Mitotic cells and their microappendages in the primitive streak of the chick embryo.

Fresh pullet eggs (White Leghorn Strain) were incubated to the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron miscropy by means of post-osmication, critical point drying and gold-palladium coating. Cells judged to be in various stages of mitosis by their surface contours were numerous on the ventral surface of the chick blastoderm. Cells which were in the late preparatory stages for mitosis had rounded up from their surroundings. Microvilli dominated the surface. The degree of separation and number of microvilli increased until late metaphase or anaphase. Mitotic cells did not completely separate themselves from adjacent cells. Ruffles and blebs were not prominent during mitotis and long filopodia were absent. A definite localization of microappendages (microvilli, blebs, ruffles) to the area of cytokinesis was evident in early telophase and persisted through daughter cell formation.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

Role of microvilli in surface changes of synchronized P815Y mastocytoma cells

The surface morphology of synchronized P815Y mastocytoma cells has been examined by scanning electron microscopy. Early G1 cells are comparatively smooth or light villated, whereas at later stages the surface becomes progressively more villated. In G1 cell most microvilli have a uniform diameter, whereas in S and G2 cells, many microvilli show branching and often originate from much larger surface protuberances. Small "blebs" are seen on the surface of many cells but these structures do not appear to be a characteristic feature of cells at any one stage of the cycle. The presence of microvilli increases the total surface of the cell to such an extent that the ratio of volume to surface area remains constant throughout the cell cycle. The mechanism of cytokinesis is thus a physical one, involving the unfolding of previously accumulated microvilli.     

Scanning microscopy of the developing vagina in postnatal gerbils

Scanning electron microscopy of postnatally developing gerbil vagina (birth to maturity) shows that longitudinal folds form prior to transverse folds; the process of fold formation is initiated on the dorsal wall and proceeds ventrally. From days 1 to 7 postnatally, the vaginal epithelium is composed of either flat or bulging cells, depending on the vaginal region. The luminal cell surface is covered with uniform stubby microvilli and solitary cilia. Between days 9 and 20, the flat cells with distinct cell boundaries spread toward more proximal areas, leading to the formation of mixed patches of cells with flat or rounded apices. Individual elongated microvilli or tufts of forked microvilli may sprout from their surfaces. Solitary cilia gradually disappear. The transition from immature to mature vaginal epithelium starts around day 20, when individual cells recess below the level of neighboring cells. This process spreads throughout the vagina during the following days, reflecting local changes in the subsurface layers of the epithelium preparatory to exfoliation. Around day 40 the actual exfoliation of the luminal cell layer starts. By this time the surface characteristics of many of the desquamating cells have changed. In addition to microvilli, microridges are being formed. The process of exfoliation is finished by about day 60. The newly appearing cell layers now transform into typical cornified cells of the cycling vaginal epithelium.     

Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine.

Dividing epithelial cells in the mouse small intestine were examined by thin-section electron microscopy with special attention given to the mode of cytokinesis. As the columnar epithelial cells entered mitosis in the crypt, they became rounded, maintaining their junctional complexes with neighboring cells while detaching themselves from the basal lamina. In such rounded cells the mitotic apparatus was formed with its long axis parallel to the luminal surface. Replicated centrioles moved down from the apical region to locate themselves lateral to the nucleus, where they served as the poles of the mitotic spindle. During mitosis the cell retained microvilli on its luminal surface, though the terminal web became much thinner. At telophase the formation of a cleavage furrow proceeded asymmetrically from the basal side alone, and thus the contractile ring which was prominent at the base of the furrow, merged with the terminal web. Eventually, an intercellular bridge with a midbody was formed on the luminal surface. The space in the furrow was occupied by the flattened cytoplasmic processes of the neighboring cells. The tight junction was also seen on the basolateral surface of the intercellular bridge with the underlying neighboring cells. At very late telophase the intercellular bridge was disconnected from the neighboring cells and protruded into the lumen. These observations have led us to propose a mode by which the simple columnar epithelium maintain the tight junctional seal during cell division in the crypt of the small intestinal epithelium.     

Studies on corneal endothelial growth and repair. IV. Changes in the surface during cell division as revealed by scanning electron microscopy

Changes in the surface morphology of regenerating rabbit, rat and frog corneal endothelial cells in vivo have been investigated by scanning electron microscopy. In adult tissue these cells do not normally divide unless given a stimulus, such as injury. Surfaces of quiescent rabbit and rat cells are devoid of microvilli but display globular projections and surface pits up to 300 nm in diameter. However, regenerating endothelia are characterized by the appearance of microvilli which attain their greatest length when the cells are rounded. At this stage, cells also possess filopodia and broad processes. In cytokinesis, the microvilli have shortened and blebs and ruffles appear for the first time. In contrast to rabbits and rats, frog endothelial cells of noninjured tissue are covered by microvilli and smaller surface pits of 60-70 nm diameters. During regeneration, these cells have reduced numbers of microvilli and extensive foldings of the membrane. Neither blebs nor filopodia occur during the mitotic cycle and ruffles are not detected until cytokinesis.     

