A scanning electron microscope study of early sea urchin reaggregation

Sea urchin embryos were observed with SEM during the first 2 h of reaggregation, following dissociation of the 16-cell stage. A dense meshwork, composed of elongated microvilli embedded in the hyaline layer, surrounds the egg during early development. The dissociation procedure strips off some of the meshwork layer leaving fewer and smaller microvilli on the cell surface. Shortly after reaggregation has begun, several types of cell extensions are formed, including filopodia, which anchor the cells to the substrate, and ruffles and pseudopods, which enable the cells to move. Possible factors involved in the behavior of dissociated cells are discussed with regard to (1) the source of additional membrane in the formation of new cell extensions; (2) the ability of the cells to move.     

Microscopic observations on the filopodia of Entamoeba histolytica.

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter micrometer thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 micrometers in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called "surface-active lysosomes with trigger," "dendritic plasmalemmal extensions," and "extra-amebic vesicles" by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.     

Microscopic Observations on the Filopodia of Entamoeba histolytica*

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter m? thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 pan in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called “surface-active lysosomes with trigger,”“dendritic plasmalemmal extensions,” and “extra-amebic vesicles” by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.     

Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella

Filopodia are rodlike extensions generally attributed with a guidance role in cell migration. We now show in fish fibroblasts that filopodia play a major role in generating contractile bundles in the lamella region behind the migrating front. Filopodia that developed adhesion to the substrate via paxillin containing focal complexes contributed their proximal part to stress fiber assembly, and filopodia that folded laterally contributed to the construction of contractile bundles parallel to the cell edge. Correlated light and electron microscopy of cells labeled for actin and fascin confirmed integration of filopodia bundles into the lamella network. Inhibition of myosin II did not subdue the waving and folding motions of filopodia or their entry into the lamella, but filopodia were not then integrated into contractile arrays. Comparable results were obtained with B16 melanoma cells. These and other findings support the idea that filaments generated in filopodia and lamellipodia for protrusion are recycled for seeding actomyosin arrays for use in retraction.     

Vesicle traffic through intercellular bridges in DU 145 human prostate cancer cells

We detected cell-to-cell communication via intercellular bridges in DU 145 human prostate cancer cells by fluorescence microscopy. Since DU 145 cells have deficient gap junctions, intercellular bridges may have a prominent role in the transfer of chemical signals between these cells. In culture, DU 145 cells are contiguous over several cell diameters through filopodial extensions, and directly communicate with adjacent cells across intercellular bridges. These structures range from 100 nm to 5 microm in diameter, and from a few microns to at least 50-100 microm in length. Time-lapse imagery revealed that (1) filopodia rapidly move at a rate of microns per minute to contact neighboring cells and (2) intercellular bridges are conduits for transport of membrane vesicles (1-3 microm in diameter) between adjacent cells. Immunofluorescence detected alpha-tubulin in intercellular bridges and filopodia, indicative of microtubule bundles, greater than a micron in diameter. The functional meaning, interrelationship of these membrane extensions are discussed, along with the significance of these findings for other culture systems such as stem cells. Potential applications of this work include the development of anti-cancer therapies that target intercellular communication and controlling formation of cancer spheroids for drug testing.     

Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C _m = 1.9 µF/cm². In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C _m values of 3.7–4.0 µF/cm², which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.     

The Dictyostelium discoideum 30,000-dalton protein is an actin filament- bundling protein that is selectively present in filopodia

The interaction with actin and intracellular localization of the 30,000-D actin-binding protein from the cellular slime mold Dictyostelium discoideum have been investigated to analyze the potential contributions of this protein to cell structure and movement. The formation of anisotropic cross-linked filament networks (bundles) containing actin and the 30,000-D protein has been observed by electron microscopy, light scattering, viscometry, and polarization microscopy. Cosedimentation experiments indicate that a maximum of one molecule of the 30,000-D protein can bind to 10 actin monomers in filaments with an apparent association constant of 1 X 10(7) liters/mol. Inhibition of the interaction of the 30,000-D protein with actin by either magnesium or calcium was observed by viscometry, light scattering, polarization microscopy, and direct binding assays. However, the concentration of magnesium required to diminish the interaction is greater than 100 times greater than that of calcium. The association constant of the 30,000-D protein for actin is 4.2 X 10(6) liters/mol, or less than 1 X 10(5) liters/mol in the presence of increased concentrations of either Mg2+ or Ca2+, respectively. Enzyme-linked immunoassays indicate that the 30,000-D protein comprises 0.04% of the protein in D. discoideum. Extensive interaction of the 30,000-D protein with actin in cytoplasm is predicted from these measurements of the concentration of this protein and its affinity for actin. The distribution of the 30,000-D protein was analyzed by immunofluorescence microscopy using mono-specific affinity-purified polyclonal antibody. The 30,000-D protein exhibits a diffuse distribution in cytoplasm, is excluded from prominent organelles, and is quite prominent in fine extensions protruding from the cell surface. The number, length, and distribution of these extensions containing the 30,000-D protein are similar to those of filopodia observed by scanning electron microscopy. To analyze the effects of cell thickness and the distribution of organelles on the immunofluorescence localization, fluorescein-labeled BSA was incorporated into the cytoplasm of living cells before fixation and staining using a sonication loading technique. The results indicate that the 30,000-D protein is selectively incorporated into filopodia. These results provide a clear distinction between the multiple actin-cross-linking proteins present in D. discoideum, and suggest that the 30,000-D protein contributes to organization of bundles of actin filaments in filopodia.     

