On the flexibility of surface structures of tumor cells in vitro

When HeLa cells are detached from their support by trypsin, trypsin-EDTA, or by mechanical means large zeiotic blebs are formed. After reseeding the cells onto glass these blebs shrink. Those blebs near the support collapse completely to form lobes in the neighborhood of lobopodia. By scanning and transmission electron microscopy we could show the transformation of these zeiotic blebs into true lobopodia. This implies a repair of cell surface structures and components as well as of the subcortical cytoskeleton.     

Freeze-fracturing and surface labelling of embryonic neural retina cells

Freeze-fracturing of dissociated and aggregating neural retina cells from 7-day chick embryos revealed on the inner faces (PF) of the cell membrane numerous particles 6–20 nm in size. In contrast, the PF faces of blebs and some of the lobopodia that project from the cell surface were practically devoid of such particles. However, the elongated filopodia that abound on these cells showed numerous particles on their PF faces. These regional differences in the distribution of particles on PF faces of these cells are interpreted as reflecting membrane activity that leads to the formation of blebs and lobopodia. The frequent presence of “pits” at the basis of blebs and lobopodia is described. It is suggested that the “pits” are associated with the formation of these membrane projections; they may represent anchoring sites for microfilaments and for microtubules involved in the dynamic structure of the cell surface. ConA-binding sites on these cells were studied by scanning electron microscopy, using labeling with hemocyanin. The distribution of these sites on different regions of the cell surface coincided with the regional differences in the distribution of the inner membrane particles.     

Surface activity and locomotion of Fundulus deep cells during blastula and gastrula stages

The surface activity and locomotion of deep cells of the Fundulus blastoderm were studied in vivo with time-lapse cinemicrography. During late cleavage, the surfaces of the blastomeres begin to undulate gently. By early blastula, these undulations increase gradually in amplitude and hemispherical surface protrusions called blebs appear. These blebs form and retract rapidly, and at mid blastula some may be seen adhering to the surfaces of other cells. At the same time, they often expand into elongate lobopodia. Cell locomotion is first evident in mid blastula and continues throughout gastrulation. During locomotion, the leading edge of a deep cell behaves in various ways. When blebs and lobopodia adhere to a substratum (other deep cells, the undersurface of the enveloping layer, or the periblast) and retract, the cell may move in the direction of the shortening cell process. Alternatively, blebs and lobopodia may adhere, but not shorten. Locomotion is accomplished rather by protoplasmic flow into the protrusion. Blebs and lobopodia also may flatten and spread on the substratum as lamellipodia. Variations in the contact and locomotory behavior of deep cells and in the rate of their movement during blastula and gastrula stages are described in detail.     

The reversible action of alpha-amanitin on nuclear structure and molecular composition

The effects of brief trypsin treatment on cell shape, surface topography, cytoskeletal components and the freeze-fracture distribution of plasma membrane intramembranous particles (PMP) are examined in contact-inhibited 3T3 cells. Untreated 3T3 cells are flat without many surface modifications, have highly organized microfilaments and microtubules and have clusters of PMP. Within seconds of trypsin treatment using a concentration of trypsin used for routine passage numerous zeiotic blebs appear on the cell surface which appear to “cap” or coalesce on the central superior aspect of the cell. In later stages retraction fibers are observed and the cells then round up and detach from the substrate. Using thin section transmission electron microscopy (TEM) the zeiotic blebs are observed and various constituents of the cytosol, including lysosomes, ribosomes, etc. pinch off and are surrounded by plasma membrane. In general there is a profound disruption of microfilaments and cortical microtubules. There is, however, a focal coalescence of microfilaments subjacent to the zones of the “capped” zeiotic blebs and some relative resistance of centriolar associated microtubules to tryptic degradation. Trypsin also causes profound changes in the nucleus which transforms from the normal round shape to a very convoluted and irregular shape. In addition to the zeiotic bleb formation which can be visualized by freeze-fracture the distribution of PMP goes from the normally clustered state to a more uniform distribution following treatment with trypsin. These studies suggest that there may be some coordinated control of the changes in cell surface topography, cytoskeletal components and intramembranous particle distribution.     