Cytocortical organization during natural and prolonged mitosis of mouse 8-cell blastomeres

Late 8-cell blastomeres were harvested within the first 45 min after entering mitosis. Some mitotic cells were analysed within the ensuing 2 h for the organization of their surface in relation to their progress through mitosis. Whereas in most late interphase cells microvilli were restricted to a discrete polar region, in mitotic cells at all stages from early metaphase to immediately postcytokinesis microvilli were found to be present over more of the cell surface. Other mitotic cells were placed in nocodazole to arrest them in M-phase for up to 10 h. They were found to show an even more extensive distribution of microvilli over the whole surface, the longer periods of incubation yielding more extended coverage such that many cells no longer appeared to have any residual surface polarity. Removal from nocodazole at all time points from 1 to 10 h resulted in most cells completing mitosis to yield pairs of cells which, in most cases, resembled pairs derived from nonarrested blastomeres and in which a defined polar area of microvilli was restored. However, the percentage of differentiative divisions decreased after 6 h arrest. If, instead of removing cells from nocodazole, they were placed in both nocodazole and cytochalasin D (CCD) for periods of up to 3 h, most microvilli retracted to reveal a tight polar zone of CCD-resistant microvilli. This result suggests that a heterogeneity of cytocortical organization may still exist within the arrested mitotic cell. We propose a model to explain the origin of this heterogeneity of organization and its relationship to the generation of cell diversity.     

CHANGES IN SURFACE MORPHOLOGY OF CHINESE HAMSTER OVARY CELLS DURING THE CELL CYCLE

Synchronized populations of Chinese hamster ovary (CHO) cells in confluent culture have been examined by scanning electron microscopy and their surface changes noted as the cells progress through the cycle. During G₁ it is characteristic for cells to show large numbers of microvilli, blebs, and ruffles. Except for the ruffles, these tend to diminish in prominence during S and the cells become relatively smooth as they spread thinly over the substrate. During G₂ microvilli increase in number and the cells thicken in anticipation of rounding up for mitosis. It appears that the changes observed here reflect the changing capacity of CHO cells during the cycle to respond to contact with other cells in the population, because, as noted in the succeeding paper (Rubin and Everhart), CHO cells in sparse nonconfluent cultures do not show the same wide range of changes during the cell cycle. Normal, nontransformed cells of equivalent type in confluent culture are essentially devoid of microvilli, blebs, and ruffles. The relation of these surface configurations to the internal structure of the cell is discussed.     

Retention of lens specificity in long-term cultures of diploid rabbit lens epithelial cells

The ventral surface of the deep layer of gastrulating quail and chick embryos was examined using scanning electron microscopy. On the basis of cell protrusions, three or four different cell types were recognized. Cells covered with microplicae were found in the caudal region of the germ and as a narrow band extending along the lateral and anterior borders of the area pellucida. Cells covered with microvilli were found in a horseshoe-shaped zone in the anterior part of the germ. Beneath the rostral end of the primitive streak, the flattened deep-layer cells exhibited intercellular ridges and few microvilli. This area was surrounded by cells that usually had extended microvilli. The pattern of these cell types is discussed in relation to the formation of the different tissues that compose the deep layer in gastrulating embryos.     

Changes in the surface and cytoskeletal apparatus of the endotheliocytes of the rat aorta during division (based on scanning electron microscopic data)

Endothelium of the abdominal aorta of 32 KWR-line rats was exposed to freeze injury. En face preparations were made to look for endothelial cells (EC) on different stages of mitosis. Specimens were dried by critical point technique, and the grid was placed on their surface. Then specimens were investigated in succession with light and scanning electron microscopes. The cytoskeleton of EC was investigated on detergent-extracting preparations. It is shown that the end of phase S of the cell cycle and the beginning of prophase are characterized with the lifting of the nuclear-containing zone. Fine microvilli appear on the EC surface during prophase. The cytoskeleton becomes more structured and polarized. During metaphase, EC becomes spherical, its microvilli are shortened. Fine cytoplasmic shoots are seen to extend from the cell poles to the substratum. The density of the fibrillar structures swiftly rise. During anaphase the EC surface is covered with blebs. During late telophase the surface of dividing EC becomes flatter. Their nuclei are connected with rare bundles of fibrillar structures. Mechanisms of EC surface changes during mitosis and the role of cytoskeletal elements are discussed.     

