymerization. Progressive addition to this structure at its base, by increasing its length, would carry outward with it a sheath of cell membrane and ectoplasm. Forming a second category of microextensions evident in the electron micrographs are those which contain no solid core. Some of these were greatly elongated and presumably were fixed while their distal ends were still anchored to the substratum (Fig. 8, a and Fig. FIGS. 8 and 9. Electron cells with retraction fibrils attached (a); detached and which may be remains of nification: x 8700.
micrographs showing portions of human conjunctiva which are without cores. Long fibrils, presumably partially collapsed fibrils ( b ); terminal enlargements fibril attachments (c). Chromium shadowed. Mag-
669
670
TAYLOR
AND
ROBBINS
9). Others appeared to have become detached from the substratum and to be collapsed and in the process of resorption (Fig. 8, b). In general these all have much less regular contours than the microspikes. Irregular swellings, sometimes present along their length, appear to contain granular cytoplasm, while the tips of some were observed to bear enlargements which might represent the remains of their terminal attachments to the substratum (Fig. 8, c, and Fig. 9, a). That these are the retraction fibrils seen in living cells is indicated by their occurrence in great numbers on cells treated with medium at pH 9 for 10 minutes before fixation, a procedure which induces retraction of cell processes (Taylor, 1962). The restless probing behavior of the microspikes as seen in living cells suggests a mechanism by which a cell, experimentally deprived of contact with solid environment, may seek out appropriate interfaces for contact relationships. Preliminary attempts were made to observe this behavior by confronting cells from primary dissociates of different chick organs. The results are still too few to warrant significant conclusions; it is notable, however, that, in electron microscopic observations of numerous encounters between embryonic liver and kidney epithelial cells, overlaps frequently were seen but there was no evidence of structural modification indicative of selective adhesions. In several cases, however, when two kidney cells confronted each other, thickenings seen at several contact points indicated the development of areas of adhesion between processes which apparently could withstand considerable retractive tension (Figs. 10 and 11). Whether these points of contact originated as microspikes has not yet been determined. DISCUSSION
Our observations on living cells have indicated that retraction fibrils are passively drawn out from the cell surface; although they may be numerous and conspicuous, they are, with respect to cell behavior, the less interesting of the two types of microextensions. Microspikes, on the other hand, despite their apparent structural simplicity and their short life span, are real cell organelles which are FIGS. 10 and 11. Electron micrographs of chick embryo kidney thickenings at points of cell-to-cell attachment. A portion of Fig. is shown at higher magnification in Fig. 11 ( X 16,800).
cells showing 10 ( x2800)
OBSERVATIONS
ON
MICROEXTENSIONS
671
672
TAYLOR
AND
ROBBINS
admirably adapted to provide for tactile exploration of and selective attachment to solid structures within the immediate fluid environment of a cell process, However, there is yet little direct evidence for assuming this function of the microspike. Such an assumption can, at this time, be based on only three facts: (1) many cells have, at least in vivo, the capacity to select for adhesion those interfaces in their environment for which an inherent contact affinity exists. (Weiss, 1941; Taylor, 1944); (2) th e structure of the microspike and its observed behavior are remarkably adapted to accomplish this function; (3) there are a few instances, here reported, in which an active microspike was observed to attach and adhere to the substratum. Evidence that this is the function of microspike activity would be greatly strengthened by the accumulation of further instances of contact and attachment between microspikes and different solid surfaces as well as other cells observed under experimental conditions. Little, however, is known regarding the conditions requisite for the expression of selective adhesion by cells which have been isolated from the organism, and a failure to demonstrate the reaction at this time may only reflect this ignorance. SUMMARY
Microextensions from the surface of tissue cells in vitro were examined by light and electron microscopy, and their activities were recorded by time-lapse motion pictures. Two types of these extensions were described on the basis of their structure and behavior. Those of one type are strands of cytoplasm spun out from the retreating borders of retracting cell processes. These retraction fib& were immobile and showed no special structure in electron micrographs. Microextensions of the second type were actively projected from the surface of the cells and, during their short existence, were in constant waving motion. High resolution pictures showed these to be supported by rigid rodlike cores. The structure and behavior of these microspikes suggest their role as organelles for selective attachment to solid surfaces in the environment. REFERENCES DEROBERTIS, E., and SOTELO, J. R. (1952). Electron microscope study tured nervous tissue. Exptl. Cell Research 3 (Suppl. 2), 433-452.
