Limitation of substratum size alters cytoskeletal organization and behaviour of Swiss 3T3 fibroblasts

Cell spreading and adhesion formation in Swiss 3T3 cells was studied on circular adhesive islands of size 400-500 microns 2 made by evaporating palladium through a mask onto an underlying non-adhesive surface. Cell spreading was limited since focal contacts were restricted to the palladium. On islands less than 2000 microns 2, focal contacts and actin bundles were arranged at the cell periphery. On islands less than 1000 microns 2, the size and number of focal contacts were reduced. Focal contacts may be important regulators of proliferation, but they do not seem to form a deterministic link between substratum contact and proliferative stimulus.     

Cell membrane polarity in rat ascites hepatoma cells

Summary As previously described, a cell surface-associated adhesive factor (AF) was separated from differentiated rat ascites hepatoma AH136B cells (forming cell islands in vivo) and highly purified by chromatography. AF induces not only aggregation of dissociated AH136B cells or undifferentiated rat ascites hepatoma AH109A cells (present as free cells in vivo), but also adhesiveness characterized by the development of junctional complexes. The localization of AF on the surfaces of AH136B cell islands was investigated using anti-AF IgG (Fab fragment) coupled to peroxidase. AF was detected in the contact region of the lateral surfaces of the AH136B cells and in the intercellular spaces. In contrast, no AF was detectable on the apical non-contacted free cell surfaces of AH136B cells. Fluorescence studies revealed that biotin-labeled AF did not bind to the apical surface of AH136B cell islands. These results indicate a distinct difference between apical and lateral surfaces of AH136B cells; neither AF nor binding site for AF were localized on the apical surface of AH136B cells, whereas both were localized on the lateral surface. On the other hand, AH136B cells detached from the cell islands, or during the process of partial dissociation from them, showed the loss of the AF localization and binding site of AF on the surfaces. The results suggest that AH136B cell surfaces may be polarized in terms of the AF localization, and this polarization may be lost after cell dissociation.     

Migration of polarized epithelial cells through permeable membrane substrates of defined pore size.

We have observed that cells of various epithelial lines exhibit the ability to migrate through permeable membrane substrates containing 3.0 microns pores. Scanning and transmission electron microscopic observations of Vero C1008 and Caco-2 cell lines grown on polycarbonate membranes containing 3.0 microns pores revealed extensive penetration of the filter and the establishment of virtually complete monolayers on the opposing surface. The migration of MDCK cells was also observed to occur under the same conditions; however, the extent of MDCK cell growth on the opposing surface was significantly less than observed for Vero C1008 and Caco-2 cells. Morphological differences were apparent between cells growing on the upper and lower faces of the filter membrane, although cells growing on both surfaces exhibited a polarized phenotype. The cells which invaded the filter were collected and maintained by serial passage. The passaged cells exhibited morphological differences and an altered rate of differentiation in comparison to the parental cell type, suggesting that the invasive cells represent a variant of the parental cell population. Studies using filters of different pore sizes indicated that cellular migration also occurs through pores of 2.0 microns diameter, but not through 1.0 micron (or smaller) pores. These observations have significant implications for studies involving the growth of epithelial cells on permeable membrane substrates containing large pores.     

Surface ultrastructure of the gill arch of the killifish, Fundulus heteroclitus, from seawater and freshwater, with special reference to the morphology of apical crypts of chloride cells

The surface ultrastructure of the gill arches of the killifish, Fundulus heteroclitus, adapted to seawater or freshwater, was found to be similar to that reported for other euryhaline teleosts. Two rows of gill filaments (about 42 filaments per row) extended posterolaterally, and two rows of gill rakers (about 10 rakers per row) extended anteromedially from each arch. Leaf-like respiratory lamellae protruded along both sides of each filament, from its base to its apex. The distributions, sizes, and numbers of various surface cells and structures were also determined. All surfaces were covered by a mosaic of pavement cells, which measured about 7 X 4 microns and exhibited concentrically arranged surface ridges. Taste buds were especially prominent on the rakers and the pharyngeal surfaces of the first and second gill arches, but were often replaced by horny spines on the third and fourth gill arches. Apical crypts of chloride cells occurred mostly on the surfaces of the gill filaments adjacent to the afferent artery of the filament. In seawater adapted killifish, crypts resembled narrow, deep holes along the borders of adjacent pavement cells, had openings of about 2 microns2, and occurred at a frequency of about 1 per 70 microns2 of surface area. In freshwater fish, the crypts usually had larger openings (about 10 microns2), occurred less frequently (1 per 123 microns2), and exhibited many cellular projections in their interiors. Changes in crypt morphology may be related to the ion transport function of chloride cells.     