Scanning electron microscopy of attached trophozoites of pathogenic Entamoeba histolytica

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2-8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

Imaging filopodia dynamics in the mouse blastocyst

During mammalian development, the first cell lineage diversification event occurs in the blastocyst, when the trophectoderm (TE) and the inner cell mass (ICM) become established. Part of the TE (polar) remains in contact with the ICM and differs from the mural TE (mTE) which is separated from the ICM by a cavity known as the blastocoele. The presence of filopodia connecting ICM cells with the distant mural TE cells through the blastocoelic fluid was investigated in this work. We describe two types of actin-based cell projections found in freshly dissected and in vitro cultured expanding blastocysts: abundant short filopodia projecting into the blastocoelic cavity that present a continuous undulating behavior; and long, thin traversing filopodia connecting the mural TE with the ICM. Videomicroscopy analyses revealed the presence of vesicle-like structures moving along traversing filopodia and dynamic cytoskeletal rearrangements. These observations, together with immunolocalization of the FGFR2 and the ErbB3 receptors to these cell extensions, suggest that they display signal transduction activity. We propose that traversing filopodia are employed by mitotic mTE cells to receive the required signals for cell division after they become distant to the ICM.     

Scanning Electron Microscopy of Attached Trophozoites of Pathogenic Entamoeba histolytica1

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2–8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

Cell adhesion during gastrulation : A new approach

In gastrulating sea urchin embryos, secondary mesenchyme cells at the tip of the advancing archenteron extend long narrow filopodia which probe the inner surface of the blastocoele wall, rejecting some surface contacts before adhering to other cells. After specific cell adhesions are made, contractions of the filopodia pull the leading tip of the archenteron to the opposite wall of the blastocoele with an accompanying elongation of the archenteron. A study was made of the biochemistry and morphology of the specific adhesions of filopodial extensions by injecting a variety of compounds into the blastocoele of living sea urchin gastrulae and observing their effects on filopodia and cell movements. A number of agents (proteases, lectins) caused specific filopodial detachment and subsequent archenteron regression. Fluorescein-conjugated lectins, including concanavalin A (conA) and wheat germ agglutinin (WGA) exhibited marked specificity of cell surface binding to specific regions (primary mesenchyme cells, blastocoele wall, etc.) of the embryo.     

Contact guidance in vitro : A light, transmission, and scanning electron microscopic study

Contact guidance was studied in cultures of chick heart fibroblasts and kidney epithelium by observing the relation of these cells to fine grooves ruled in plastic culture dishes, and also to ridges or grooves in plastic replicas moulded from rulings made in metal. The relation of the cells to the regularly arranged collagen fibers of fish scales was also studied by scanning and transmission electron microscopy (SEM and TEM). On the rulings with groove periodicity in the range of 5 μm about 75% of the cells were aligned, but on grooves separated about 30 μm only 60% of cells were aligned. Cytoplasmic components of the cells such as microfilaments maintained a constant relation to the axis of the cell as a whole, but they, and also any cytoplasmic extensions, such as filopodia, bore no consistent relation to any features of the substratum, whether or not the cells were aligned. The cells were not guided to become aligned by filopodia or lamellipodia. The most remarkable and consistent finding was that cells bridged over grooves without contacting their surfaces, whether the grooves were 2 or 10 μm wide. The bridging was a characteristic of cells growing on any of the substrates, including those with grooves or ridges, and also of collagen substrates made from fish scales. A hypothesis is proposed to explain the contact guidance seen on ridged or grooved substrata and on the orientated collagen fibers involving the observed cell bridging and the fact that linear cell-to-substrate contacts (focal contacts) are known to be vital for cell movement. The cell is considered to be stiff so that as it bridges over much of the substratum there is only a limited area available for contact. Assuming that focal contacts need to be of a certain length to provide adhesion, a cell orientation that presents the maximum linear contact would be favoured. An examination of the results of this study and of the reports in the literature shows that cells on these types of substrata take on an orientation such that linear contacts would be expected to predominate.     