of cul-
OBSERVATIONS
ON MICROEXTENSIONS
673
GEY, G. 0. ( 1956). Some aspects of the constitution and behavior of normal and malignant cells maintained in continuous culture. Haroey Lectures Ser. 50, 154-229. LEWIS, W. H., and LEWIS, M. R. ( 1924). Behavior of cells in tissue cultures. In “General Cytology” (E. V. Cowdry, ed.), pp. 385-447. Univ. of Chicago Press, Chicago, Illinois. PORTER, K. R., CLAUDE, A., and FULLAM, E. F. (1945). A Study of tissue culture cells by electron microscopy. J. Exptl. Med. 81, 233-246. ROBBINS, E. (1961). Some theoretical aspects of osmium tetroxide fixation. J. Biophys. Biochem. Cytol. 2, 445-455. TAYLOR, A. C. (1944). Selectivity of nerve fibers from the dorsal and ventral roots in the development of the frog limb. J. Exptl. 2001. 96, 159-185. TAYLOR, A. C. ( 1962). Responses of cells to pH changes in the medium. J. Cell Biol. 15, 201-209. WEISS, P. (1941). Nerve patterns: the mechanics of nerve growth. Growth 5, 163-203. WEISS, P. ( 1961). From cell to molecule. In “The Molecular Control of Cellular Activity” (J. M. Allen, ed.), pp. l-72. McGraw-Hill, New York.
BIOLOGY,
Observations
7, 660-673
(1963)
on Microextensions of Isolated
from
Vertebrate
the Surface
Cells1
A. CECIL TAYLOR AND ELLIOTT ROBBINS The Rockefeller
Institute,
New York, and the Albert Einstein New York, New York Accepted
November
College
of Medicine,
30, 1962
INTRODUCTION
With the advent of the phase contrast microscope a new tool for visualization and photographic recording of living cells became available. Although certain minute projections from the surface of living cells had often been seen in ordinary light microscopy or with dark field illumination (Lewis and Lewis, 1924), their form and particularly their activities were first observed and described in detail by Gey (review, 1956) with phase contrast. In an early electron microscope study Porter and his collaborators (1945) observed similar projections from unsectioned, thinly spread cells which were mounted whole on Formvar-coated grids and examined with the benefit of the increased resolution afforded by this instrument. It was pointed out that in some instances the electron microscope revealed surface processes not visible in ordinary light. The microprocesses described were subsequently reported on different cells by other investigators (DeRobertis and Sotelo, 1952; Gey, 1956). Because of their possible bearing on problems of cell contact and selective adhesion, the characteristics and behavior of these microirregularities, which seem to appear on the surface of most cells, deserve more attention than they have received. To this end the ’ These investigations form Dr. Paul Weiss as principal American Cancer Society and of Health of the U. S. Public Supported in part also by
part of a program of study under the direction of investigator, aided in part by grants from the the National Cancer Institute (National Institutes Health Service). NIH grant no. GM07348-03. 660
OBSERVATIONS
ON
MICROEXTENSIONS
661
present preliminary report adds some light- and electron-microscopic observations made upon these cell extensions. METHODS
Light microscopy. Observations were made on two commercially obtained cell strains of human origin (liver and conjunctiva) and on freshly dissociated chick embryo liver and kidney. The cells were dispersed with trypsin or Versene and allowed to spread on a glass surface in liquid medium consisting of Eagle’s basal medium + 10% beef embryo serum. Spreading occurred during incubation for periods of from 6 to 24 hours, after which the cells were examined directly and photographed with 40~ and 100x oil immersion, phase contrast objectives or with dark field optics. Time-lapse motion pictures were also made at the same magnifications and at lo-second intervals. Electron microscopy. Glass slides were