Study of the surface of cytolytic T-lymphocytes by scanning electron microscopy

Scanning electron microscopy revealed three types of cytolytic T lymphocytes (CTL) interacting with target cells (TC). The type I cells occurred as smooth spherical lymphocytes with single microvilli; the type II rounded or oval cells were uniformly covered with microvilli; the type III cells were marked by an irregular shape, were densely covered with microvilli, while their surface was folded or tuberous. Within the first several minutes after absorption the surface of TC was largely covered by the type I lymphocytes. The proportion of the type III cells rose with the time of interaction. At the beginning it was 8-9%, reaching 30-71% after 30-60 minutes of incubation. It is assumed that the 3 types of the cells described mirror 3 stages of CTL activation. The increase in number of microvilli and appearance of the membranous folds may be a consequence of exocytosis and incorporation of the membrane of secretory granules into the plasma membrane of lymphocytes. The data obtained support the authors' assumption about the secretory mechanism of the action of CTL, whose contact with TC stimulated secretion activation.     

Exocytosis in human salivary glands visualized by high-resolution scanning electron microscopy

The luminal membrane of salivary acinar cells creates a specialized cell surface area that accepts exocytosis and undergoes dynamic changes during secretion. These changes were visualized three-dimensionally from both the inside and outside of the cell in human parotid and submandibular glands, by application of in vitro secretory stimulation and then of OsO₄ maceration to remove cytoplasmic organelles by varying degrees. In control glands treated without secretagogues, the luminal surface of serous acinar cells bore well-developed microvilli with only an occasional incidence of exocytotic profiles. Following treatment with the β-adrenergic agonist, isoproterenol, considerable shortening and loss of microvilli occurred along the luminal membrane where, on its cytoplasmic side, many protuberances of sizes similar to or smaller than those of single secretory granules (～1 μm in diameter) appeared. The cytoplasmic surface of these protuberances exhibited small vesicles (～100–150 nm in diameter) that, by transmission electron microscopy, were shown to be coated pits or vesicles present on or around the exocytosed granule membranes. Treatment of tissues with the muscarinic agonist carbachol also caused a decrease of microvilli and the appearance of protrusions at the luminal membrane. However, unlike isoproterenol treatment, many of these protrusions were devoid of small pits or vesicles and were much larger than a single secretory granule. These results indicate that (1) secretory stimulation causes the dynamic transformation of microvilli at the luminal membrane, where granule docking and membrane fusion take place, and (2) after fusion, the exocytosed membranes are processed differently, by coated pit/vesicle mediated or non-mediated mechanisms, according to the autonomic receptor control.     

The protein kinase kin1, the fission yeast orthologue of mammalian MARK/PAR-1, localises to new cell ends after mitosis and is important for bipolar growth

The kin1 protein kinase of the fission yeast Schizosaccharomyces pombe is a member of the PAR-1/MARK (partitioning-defective 1/microtubule-associated protein/microtubule affinity-regulating kinase) family important in eukaryotic cell polarity and cytoskeletal dynamics. We show here that kin1 plays a role in establishing the characteristic rod-shaped morphology of fission yeast. Cells in which kin1 was deleted are viable but are impaired in growth, and are rounded at one end or both ends. They are monopolar because after mitosis they fail to activate bipolar growth, and are delayed in cytokinesis, resulting in a high proportion of septated cells often with multiple septa. This phenotype can be partially rescued by heterologous expression of human MARKs, which restore bipolar growth in most cells, but do not correct the delay in cytokinesis. Using chromosomal epitope tagging, we show that kin1p localises to the cell ends, except during mitosis when it disappears from cell ends. After mitosis, kin1p first reappears at the new cell end. Overexpression of kin1 results in a loss of polarity, with partially or fully rounded cells. From these results we suggest that kin1 is required to direct the growth machinery to the cell ends.     

Surface morphology and agglutinability with concanavalin A in normal and transformed murine fibroblasts

The surface morphology of attached and suspended normal and transformed fibroblasts has been studied with the scanning electron microscope. Normal murine fibroblasts (3T3) grow in vitro with widely extended leading lamellae. During most parts of the cell cycle the surfaces of these cells are practically free of microvilli. When the cells round up for mitosis, their cell surfaces become adorned with many microvilli. In contrast, simian virus 40-transformed fibroblasts (SV3T3) grow more compact, and their cell surfaces remain smooth throughout the life cycle. When confluent 3T3 and SV3T3 cells are suspended with ethylenediaminetetraacetic acid (EDTA) for agglutination assays, similar differences in surface morphology are found: 3T3 cells always bear many microvilli, whereas most SV3T3 cells are essentially free of microvilli. The addition of concanavalin A (Con A) does not influence the surface morphology of the suspended cells. The morphological differences described here may be important for the agglutination process of the normal and transformed 3T3 cells, because they affect the real cell surface area and thus the density of Con A-binding sites.     