Shear stress gradient over endothelial cells in a curved microchannel system.

Our purpose was to test a scale model of the microcirculation by measuring the shear forces to which endothelial cells were exposed, and comparing this to computer simulations. In vitro experiments were performed to measure the 2-dimensional projected velocity profile along endothelial cell lined microchannels (D-shaped, 10-30 microns radius, n = 15), or in microchannels without endothelial cells (n = 18). Microchannels were perfused with fluorescently labeled microspheres (0.5 micron dia., < 1%) suspended in cell culture media. The velocity of individual microspheres was obtained off-line (videorecording), using an interactive software program; velocity was determined as the distance traveled in one video field (1/60 s). Mass balance was verified in the microchannels by comparing the microsphere velocities to the perfusion pump rate. In confluent endothelial cell lined microchannels, a velocity profile was obtained as microspheres passed an endothelial cell nucleus (identified by fluorescent dye), and again, for a paired region 100 microns away without nuclei (cytoplasm region). The velocity profile was significantly shifted and sharpened by the endothelial cell nucleus, as anticipated. Over the nucleus, data are consistent with a normal sized nucleus extending into the lumen, further confirming that this scale model can be used to determine the wall shear stress to which endothelial cells are exposed. Using the experimental bulk phase fluid parameters as boundary conditions, we used computational fluid dynamics (CFD) to predict the expected wall shear stress gradient along an endothelial cell lined D-shaped tube. The wall shear stress gradient over the nucleus was 2-fold greater in the radial versus axial directions, and was sensitive to lateral versus midline positioned nuclei.     

Spreading of mouse fibroblasts on the substrate with multiple spikes

Mouse embryo fibroblasts were cultivated on special substrates with discontinuous surfaces. The substrates were silicon plates with multiple vertical (65-90 microns height) spike-like silicon microcrystals evenly distributed on the plate surfaces. It was shown that the cells were successfully spread and flattened on these substrates. The spread cells formed several discrete attachment zones at the tops and side surfaces of the spikes; these zones were separated from one another by distances considerably greater than the diameter of the unspread cell. At early stages of spreading the unspread cells attached to the tops of single spikes and extended long filopodia attached to the distant spikes. At later stages the lamellae were formed between the filopodia: probably these filopodia served as guidelines for extension of lamellae and progressive cell spreading. These experiments demonstrated that continuity of substrate surface is not a necessary condition for advanced cell spreading.     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 microns2, of which 34.5 microns2 was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 micron2 of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 microns2 (6.2%). Mitochondria occupied 7.3 microns2 (3.3%) and the Golgi network 5.49 microns2 (2.5%). Decidual secretory bodies occupied 0.91 micron2 (0.42%) and cytoplasmic processes 1.89 micron2 (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 microns of plasma membrane. The mean nuclear membrane measured 28.3 microns (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 microns (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 microns (400% pm, 52.6% cm); Golgi membrane measured 30.77 microns (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 microns (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 microns or 2.9% cm). A mean of seven such processes was observed per 87.8 microns of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

A colloidal gold labeling technique for the direct determination of the surface area of eukaryotic cells

We have developed a colloidal gold labeling technique for the direct quantitation of the cell surface area. The method is based on coating the cell surface with [195Au] colloidal gold-protein complexes followed by morphometric determination of the labeling density (gold particles/micron2 cell surface) and radiometric determination of the total number of gold particles bound per cell. The ratio of both values directly gives the cell surface area. The accuracy of the method was shown using Staphylococcus aureus cells as a model system, where the cell surface area determined with our assay (4.0 microns2) corresponded well to the value calculated from the radius of the cells (3.6 microns2). In a more complex model system J-774 mouse macrophages were labeled with different amounts of [195Au] gold-protein complexes to show that the assay is independent of the degree of saturation of the cell surface binding sites. Both high (135 Au/microns2) and low (65 Au/microns2) labeling densities resulted in a surface area of about 1200 microns2. The technique finally was applied to L-929 fibroblasts to determine the increase of the cell surface area when the cells change from a spherical to a flat monolayer state. We found that the cell surface area increased 3-fold during the spreading process. The results show that the colloidal gold labeling technique allows the direct determination of the surface area of complex eukaryotic cells. The technique is suitable for the quantitation of changes in the surface architecture known to occur in different functional states of eukaryotic cells.     