coated with 0.1% collodion or Formvar. One-half milliliter of a monodisperse cell suspension (human conjunctiva and HeP strain or various 7- to g-day chick embryo organs in Eagle’s basal medium plus 15% fetal calf serum) was carefully placed on one end of the coated slide so as to form a large drop. The cells were incubated in an atmosphere of 5% CO, + 95% air and 100% humidity until they were spread. Fixation was carried out on the glass slide with either 2% 0~0, in isotonic Tyrode’s solution at pH 7 or in a hypertonic solution similar to that already described (Robbins, 1961) except that 1% formalin was added. In some instances cells were postfixed with 0.1% phosphotungstic acid. After rinsing with distilled water, the collodion was floated off the slide and mounted on 200-mesh grids. The cells were allowed to dry slowly. Some were then shadowcast with either chromium or palladium at an angle of 20 degrees and carbon coated. Examination was carried out in an RCA EMU 3G at an accelerating voltage of XL 106 kv. OBSERVATIONS
In this report the term microextension will be employed as a general name for cell processes near or below the limits of light microscope resolution. Two distinct types of microextensions were identified on the basis of their characteristics as seen both in the living cell and under the electron microscope. One of these types will here be referred to as microspikes, a term first suggested by Weiss (1961) to designate
662
TAYLOR
AND
ROBBINS
microextensions with a rigid core; the other type will be called retraction fibrils, which seems an appropriate name for the strands of cytoplasm spun out when a cell border retreats from its attachment to the substratum. Light Microscopy
of Living
Cells
In general, our observations of microextensions made with the light microscope are in agreement with earlier descriptions. Certain features, however, require emphasis here because of their bearing upon electron microscope studies to be discussed later. A brief summary of observations made by light microscopy, including the study of time-lapse motion pictures, can be organized under the following headings: Form and dimensions. While significant measurements of the thickness of these microprocesses seen with the light microscope were difficult to make, they appeared to range in diameter from about 0.5 ,LLdownward to filaments barely visible with a 100 x oil phase objective (about 0.1 p). The length of most microspikes did not exceed 15 ,L whereas retraction fibrils were frequently longer, particularly when associated with extreme cell retraction. Most microspikes, particularly the finer ones, were of uniform diameter throughout their length (cf. a in Figs. 1 and 3). Some, however, appeared thicker near the base (Fig. 3, b) or were forked at the distal end (Fig. 3, c). Unlike microspikes, the retraction fibrils often did not have a uniform diameter and frequently did not extend along a straight line (Fig. 1, b). This may be due to irregular attachments to the substratum
along
their
length.
When
they
were
anchored
only
at their
tips and were under tension, they appeared as straight strands. FIG. 1. A spread HeLa cell showing an active border above bearing microspikes (a), and a retreating border below with retraction fibrils attached to the substratum ( b ). Phase contrast, oil immersion. Magnification: x 1000. FIG. 2. HeLa cells spread on glass and photographed in dark field illumination. Processes extend between the cells which presumably originated as microspikes (a). Magnification: x 1000. FIG. 3. Frames from a motion picture film of a human conjunctiva cell showing the rapid movements of microspikes. Long microspikes with uniform diameter (a); tapered microspike (b ) ; forked microspike (c). Oil immersion; Ten-second interval between frames. Magnification: X 660.