Changes in the cell surface and cytoskeleton of A-431 cells under the action of epidermal growth factor

Certain changes in human carcinoma A-431 are found by scanning electron microscopy. The early cell response to growth factor (after 10 minutes) involves a disappearance of microvilli, an appearance of ruffles and rounded cells, along with a decrease in cell attachment to the substrate. The cell surface changes correlate with the state of cytoskeleton elements: the material stained with iron hematoxylin is accumulated in ruffle formation sites. Retractional fibrils filled with the cytoskeleton material result from a decrease in the cell area.     

Cell Surface Morphology After Trypsinisation Depends on Initial Cell Shape

Cells growing in tissue culture exhibit constant variation in shape and surface morphology, particularly during the process of mitosis, where the cell rounds up exhibiting an intensely microvillous surface prior to cytokinesis. During routine subculturing, cells are induced to round up and relinquish contact with the substratum. Although the cells retain their viability throughout trypsinisation, their surface morphology demonstrates a variety of changes between finger-like microvillous projections, and spherical protruberances termed blebs.
The reaction of individual cells to cell rounding, in the presence of trypsin appears to be dependent on cell shape, which may be modulated naturally or altered by experimental agents. Cells of bipolar morphology, termed fibroblasts, produce a blebbed surface morphology in response to trypsin, whereas isometric, 'epithelioid' cells respond by the formation of a microvillous cell surface.
Blebbed cells subsequently undergo membrane reorganisation towards a more organised, and more permanent microvillous cell surface, even in the continued presence of trypsin. Naturally occurring spherical cells, for example, mitotic or suspension cultures, are microvillous and trypsin has no effect on their surface morphology. It would appear that blebs are the cells response to experimentally induced rapid change of shape of well spread cells, and thus represent a pathological response for prevention of membrane loss in conditions which produce a rapid assumption of a minimum surface area configuration, i.e. a sphere, which occurs too quickly for membrane resorption, or normal storage in the form of microvilli.     

Scanning Electron microscopic Study on the Early Development of Silkworm Eggs (Bombyx mori L.)

Morphological changes of the surface of eggs of the silkworm Bombyx mori L. were studied during early developmental stages by scanning electron microscopy. The egg surface was covered with numerous microvilli at least until 4 h after oviposition. At 6 h the microvilli were replaced by ruffle-like microprojections. This suggests that developmental changes of the surface structure may occur without direct influence of cleavage nuclei. Immediately before blastoderm cell formation, microvilli reappeared in the presumptive groove area. The ruffles seen on the apical portion of newly-formed blastoderm cells gradually became flattened, while microvilli developed on the lateral side of the cells. The mode of blastoderm cell formation is different from the typical one seen in most species of insects.     

Surface and shape changes during cell division

Summary Rat kangaroo cells (PtK₂) were studied with scanning and transmission electron microscopy in order to correlate shape changes during the cell cycle with the presence or absence of microvilli and stress fibers. During interphase, bundles of actin are prominent in the cytoplasm, and microvilli are localized over and around the centrally positioned nucleus. As mitosis begins, the interphase bundles of actin and the microvilli disappear, but the mitotic cells maintain a flattened shape. At metaphase the cell is still so flat that both the chromosomes and spindle apparatus are visible through the intact cell membrane. Microvilli reappear in late anaphase above the chromosomes and poles. Before cleavage begins, microvilli increase in number until they cover the apical surface of the cell. At the same time, the cell increases in height so that the chromosomes and mitotic apparatus can no longer be detected through the cell membrane. During cleavage, microvilli continue to cover the cell in a uniform manner but become greatly diminished in number after cytokinesis is completed and the cells flatten and enter interphase. It is suggested that the microvilli organize a network of actin filaments which interact with cortical myosin to produce the cell rounding prior to cleavage.     

Characteristics of the upper cell surface in cultures of normal and SV-40 virus-transformed epithelium of mouse kidney. Study with the aid of scanning electron microscopy]

Central cells of the normal epithelial sheet are sparsely covered by microvilli. Numerous microvilli were seen in the regions of intercellular contacts. Marginal cells of sheets had a finely developed lamellar cytoplasm (lameloplasm) with smooth upper surface at their free margins. A transformed cell line (MPTR) resembled normal parent cells by its ability to form monolayered sheets in cultures. More microvilli of increased length appeared on the upper surface of central MPTR cells. The normal structure of lamelloplasm was changed at the free edge of the MPTR sheets. It is suggested that abnormal cell attachment to the substratum may be responsible for the altered cell surface morphology (increased length of microvilli, defective, structure of lamelloplasm) in the MPTR cultures.