TRPV1 expression-dependent initiation and regulation of filopodia

Transient receptor potential vanilloid subtype 1 (TRPV1), a non-selective cation channel, is present endogenously in dorsal root ganglia (DRG) neurons. It is involved in the recognition of various pain producing physical and chemical stimuli. In this work, we demonstrate that expression of TRPV1 induces neurite-like structures and filopodia and that the expressed protein is localized at the filopodial tips. Exogenous expression of TRPV1 induces filopodia both in DRG neuron-derived F11 cells and in non-neuronal cells, such as HeLa and human embryonic kidney (HEK) cells. We find that some of the TRPV1 expression-induced filopodia contain microtubules and microtubule-associated components, and establish cell-to-cell extensions. Using live cell microscopy, we demonstrate that the filopodia are responsive to TRPV1-specific ligands. But both, initiation and subsequent cell-to-cell extension formation, is independent of TRPV1 channel activity. The N-terminal intracellular domain of TRPV1 is sufficient for filopodial structure initiation while the C-terminal cytoplasmic domain is involved in the stabilization of microtubules within these structures. In addition, exogenous expression of TRPV1 results in altered cellular distribution and in enhanced endogenous expression of non-conventional myosin motors, namely myosin IIA and myosin IIIA. These data indicate a novel role of TRPV1 in the regulation of cellular morphology and cellular contact formation.     

A requirement for filopodia in the adhesion of pre-aggregative cells of Dictyostelium discoideum

The adhesion of pre-aggregative cells of Dictyostelium discoideum was found to be partly dependent upon the integrity of filopodial extensions of the cells. Removal of filopodia by cytochalasin B (CB) results in reduced adhesiveness in stationary phase cells. The relationship between filopodia and cell adhesion is discussed.     

Wound healing in the primitive deep layer of the young chick blastoderm.

Wound healing in the primitive deep layer of stage 4 chick blastoderms was studied in vitro by cinemicrophotography of living cultures and by photomicroscopy, scanning- and transmission electron microscopy after fixation. Experimental wounds with an average diameter of 0.3 mm healed completely within 2 to 4 h through migration of the cells at their rims. Healing occurred in mesenchyme-free areas, providing us with a purely epithelial reaction. The rim cells of the primitive deep layer formed extensions at their free flank, described as fila, filopodia, lamellae and lamellipodia. They were already present in blastoderms fixed at the earliest after the intervention. This reaction was ascribed to the elimination of a normal fellow cell at the side of the rim cell facing the defect. Movement of the rim cell ceased upon meeting another cell with the same polarity. At this moment lamellipodia disappeared as suddenly as they had formed, and the number of fila and filopodia decreased. We believe that the chick blastoderm's primitive deep layer might be appropriate for analysis of the factors governing primary epithelial wound healing.     

Lamellipodia nucleation by filopodia depends on integrin occupancy and downstream Rac1 signaling

Time-lapse video-microscopy unambiguously shows that fibroblast filopodia are the scaffold of lamellipodia nucleation that allows anisotropic cell spreading. This process was dissected into elementary stages by monitoring cell adhesion on micropatterned extracellular matrix arrays of various pitches. Adhesion structures are stabilized by contact with the adhesive plots and subsequently converted into lamellipodia-like extensions starting at the filopodia tips. This mechanism progressively leads to full cell spreading. Stable expression of the dominant-negative Rac1 N17 impairs this change in membrane extension mode and stops cell spreading on matrix arrays. Similar expression of the dominant-negative Cdc42 N17 impairs cell spreading on homogenous and structured substrate, suggesting that filopodia extension is a prerequisite for cell spreading in this model. The differential polarity of the nucleation of lamellipodial structures by filopodia on homogenous and structured surfaces starting from the cell body and of filopodia tip, respectively, suggested that this process is triggered by areas that are in contact with extracellular matrix proteins for longer times. Consistent with this view, wild-type cells cannot spread on microarrays made of function blocking or neutral anti-beta 1 integrin antibodies. However, stable expression of a constitutively active Rac1 mutant rescues the cell ability to spread on these integrin microarrays. Thereby, lamellipodia nucleation by filopodia requires integrin occupancy by matrix substrate and downstream Rac1 signaling.     