OBSERVATIONS
ON
MICROEXTENSIONS
663
664
TAYLOR
AND
ROBBINS
Movements. The most striking distinction between the two types of microextensions, as seen on the living cell, was their difference in mobility. When first formed, the microspikes project from the cell surface into the liquid medium and, being unattached peripherally, are free to wave about in continuous activity. In these movements they remain rigid throughout their length, bending back and forth like rodlets hinged to the cell surface. Their action thus resembles that of the spines of a sea urchin rather than the tentacles of a coelenterate. These movements may be quite rapid, as illustrated by the changes visible in the reproduced frames from a motion picture strip (Fig. 3). The life of a single spike may last only a few minutes, for spikes are constantly disappearing and new ones are being formed nearby to take their place. In time-lapse films the formation of new microspikes was difficult to observe since they seemed at first appearance to be full-length structures. The regression of a spike, however, frequently was observed. This might occur by the folding back of a spike against the plasmalemma, with which it then appeared to fuse, or it might simply collapse, as if some rigid support had dissolved. Only rarely were microspikes seen in the process of forming an attachment to the substratum by their distal ends. When this was observed in two instances on a motion picture record, the anchored microspikes served as guides along which ectoplasm flowed out to form new cell processes. Figure 2 is a photograph of spreading HeLa cells viewed in dark field illumination which shows what appear to be microspikes that have formed connections between adjacent cells. Some of these have already become enlarged by the outflow of cytoplasm, When the same preparation was examined an hour later two of these contact points had become greatly broadened. Retraction fibrils, unlike the microspikes, were, from their beginning, anchored at the tips and showed no motion when stretched taut. When slack, or if detached, they were seen to flutter passively as they were buffeted by particles in Brownian motion. Detached retraction fibrils eventually coiled up and were slowly resorbed. FIG. 4. Electron micrograph of portions of a human conjunctiva cell showing microspikes and their cores. Composite microspikes ((I and c); unit microspikes ( b ) . Chromium shadowed. Magnification: X 7200. FIG. 5. Electron micrograph of part of a human conjunctiva cell with microspikes. The deformation of the cores at their tips (a and b) indicate a protrusive thrust of the core. Chromium shadowed. Magnification: x7200.
OBSERVATIONS
ON
MICROEXTENSIONS
665
666
TAYLOR
AND
ROBBINS
After the retreat of a cell process and the formation of retraction fibrils, the cell border was frequently observed to advance again, flowing out along the anchored retraction fibrils it had previously formed, apparently reincorporating them into its substance. Distribution. Microspikes appeared larger, longer, and more conspicuously motile on the advancing borders of a cell than they did on more inactive portions of its surface. Although they were more obvious when projecting radially from the cell borders, and hence appearing in profile, their presence elsewhere over the surface of the flattened process could be verified by focusing directly upon them. Nearly as active, although somewhat smaller, microspikes were often observed on surfaces with no undulating membrane or microfrills. They apparently can arise from an otherwise inactive and consolidated surface membrane. Even completely contracted and rounded cells were sometimes covered with a fur of mixed microspikes and retraction fibrils. When cells were spread on glass, retraction fibrils were observed only at their margins or between two cells. These fibrils could be readily produced by any treatment, such as cold or high pH, which would initiate contraction of the flattened cell borders. Electron Microscopic
Observations
When cells of the same types as those studied in the living state were fixed and dried for electron microscope examination some distortion of the freely projecting microextensions was to be expected. However, examination at this stage by phase contrast showed them to be essentially the same in appearance as cells fixed and mounted for light microscopy. In accord with observations by Porter et al. (1945), structural differences revealed by the higher resolving power of the electron microscope substantiate the presence of the two types of microextensions as assumed from observing their behavior in vitro. Electron micrographs showed that one type of microextension is characterized by the presence of one or more cylindrical cores of uniform diameter extending from within the marginal cytoplasm to the tip of the process (Figs. 47). That this core is a rigid structure is evidenced by the straightness of the microextension itself, by the tentlike configuration of the plasmalemma where it is carried outward, by protruding core structures (Fig. 4, a), and by the bending or spiraling deformation of the distal ends of some cores where their protrusive
$
I