MOTILITY AND LOCOMOTION OF EMBRYONIC CELLS OF THE MEDAKA, ORYZIAS LATIPES, DURING EARLY DEVELOPMENT

The motility and locomotion of embryonic cells of the medaka, Oryzias latipes , were studied in situ with time-lapse cinematography.
In the early morula (128-cell stage), the surface of the blastomeres begins to undulate gently. By the early blastula stage, these undulations increase gradually in amplitude, and blebs appear. These blebs protrude and retract rapidly. In the mid-blastula stage they are found in most of the blastomeres. Some are found adhering to the surfaces of other cells. Blebs often expand into elongate lobopodia. Cell locomotion is first evident in the mid-blastula stage and continues throughout gastrulation and afterward. The cells move in the direction of the protrusion. In the late blastula a number of blastomeres locomote in random directions. In the thickening stage, when blastoderm epiboly begins, the cells with lamellipodia or elongate filopodia increase gradually in number, and in the early gastrula most cells change into this form. The motility, rate of movement, and mode of locomotion of embryonic cells during early development are described in detail.     

A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts.

We suggest a method of quantitating the motile actions of surface protrusions in spreading animal cells in culture. Its basis is the determination of the percentage of freshly plated cells which produce particle-free areas around them on a gold particle-coated glass cover slip within 50 min. Studying 3T3 cells with this assay, we found that the presence of Na+, K+, Cl-, and Mg++ or Ca++ in a neutral or slightly alkaline phosphate or bicarbonate buffered solution is sufficient to support the optimal particle removal by the cells for at least 50 min. Two metabolic inhibitors, 2,4-dinitrophenol and Na-azide, inhibit the particle removal. If D-glucose is added along with the inhibitors, particle removal can be restored, whereas the addition of three glucose analogues which are generally believed to be nonmetabolizable cannot restore the activity. Serum is not required for the mechanism(s) of the motile actions of surface protrusions in spreading 3T3 cells. However, it contains components which can neutralize the inhibitory actions of bovine serum albumin and several amino acids, particularly L-cystine or L-cystein and L-methionine. Furthermore, serum codetermines which of the major surface extension, filopodia, lamellipodia, or lobopodia, is predominantly active. We found three distinct classes of extracellular conditions under which the active surface projections are predominantly either lamellipodia, (sheetlike projections), lobopodia (blebs), or filopodia (microspikes). The quantitated dependencies on temperature, pH and the inhibition by cytochalasin B or the particle removal are very similar in all three cases. Preventing the cells from anchoring themselves for 15-20 min before plating in serum-free medium seems to stimulate particle removal threefold.     

Ultrastructural features of rectal epithelium of the mouse during the early phases of migration to repair a defect

Observations were made by scanning and transmission electron microscopy on the migrating epithelial cells of the mouse rectum at intervals up to 24 h after stripping the epithelium off the mucosa. Resurfacing of the denuded basal lamina proceeded by the centrifugal migration of the columnar cells of the crypts. Changes in these cells occurred very rapidly. In less than 20 min a flat leading lamella developed and extended out on the basal lamina. The leading lamella could be recognized easily in scanning electron micrographs by the absence of microvilli, although these were retained on the cell body, gradually getting less regular and sparser than normal. Many zeiotic blebs appeared on the free margin of these cells. The features of migrating epithelium which are displayed in the in vivo repair of rectal mucosa are shared with migrating epithelia cultured in vitro. Goblet cells appeared not to be active in resurfacing the lesions. They disappeared from the surface epithelium, but were evident again by 18 and 24 h. The method of producing these lesions can also be used to study the cells that are removed.     

Circus movement in dissociated embryonic cells of a teleost, Oryzias latipes.

The dissociated early embryonic cells of the fresh water fish, Oryzias latipes, protrude hyaline lobopodia, which tend to rotate around the cell circumference in a propagating wave. Cells from late blastula or gastrula continuously show this "circus movement", while most cells up to early blastula are rounded. The linear velocity of the lobopodium was estimated by means of time-lapse cinemicrography. The velocity increases slightly as cell diameter increases. The effects of pH, temperature and osmotic pressure of the immersion media on the movement were also quantitatively investigated. Cells become rounded and do not form lobopodial blebs when immersed in media below pH 5. The velocity is reduced by decreasing temperature, but the movement continues even at 5 degrees C. Cells placed in hypertonic salt solutions become crenated and do not continuously demonstrate the circus movement.     