Cellular migration through the cardiac jelly matrix: A stereoanalysis by high-voltage electron microscopy

Formation and migration of cushion tissue in the developing chick heart was analyzed by scanning and high-voltage electron microscopic stereoanalysis. Two methods of fixation which enhance the preservation of water-soluble components of the extracellular matrix (cardiac jelly) were employed: 1% tannic acid in 3% glutaraldehyde (TAG) and 1% cetylpyridinium chloride (CPC) in 3% glutaraldehyde. Our results indicated that the preservation of the cell: matrix interaction exhibited by endocardial cells and migrating cushion tissue is dependent upon the method of fixation. In TAG-fixed embryos, filopodial extensions from the endocardium as well as filopodia of pioneering cells are most often associated with microfibrillar components of the matrix, whereas in CPC-fixed material these same cellular extensions are found in association with pleomorphic anastomosing strands rich in hyaluronate. Following these initial cell:matrix interactions by both the endocardium and pioneering cells, trailing cells invade the extracellular matrical region and clearly encounter in both types of fixation a different microenvironment in which to engage in cell:matrical associations. These observations support the hypothesis that filopodial probing by endocardial cells and pioneering cells results in macromolecular reorderings of the matrix and thus suggest an additional function for filopodia beyond translocation of the cells.     

Fluorescence microscopy study of polymorphonuclear leukocyte substrate attached materials

We describe a technique to visualize substrate-attached materials (SAM) of polymorphonuclear leukocytes (PMN) using the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3',-tetramethylindocarbocyanine-perchlorate (DiC18Icc). DiC18Icc was incorporated into the membranes of living cells or SAMs. Since cell preparation does not require fixation, SAMs can be rapidly visualized by fluorescence microscopy. SAMs are generated by subjecting attached cells to a shearing force by rinsing with phosphate-buffered saline (PBS). The SAM-labeling protocol identified a membrane compartment as shown by detergent extraction. The SAMs of PMN leukocytes observed with this technique display complex patterns of interconnecting filaments, foci with radiating filaments, and smooth membranous areas with interconnecting filaments. The sensitivity and nondestructive nature of the DiC18Icc-labeling procedure have allowed us to observe filopodia of motile cells. The results are consistent with the hypothesis that locomotion involves a series of attachment and detachment steps. After 60 minutes of locomotion, these trailing filopodia have been measured at lengths up to 100 micron. The amount of membrane associated with these filopodia accounts for roughly 10% of the total membrane area of resting cells. These data set limits for models of membrane flow during chemotaxis.     

Internal structure of the postcapillary high-endothelial venules of rodent lymph nodes and Peyer's patches and the transendothelial lymphocyte passage

Scanning electron microscopy (SEM) shows that the postcapillary high-endothelial venules of lymph nodes and Peyer's patches consist of two segments each with a different surface relief: a proximal segment with a cobblestone surface pattern and a distal segment of interlacing cytoplasmic plates. Both segments have deep adluminal crevices in which lymphocytes are lodged. The internal structural configuration of this endothelium has been examined by transmission electron microscopy (TEM) of serial sections of lymph nodes and Peyer's patches of mice, rats, and guinea pigs. The serial sections revealed that the endothelial cell bodies and their cytoplasmic extensions were disposed in a direction generally lateral to the luminal surface and intruded into the intercellular spaces of similarly disposed neighboring endothelial cells, resulting in a complex interlacing cellular pattern. Lymphocytes penetrated the endothelial cell body and secondarily followed an intracellular pathway through which they entered the extravascular compartment. At the exposed surfaces of the adluminal venule wall, recirculating lymphocytes were seen in SEM images to enter the endothelium by penetrating the endothelial cell body. The mode of migration of lymphocytes lodged in the endothelial crevices could be determined by SEM and has been examined by TEM of serial sections. At these locations as at the exposed surfaces, lymphocytes also entered the venule by penetrating the endothelial cell body. At both sites this transcellular pathway was followed by lymphocyte entry into the intercellular spaces from which they migrated into the extravascular compartment.     

The morphology of the denuded epidermal basal cell layer of the hairless mouse after different preparation methods. A scanning and transmission electron microscopical study

The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study. The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion. Trypsinization before removal of the suprabasal cells caused the basal cells to shrink. Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved. SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized. Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations. However, cell bridges, and occasional desmosomes were present. The cell interior demonstrated important cellular injury. If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated "regenerative" morphology, without junctions, similar to that observed in wound healing. Epidermal non-keratinocytes were seen only after trypsinization. Control experiments revealed that they adapted poorly to organ culture conditions. By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes. This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis. We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced "clear cells", whereby the neighboring cells appeared darker. This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells. The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation. The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with "foot pads". Our study also shows that SEM of the epidermal basal layer might be feasible. Various artifacts, however, must be considered, depending on the denudation method used. We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis. Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry. Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t