FIG. 6. Electron micrograph of 4 unit microspike at the margin of a human conjunctiva cell showing clearly the coarsely granular structure of the cores and their lack of periodicity. Chromium shadowed. Magnification: x 32,000. FIG. 7. Electron micrograph of a portion of a chick embryo kidney cell showing many unit microspikes (a). Magnification: X 3200. 667
668
TAYLOR
AND
ROBBINS
thrust has encountered an excessive resistance of the enveloping plasmalemma sheath (Fig. 5, a and b). These and other considerations strongly suggest that such processes are actively projected from within the cytoplasm rather than being protracted from without. The unit microextension of this type appears to be a single axial core surrounded by a thin sleeve of plasmalemma and a small amount of ectoplasm (Figs. 4 b, 6, and 7 a). The core itself measures between 500 and 800 A in diameter, and appears to have a rough granular structure with no evidence of a regular periodicity (Fig. 6). The unit process measures approximately 800-1006 A in diameter. Frequently, however, a single microextension contains more than one core. Whether such composite extensions are the result of lateral fusions of two or more unit extensions or the simultaneous protrusion of several cores, as may be occurring at a in Fig. 4, cannot be determined. Whereas the dimensions of a unit microextension would place it at the lower limit of light microscope visibility, composite structures, such as that at c in Fig. 4, are easily visible. This type of axiate microextension fits well into the category of the microspike as described from observations on the living cells. The rigid axial core would account for the observed uniform diameters and characteristic motions like those of a hinged rod. The fusion of unit spikes of unequal length or the extension into the same sleeve of several cores at different rates (Fig. 5, a) could explain the appearance of tapered composite microspikes, and forked extensions could result from an incomplete fusion (Fig. 4, c). A microspike would disappear rapidly after the collapse of its core by a solation process, and the formation of new spikes might occur through development of a core by rapid cytoplasmic gelation, or polymerization. Progressive addition to this structure at its base, by increasing its length, would carry outward with it a sheath of cell membrane and ectoplasm. Forming a second category of microextensions evident in the electron micrographs are those which contain no solid core. Some of these were greatly elongated and presumably were fixed while their distal ends were still anchored to the substratum (Fig. 8, a and Fig. FIGS. 8 and 9. Electron cells with retraction fibrils attached (a); detached and which may be remains of nification: x 8700.
micrographs showing portions of human conjunctiva which are without cores. Long fibrils, presumably partially collapsed fibrils ( b ); terminal enlargements fibril attachments (c). Chromium shadowed. Mag-
669
670
TAYLOR
AND
ROBBINS
9). Others appeared to have become detached from the substratum and to be collapsed and in the process of resorption (Fig. 8, b). In general these all have much less regular contours than the microspikes. Irregular swellings, sometimes present along their length, appear to contain granular cytoplasm, while the tips of some were observed to bear enlargements which might represent the remains of their terminal attachments to the substratum (Fig. 8, c, and Fig. 9, a). That these are the retraction fibrils seen in living cells is indicated by their occurrence in great numbers on cells treated with medium at pH 9 for 10 minutes before fixation, a procedure which induces retraction of cell processes (Taylor, 1962). The restless probing behavior of the microspikes as seen in living cells suggests a mechanism by which a cell, experimentally deprived of contact with solid environment, may seek out appropriate interfaces for contact relationships. Preliminary attempts were made to observe this behavior by confronting cells from primary dissociates of different chick organs. The results are still too few to warrant significant conclusions; it is notable, however, that, in electron microscopic observations of numerous encounters between embryonic liver and kidney epithelial cells, overlaps frequently were seen but there was no evidence of structural modification indicative of selective adhesions. In several cases, however, when two kidney cells confronted each other, thickenings seen at several contact points indicated the development of areas of adhesion between processes which apparently could withstand considerable retractive tension (Figs. 10 and 11). Whether these points of contact originated as microspikes has not yet been determined. DISCUSSION
Our observations on living cells have indicated that retraction fibrils are passively drawn out from the cell surface; although they may be numerous and conspicuous, they are, with respect to cell behavior, the less interesting of the two types of microextensions. Microspikes, on the other hand, despite their apparent structural simplicity and their short life span, are real cell organelles which are FIGS. 10 and 11. Electron micrographs of chick embryo kidney thickenings at points of cell-to-cell attachment. A portion of Fig. is shown at higher magnification in Fig. 11 ( X 16,800).