Fine needle aspiration biopsy of the breast. Value of nuclear morphometry after different sampling methods

OBJECTIVE: To study the potential of nuclear morphometry in supporting the interpretation of fine needle aspiration biopsy (FNAB) samples of the breast fixed in 50% ethanol and centrifuged on slides. STUDY DESIGN: Computerized morphometry was used to outline the nuclei of breast epithelial cells in breast cancer, fibroadenoma and fibrocystic disease. The diagnoses were histologically confirmed. We applied 2 different sampling methods (measurements done on cell groups and on free cells). RESULTS: The mean nuclear area of cell groups of malignant samples (23) varied from 42 to 125 microns 2, in fibroadenomas from 30 to 50 microns 2 and in fibrocystic disease from 26 to 57 microns 2. The mean nuclear area of free cells varied as follows: cancer, 66-181 microns 2; fibroadenoma, 33-70 microns 2; fibrocystic disease, 35-60 microns 2. Apocrine metaplasia was excluded from comparison on a morphologic basis. CONCLUSION: The study suggests that if the mean nuclear area of cell groups is < 42 microns 2, the lesion is probably benign; if > 57 microns 2, and apocrine metaplasia is excluded, malignancy should be considered. The differential diagnosis between carcinoma and fibroadenoma could be based on free cells: mean area of free cell nuclei < or = 65 microns 2 suggested a benign lesion, and of > or = 71 microns 2 suggested a malignant lesion. Morphometric nuclear size features (exemplified by nuclear area) appeared efficient in distinguishing between malignant and benign lesions when measured from free cells and cell groups.     

Maintenance of the 2 microns circle plasmid in populations of Saccharomyces cerevisiae.

The 2 microns circle plasmid is maintained at high frequencies in populations of yeast cells. To find out how the plasmid is maintained, three forces were measured: the selective advantage or disadvantage conferred by 2 microns circles, the rate of generation of [Cir0] cells, and the rate of illegitimate transfer of 2 microns circles from cell to cell. It was found that under the conditions used, 2 microns circles confer a selective disadvantage of about 1%, that [Cir0] cells are generated at the rate of 7.6 x 10(-5) per [Cir+] cell per generation, and that illegitimate transfer of 2 microns circles occurs at a rate less than 10(-7) per recipient cell per generation. The most likely explanation of 2 microns circle maintenance is that the plasmid is sexually transmitted at such a rate that it spreads through populations despite selection against it.     

Antifouling potential of lubricious, micro-engineered, PDMS elastomers against zoospores of the green fouling alga Ulva (Enteromorpha)

The settlement and release of Ulva spores from chemically modified, micro-engineered surface topographies have been investigated using poly(dimethyl siloxane) elastomers (PDMSe) with varying additions of non-network forming poly(dimethyl siloxane) based oils. The topographic features were based on 5 microns wide pillars or ridges separated by 5, 10, or 20 microns wide channels. Pattern depths were 5 or 1.5 microns. Swimming spores showed no marked difference in settlement on smooth surfaces covered with excess PDMS oils. However, incorporation of oils significantly reduced settlement density on many of the surfaces with topographic features, in particular, the 5 microns wide and deep channels. Previous results, confirmed here, demonstrate preferences by the spores to settle in channels and against pillars with spatial dimensions of 5 microns, 10 microns and 20 microns. The combination of lubricity and pillars significantly reduced the number of attached spores compared to the control, smooth, unmodified PDMSe surfaces when exposed to turbulent flow in a flow channel. The results are discussed in relation to the energy needs for spores to adhere to various surface features and the concepts of ultrahydrophobic surfaces. A factorial, multi-level experimental design was analyzed and a 2nd order polynomial model was regressed for statistically significant effects and interactions to determine the magnitude and direction of influence on the spore density measurements between factor levels.     

Survival of biological cells deformed in a narrow gap

Recent studies show that during slow freezing of biological cells, the cells may be also injured by not only chemical damage but also mechanical damage induced by ice crystal compression. A new experimental procedure is developed to quantify cell destruction by deformation with two parallel surfaces. The viability of cells (prostatic carcinoma cells, 17.5 microns in mean diameter) is measured as a function of gap size ranging from 3.5 microns to 16.2 microns at 0 degree C, 23 degrees C and 37 degrees C. The viability at a smaller gap size is significantly lower at 37 degrees C than at 23 degrees C, while the difference between 0 degree C and 23 degrees C is much smaller. This suggests that deformation damage is related to the deformation of the cytoskeleton rather than the mechanical properties of the lipid membrane.     