Plasma membrane specializations in resting, stimulated and phagocytosing arthropod (Limulus polyphemus, Gromphadorhina portentosa and Blattella germanica) immunocytes: structural and functional analogies with those of vertebrate macrophages and neutrophils

Using indirect immunofluorescence and scanning electron microscope techniques, we have shown that the plasma membrane specializations, such as pseudopods, filopods, lamellipods, and zeiotic blebs occur in activated and/or phagocytosing arthropod (Limulus polyphemus, Gromphadorhina portentosa, and Blattella germanica) immunocytes (hemocytes), as they do in challenged vertebrate neutrophils and macrophages. All four specializations may also be caused by some of the chemicals in the preparative media, which suggests that arthropod immunocytes, like the vertebrate neutrophils and macrophages, have evolved as highly reactive cells that are sensitive to chemical and/or biological foreign agonists/antigens. These attributes are necessary for the effectiveness of a cellular defense mechanism. The plasma membrane specializations perform the same functions in arthropod immunocytes as reported for those of vertebrates. For example, pseudopods and lamellipods are needed for spreading and locomotion and filopodia for cell adhesion and crawling. Because they are formed as a result of similar reactions to foreign antigens, as in vertebrate cells, it is suggested that similar terminologies be consistently used for them in arthropods.     

Remnant motility of macrophages treated with cytochalasin B in the presence of colchicine

We have previously observed that mouse peritoneal macrophages cultured for 48 h and treated with colchicine to depolymerize cytoplasmic microtubules become ameboid and cease to migrate by gliding on the substratum. We have now found that when such cells were further exposed to both colchicine and cytochalasin B, the induced ameboid movements were reversibly inhibited. Cells treated concomitantly with both drugs did not become motionless, but exhibited a remnant motility that took the form of zeiosis (blebbing). The zeiotic blebs contained ribosomes and fibrous material, but lacked organized microfilament arrays and rarely included other cytoplasmic organelles. Zeiosis appears to be a form of surface movement independent both of cytoplasmic microtubules and of the cytochalasin-sensitive contractile system. These observations imply an additional mechanism that can reversibly alter the form of the cell.     

Cell migration: fibroblasts find a new way to get ahead

Fibroblasts migrate on two-dimensional (2D) surfaces by forming lamellipodia-actin-rich extensions at the leading edge of the cell that have been well characterized. In this issue, Petrie et al. (2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201201124) show that in some 3D environments, including tissue explants, fibroblasts project different structures, termed lobopodia, at the leading edge. Lobopodia still assemble focal adhesions; however, similar to membrane blebs, they are driven by actomyosin contraction and do not accumulate active Rac, Cdc42, and phosphatidylinositol 3-kinases.     

Cell‐to‐cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP‐rich parasitophorous extrusions

The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long‐term‐infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. The extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro‐irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non‐irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system.     

Electron microscopic study of rapid morphogenetic processes in embryonic tissue explants of amphibia]

Submicroscopic rearrangements are described. They serve as the bases of rapid (up to 20 min) changes in the form of the common frog neurula explants: formation of filamentous layer under "naked" surface, appearance of lobopodia on "naked" surface, their "flow", cell polarization and submersion. In all these processes an active part appears to be played by microtubules and microfilaments the bundles of which are always oriented along the long axes of active cells or the directions of passive mechanical tensions. In the cells which are not yet polarized the microtubules form under the surface adjacent to the already polarized cell. This may be considered as one of the chains of cooperative cell polarization.     

Membrane tether formation from blebbing cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.     

MOTILITY OF DISSOCIATED EMBRYONIC CELLS IN XENOPUS LAEVIS: ITS SIGNIFICANCE TO MORPHOGENETIC MOVEMENTS *

Motilities of dissociated embryonic cells of Xenopus laevis were investigated in order to elucidate the role of cell motilities in gastrulation. Various shapes and motile forms of the cells were classified into six types, i.e., globular cells with large lobopodia, locomotive vermiform cells with a hyaline cap, globular cells with many bulbous processes, oval cells with filiform pseudopodia, flattened cells with membraneous processes or lamellipodia which attached to glass, and attached cells with hyaline lobopodia. Cells dissociated from the ectodermal region began to exhibit pseudopodial activity at stage 11, while isolated mesoderm cells did so at stage 10 1/2. The pseudopodial activity of cells from these two regions increased coincidently with gastrulation. Approximately 10% of the cells isolated from the dorsal part of the neurula transformed into vermiform cells. Cells dissociated from the endodermal region were less motile during gastrulation. Invaginating cells of the presumptive pharyngeal endoderm were also immotile, when they were isolated.     

The ultrastructure of blebs induced in the hamster jejunum by ethanol.