cells showing 10 ( x2800)
OBSERVATIONS
ON
MICROEXTENSIONS
671
672
TAYLOR
AND
ROBBINS
admirably adapted to provide for tactile exploration of and selective attachment to solid structures within the immediate fluid environment of a cell process, However, there is yet little direct evidence for assuming this function of the microspike. Such an assumption can, at this time, be based on only three facts: (1) many cells have, at least in vivo, the capacity to select for adhesion those interfaces in their environment for which an inherent contact affinity exists. (Weiss, 1941; Taylor, 1944); (2) th e structure of the microspike and its observed behavior are remarkably adapted to accomplish this function; (3) there are a few instances, here reported, in which an active microspike was observed to attach and adhere to the substratum. Evidence that this is the function of microspike activity would be greatly strengthened by the accumulation of further instances of contact and attachment between microspikes and different solid surfaces as well as other cells observed under experimental conditions. Little, however, is known regarding the conditions requisite for the expression of selective adhesion by cells which have been isolated from the organism, and a failure to demonstrate the reaction at this time may only reflect this ignorance. SUMMARY
Microextensions from the surface of tissue cells in vitro were examined by light and electron microscopy, and their activities were recorded by time-lapse motion pictures. Two types of these extensions were described on the basis of their structure and behavior. Those of one type are strands of cytoplasm spun out from the retreating borders of retracting cell processes. These retraction fib& were immobile and showed no special structure in electron micrographs. Microextensions of the second type were actively projected from the surface of the cells and, during their short existence, were in constant waving motion. High resolution pictures showed these to be supported by rigid rodlike cores. The structure and behavior of these microspikes suggest their role as organelles for selective attachment to solid surfaces in the environment. REFERENCES DEROBERTIS, E., and SOTELO, J. R. (1952). Electron microscope study tured nervous tissue. Exptl. Cell Research 3 (Suppl. 2), 433-452.
of cul-
OBSERVATIONS
ON MICROEXTENSIONS
673
GEY, G. 0. ( 1956). Some aspects of the constitution and behavior of normal and malignant cells maintained in continuous culture. Haroey Lectures Ser. 50, 154-229. LEWIS, W. H., and LEWIS, M. R. ( 1924). Behavior of cells in tissue cultures. In “General Cytology” (E. V. Cowdry, ed.), pp. 385-447. Univ. of Chicago Press, Chicago, Illinois. PORTER, K. R., CLAUDE, A., and FULLAM, E. F. (1945). A Study of tissue culture cells by electron microscopy. J. Exptl. Med. 81, 233-246. ROBBINS, E. (1961). Some theoretical aspects of osmium tetroxide fixation. J. Biophys. Biochem. Cytol. 2, 445-455. TAYLOR, A. C. (1944). Selectivity of nerve fibers from the dorsal and ventral roots in the development of the frog limb. J. Exptl. 2001. 96, 159-185. TAYLOR, A. C. ( 1962). Responses of cells to pH changes in the medium. J. Cell Biol. 15, 201-209. WEISS, P. (1941). Nerve patterns: the mechanics of nerve growth. Growth 5, 163-203. WEISS, P. ( 1961). From cell to molecule. In “The Molecular Control of Cellular Activity” (J. M. Allen, ed.), pp. l-72. McGraw-Hill, New York.