THE RELATIVE EXTENSIBILITY OF CELL SURFACES

Observations have been made on the response, in vitro, of cultured and freshly dissociated cells to mechanical deformation. Large numbers of individual cells were studied by means of a special culture chamber bounded by two parallel glass coverslips whose spacing could be reduced from 140 to 2 microns in steps of roughly 0.5 micron. The degree of deformation required for herniation of the cell surface was measured. These measurements lead to the definition of a statistical index characteristic of the extensibility of cell surfaces. This index has been shown to be distinctive for several types of cells; to alter with certain stages of embryonic development; and to be stable with respect to the culturing of cells and certain alterations in the method of cell culture.     

Effect of surface nanoscale topography on elastic modulus of individual osteoblastic cells as determined by atomic force microscopy

Mechanical stimulation of osteoblasts by fluid flow promotes a variety of pro-differentiation effects and improving the efficiency of these mechanical signals could encourage specific differentiation pathways. One way this could be accomplished is by altering mechanical properties of osteoblasts. In this study, murine osteoblastic MC3T3-E1 cells were cultured on surfaces covered with nanometer-sized islands to examine the hypothesis that the elastic modulus of osteoblastic cells is affected by nanoscale topography. Nanoislands were produced by polymer demixing of polystyrene and poly(bromostyrene), which leads to a segregated polymer system and formation of nanometer-sized topographical features. The elastic modulus of MC3T3-E1 cells was determined using atomic force microscopy in conjunction with the Hertz mathematical model. Osteoblastic cells cultured on nanotopographic surfaces (11-38 nm high islands) had a different distribution of cellular modulus values, e.g., the distribution shifted toward higher modulus values, relative to cells on flat control surfaces. There were also differences in cell modulus distribution between two flat controls as surface chemistry was changed between polystyrene and glass. Taken together, our results demonstrate that both surface nanotopography and chemistry affect the mechanical properties of cells and may provide new methods for altering the response of cells to external mechanical signals.     

Quantitative ultrastructure of isolated Kupffer cells of rat liver

The average volume of isolated Kupffer cells of rat liver is 821 +/- 64 microns 3, the average surface being 423 +/- 24 microns 2 (599 microns 2, with cell processes included). The surface structure (pseudopodia, lamellipodia, filopodia, microvilli) of isolated cells is much less developed than that of Kupffer cells in situ. By morphometric characterization volume densities are 0.1264 +/- 0.0077 (SE) for mitochondria and 0.3591 +/- 0.0169 for lysosomal structures. The volume of mitochondria amount to 0.79 +/- 0.04 microns 3.     

Cooperative control of Akt phosphorylation, bcl-2 expression, and apoptosis by cytoskeletal microfilaments and microtubules in capillary endothelial cells

Capillary endothelial cells can be switched between growth and apoptosis by modulating their shape with the use of micropatterned adhesive islands. The present study was carried out to examine whether cytoskeletal filaments contribute to this response. Disruption of microfilaments or microtubules with the use of cytochalasin D or nocodazole, respectively, led to levels of apoptosis in capillary cells equivalent to that previously demonstrated by inducing cell rounding with the use of micropatterned culture surfaces containing small (<20 microm in diameter) circular adhesive islands coated with fibronectin. Simultaneous disruption of microfilaments and microtubules led to more pronounced cell rounding and to enhanced levels of apoptosis approaching that observed during anoikis in fully detached (suspended) cells, indicating that these two cytoskeletal filament systems can cooperate to promote cell survival. Western blot analysis revealed that the protein kinase Akt, which is known to be critical for control of cell survival became dephosphorylated during cell rounding induced by disruption of the cytoskeleton, and that this was accompanied by a decrease in bcl-2 expression as well as a subsequent increase in caspase activation. This ability of the cytoskeleton to control capillary endothelial cell survival may be important for understanding the relationship among extracellular matrix turnover, cell shape changes, and apoptosis during angiogenesis inhibition.     

Histomoniasis and reticuloendotheliosis in a wild turkey (Meleagris gallopavo) in North Carolina

A moribund wild turkey (Meleagris gallopavo) died shortly after it was discovered in Martin County, North Carolina (USA). The 4.3-kg female turkey appeared in good condition with no visible external lesions or evidence of injury. There were 2- to 5-mm yellow-white plaques on the mucosal surfaces of the oral cavity and mid-esophagus. The liver had large, multifocal, irregular pale areas on cut and uncut surfaces. The spleen contained multifocal, pale, hard, nodules. Microscopic changes in the liver consisted of large multifocal coalescing areas of necrosis. Occasional spherical 10 to 15 microns in diameter organisms consistent with Histomonas meleagridis were present in the necrotic areas. Viable hepatic parenchyma contained multifocal infiltrations of numerous mononuclear cells interpreted as neoplastic cells resembling lymphoblasts and plasma cells. Similar neoplastic cell infiltrates, consistent with the lymphoproliferative disease reticuloendotheliosis, were present in spleen, lung, and esophageal and oral mucosa. Reticuloendotheliosis virus, subtype 2, was isolated from samples of liver and spleen.     

Micropatterned surfaces for the control of endothelial cell behaviour

Micropatterned materials were synthesised by photoimmobilising the sulphated hyaluronic acid, adequately functionalised with a photoreactive moiety, on glass substrates. Four different patterns (10, 25, 50 and 100 microns) were obtained. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask was well reproduced on the sample surface. Analysis of endothelial cell behaviour on these micropatterned materials was performed in terms of adhesion, locomotion and orientation. Decreasing the stripe dimensions a more fusiform shape of the adhered endothelial cells was observed. At the same time the cell locomotion and orientation were increased. Furthermore, a photoimmobilisation of stripes of HyalS (10 and 100 microns) was performed on a continuous HyalS layer, in turn immobilised on glass substrate. Being excluded a different chemistry between the stripe and the substrate, the influence of topography on the behaviour of endothelia cells was thus envisaged.     

Dimensionality Controls Cytoskeleton Assembly and Metabolism of Fibroblast Cells in Response to Rigidity and Shape

Background

Various physical parameters, including substrate rigidity, size of adhesive islands and micro-and nano-topographies, have been shown to differentially regulate cell fate in two-dimensional (2-D) cell cultures. Cells anchored in a three-dimensional (3-D) microenvironment show significantly altered phenotypes, from altered cell adhesions, to cell migration and differentiation. Yet, no systematic analysis has been performed that studied how the integrated cellular responses to the physical characteristics of the environment are regulated by dimensionality (2-D versus 3-D).

Methodology/Principal Findings

Arrays of 5 or 10 µm deep microwells were fabricated in polydimethylsiloxane (PDMS). The actin cytoskeleton was compared for single primary fibroblasts adhering either to microfabricated adhesive islands (2-D) or trapped in microwells (3-D) of controlled size, shape, and wall rigidity. On rigid substrates (Young''s Modulus = 1 MPa), cytoskeleton assembly within single fibroblast cells occurred in 3-D microwells of circular, rectangular, square, and triangular shapes with 2-D projected surface areas (microwell bottom surface area) and total surface areas of adhesion (microwell bottom plus wall surface area) that inhibited stress fiber assembly in 2-D. In contrast, cells did not assemble a detectable actin cytoskeleton in soft 3-D microwells (20 kPa), regardless of their shapes, but did so on flat, 2-D substrates. The dependency on environmental dimensionality was also reflected by cell viability and metabolism as probed by mitochondrial activities. Both were upregulated in 3-D cultured cells versus cells on 2-D patterns when surface area of adhesion and rigidity were held constant.

Conclusion/Significance

These data indicate that cell shape and rigidity are not orthogonal parameters directing cell fate. The sensory toolbox of cells integrates mechanical (rigidity) and topographical (shape and dimensionality) information differently when cell adhesions are confined to 2-D or occur in a 3-D space.     

Quantitation of cell area on glass and fibronectin-coated surfaces by digital image analysis.

By using digital image processing and analysis, two procedures were developed to rapidly measure the projected area of a field of adherent 3T3 fibroblasts without staining of cell borders. The cell area of newly attached and rounded cells with well-resolved borders was obtained by a gray value thresholding procedure. For cells that had undergone an appreciable degree of spreading, cell boundaries were less distinct and a nonlinear spatial Sobel filter was used, followed by thresholding. For both procedures, linear relations were observed between cell areas obtained from image analysis and cell areas obtained by tracing. The areas of a population of traced cells were not statistically different from the area distribution obtained by using the standard curves for the processed images. Uncertainty in the estimated mean area depended only upon the number of cells examined. Approximate numbers of cells required to obtain estimates of the mean are calculated. As an application of these procedures, cell areas were measured for 3T3 cells attached to glass and fibronectin-coated surfaces and were found to be significantly larger for cells spreading on fibronectin-coated glass than on glass alone. Increased cell area during spreading on fibronectin-coated surfaces was proportional to increased cell adhesivity after exposure to a shear stress of 58 dyn/cm2.