Previous light microscopic studies showed that perfusion of the hamster jejunum with 4.8% ethanol (ethanol period) in vivo produced fluid-filled subepithelial blisters (blebs) on the villi. These blebs had virtually disappeared within 45 min after the discontinuation of the ethanol perfusion (recovery period). The present study examined these ethanol-induced changes in the jejunum by scanning (SEM) and transmission (TEM) electron microscopy. TEM revealed that ethanol did not damage epithelial cells in areas where blebs were not present. In these areas the basal surfaces of the epithelial cells were attached to the basal lamina, and the lateral intercellular spaces (LIS) were open. In the areas where blebs formed, the stretched epithelial cells which covered the blebs lost their basal anchoring and so could not maintain their LIS. Both SEM and TEM indicate that there was a decrease in the quantity of glycocalyx at the surfaces of cells which covered blebs. Our findings indicate that ethanol does not directly damage epithelial cells but that the cellular damage is due to detachment over the blebs. It is likely that ethanol at first traverses the epithelial layer and then produces stasis in the villus core. Continued fluid transport by the epithelial layer in the presence of statis results in accumulation of the fluid and widely dilated LIS. With subsequent enlargement of the LIS the bases of the cells detach from the basal lamina and blebs are formed.     

Myosin II and Arp2/3 cross-talk governs intracellular hydraulic pressure and lamellipodia formation

Human fibroblasts can switch between lamellipodia-dependent and -independent migration mechanisms on two-dimensional surfaces and in three-dimensional (3D) matrices. RhoA GTPase activity governs the switch from low-pressure lamellipodia to high-pressure lobopodia in response to the physical structure of the 3D matrix. Inhibiting actomyosin contractility in these cells reduces intracellular pressure and reverts lobopodia to lamellipodial protrusions via an unknown mechanism. To test the hypothesis that high pressure physically prevents lamellipodia formation, we manipulated pressure by activating RhoA or changing the osmolarity of the extracellular environment and imaged cell protrusions. We find RhoA activity inhibits Rac1-mediated lamellipodia formation through two distinct pathways. First, RhoA boosts intracellular pressure by increasing actomyosin contractility and water influx but acts upstream of Rac1 to inhibit lamellipodia formation. Increasing osmotic pressure revealed a second RhoA pathway, which acts through nonmuscle myosin II (NMII) to disrupt lamellipodia downstream from Rac1 and elevate pressure. Interestingly, Arp2/3 inhibition triggered a NMII-dependent increase in intracellular pressure, along with lamellipodia disruption. Together, these results suggest that actomyosin contractility and water influx are coordinated to increase intracellular pressure, and RhoA signaling can inhibit lamellipodia formation via two distinct pathways in high-pressure cells.     

Surface configuration of mesothelial cells in effusions. A comparative light microscopic and scanning electron microscopic study.

Surface configuration of mesothelial cells identified by light microscopy (LM) has been studied by scanning electron microscopy (SEM). It has been shown that mesothelial cells may have a variable SEM appearance. The surfaces of a small proportion of mesothelial cells are covered by regular microvilli (MV) and show openings of the pinocytotic vesicles. The surfaces of the majority of these cells are covered by vesicles or blebs. An intermediate population of mesothelial cells, i.e., cells displaying side-by-side blebs and MV, has also been observed. The latter cells no longer display pinocytotic vesicles. Occasional mesothelial cells have smooth surfaces. It has been shown by LM and transmission electron microscopy that cells with blebs are viable and capable of mitotic activity. It is concluded that mesothelial cells, detached from their epithelial setting, lose microvilli and pinocytotic vesicles and acquire surface blebs. The possible relationship between mesothelial cells and macrophages based on surface features has been discussed.     

A Model of Blebbing in Mitotic Tissue Culture Cells

A preliminary study of blebbing in tissue cultures has been made. The tubal epithelium of fetal mouse oviduct was cultured at 37°C in Rose chambers. A cinematographic record was obtained of phase microscope observations of mitotic cells. Measurements of the size of both cells and blebs were made on the film using a “traveling” microscope. The duration and the rise and decay times of blebs were determined simply by counting frames on the film. Detailed observations are reported on blebbing in four cells undergoing mitosis. The results indicate that the frequency of blebbing as well as the duration of individual blebs exhibits a maximum during telophase. A model is proposed to account for blebbing in mitotic cells. The model attributes to local regions of the cell membrane the property of constant tension independent of stretch over some restricted range of stretch. This property implies that the cell membrane is locally unstable. Further assumptions stated explicitly in the model are that (i) cell division occurs at constant volume, (ii) the cell membrane stretches during cleavage, (iii) there is a positive pressure drop across the cell membrane. Evidence is cited in support of these assumptions as well as independent evidence that the cell membrane may be locally unstable. A physical model is described which would be expected to exhibit blebbing given the above assumptions.     

Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing

Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism.