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PubMed1994

Multiple roles of intermediate filaments.

The widespread distribution, diversity of types, and highly conserved features of intermediate filaments (IFs) and the diversity of associated proteins indicate multiple roles for IFs in the nucleus and the cytoplasm of eukaryotic cells. Traditional functions include maintenance of cell shape, cell locomotion, movement and positioning of organelles and guiding cell development. Recent studies indicate some of these functions can now be expanded. Currently available information shows that IFs are important to nuclear function and cell division, to transport between the nucleus and cytoplasm, to the mechanical integrity of cells and tissues, to reorganization of the cytoplasm and chromatin in response to intercellular signals, and to embryonic and cellular differentiation.

Animals↗

PubMed1998

Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1.

Activation of Ca2+ entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca2+ entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca2+ channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+ that was dependent on a membrane current having a net inward component of 5.45 +/- 0.90 pA/pF at -80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+ entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential (trp) gene have been proposed to form Ca2+ entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca2+ entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.

Amino Acid Sequence↗

PubMed2003

A Rho-associated kinase mitigates reperfusion-induced change in the shape of cardiac capillary endothelial cells in situ.

OBJECTIVE: We have previously demonstrated that ischaemia and reperfusion of the myocardium alter capillary dimensions and endothelial cell shape and that these changes are likely to be effected by the actomyosin contractile system in endothelial cells. Rho GTPases are involved in the regulation of cytoskeletal re-organization and in cell contraction. Rho-associated kinase regulates the sensitivity of myosin light chain to Ca(2+) in smooth muscle but not in cardiac or skeletal muscle myocytes. This study investigated the role of Rho-associated kinase in endothelial cell shape change induced by cardiac ischaemia and reperfusion. The role of Rho proteins in endothelial cell shape change in situ in the myocardial capillary bed has to date not been investigated. METHODS: Ischaemia and reperfusion were induced in Langendorff perfused rat hearts at constant flow. Electron microscopy and immunofluorescence studies localized the beta Rho-associated kinase isotype in capillary endothelial cells. Whole capillary and luminal cross-section areas, luminal and abluminal membrane lengths were measured to monitor changes in cell dimensions. We used a ROCK inhibitor, Y-27632, to investigate the role of this protein in endothelial cell shape change. RESULTS: ROCK1 localized primarily to intracellular membranes in endothelial cells. Morphometric analysis and a study of capillary lumen resin casts demonstrated that inhibition of the activity of this kinase with Y-27632 ablated the change in shape of endothelial cells induced by ischaemia and reperfusion. CONCLUSION: These results suggest that ROCK1 is involved in cardiac capillary endothelial cell shape change in situ and that targeting the contractile system in this way may be useful in ameliorating reperfusion injury.

Amides↗

PubMed2006

Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining.

BACKGROUND: Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. RESULTS: We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea) and side scatter (SSheight and SSarea). The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases cell viability but decreases the proliferative ability of the PC12 cells. CONCLUSION: We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses.

Adrenal Gland Neoplasms↗

PubMed1983

Virus replication in infected epithelial cells is coupled to cell shape-responsive metabolic controls.

The cell shape of monkey epithelial cells was varied from flat to spheroidal by gradually reducing the substrate adhesiveness with poly (HEMA) films of increasing thickness. The decrease in cell spreading is accompanied by a dramatic response in cellular macromolecular metabolism in the nucleus. Within 14 to 16 hr, DNA and RNA syntheses are inhibited by more than 95%, while the level of protein synthesis is reduced by only twofold after 24 hr in spheroidal-suspension culture. When epithelial cells, spread to various degrees, are infected with SV40 or herpes virus a parallel inhibition of virus replication and cellular macromolecular metabolism occurs. However, VSV can proliferate in the metabolically active cytoplasm of epithelial cells in which nuclear activity is inhibited owing to alterations in cell shape. The results suggest that the metabolic restrictions imposed on epithelial cells, owing to changes in cell spreading, are a dominant phenomenon that cannot be overcome by virus infection. Rather, virus replication, which is dependent on the cellular metabolic machinery, is inhibited in parallel with the inhibition of cellular macromolecular metabolism.

Animals↗

PubMed2005

Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments.

Cell shape and movements rely on complex biochemical pathways that regulate actin, microtubules, and substrate adhesions. Some of these pathways act through altering the cortex contractility. Here we examined cellular systems where contractility is enhanced by disassembly of the microtubules. We found that adherent cells, when detached from their substrate, developed a membrane bulge devoid of detectable actin and myosin. A constriction ring at the base of the bulge oscillated from one side of the cell to the other. The movement was accompanied by sequential redistribution of actin and myosin to the membrane. We observed this oscillatory behavior also in cell fragments of various sizes, providing a simplified, nucleus-free system for biophysical studies. Our observations suggest a mechanism based on active gel dynamics and inspired by symmetry breaking of actin gels growing around beads. The proposed mechanism for breakage of the actomyosin cortex may be used for cell polarization.

Actins↗

PubMed1984

Retinoic acid restores shape-dependent growth control in neoplastic cells cultured on poly(2-hydroxyethyl methacrylate)-coated substrate.

The ability of retinoic acid to modulate cell-shape-dependent growth of untransformed (human skin fibroblasts and mouse embryo Swiss 3T3 fibroblasts) and neoplastic cells (human cervical carcinoma HeLa-S3, osteosarcoma Hs791, and murine melanomas B16-F1, S91-C2 and S91-C154) was examined. The cells were plated on tissue culture dishes coated with increasing concentrations of poly(2-hydroxyethyl methacrylate), poly(HEMA) which cause a gradual decrease in substrate adhesiveness. Untreated cells as well as cells pretreated with 10 microM retinoic acid for 4 days displayed a similar graded series of cell shapes between flat and spherical on these modified substrata, with the exception of HeLa-S3 cells which were rounded and loosely attached even on uncoated plastic dishes. A marked cell-shape-dependent decrease in DNA synthesis was observed in untransformed human skin fibroblasts, Swiss 3T3 fibroblasts and neoplastic human Hs791 cells 20 h following plating of untreated cells on poly(HEMA)-coated substrates of decreasing adhesiveness. Conversely, in B16-F1, HeLa-S3 and S91-C154 cells DNA synthesis was only slightly affected by changes in cell shape. Pretreatment with retinoic acid rendered DNA synthesis in Swiss 3T3, Hs791, B16-F1 and S91-C2 cells much more sensitive to changes in cell shape. In contrast, retinoic acid exerted only marginal effects on the sensitivity of DNA synthesis to changes in cell shape in untransformed human skin fibroblasts, in HeLa-S3 cells and in the retinoic-acid-resistant S91-C154 cells. The results suggest that retinoic acid can restore in certain tumor cells the tight coupling between cell shape and DNA synthesis that exists in untransformed cells.

Cell Communication↗

PubMed2004

MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus.

The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2-peptidoglycan biosynthesis complex.

Actins↗

PubMed1981

Modulation of the shape of epithelial lens cells in vitro directed by a retinal extract factor. A model of interconversions and the role of actin filaments and fibronectin.

We have shown previously [1] that bovine epithelial lens cells can be stimulated to divide and elongate by a retinal extract (RE). In this report we show that the morphological response to the stimulatory factor is directly related to the target-cell shape, and we describe how the cell shape can be modulated into morphologically different types. If the cells are grown continuously from the explant in the presence of the RE factor, they keep a typical regular pavement-like epithelial shape (type I), even after serial passages. If the same cells are cultured in the absence of the factor, they become extremely irregular in shape and enlarge enormously (type II), and during serial passage elongate spontaneously to a fibroblast-like pattern. However, when type II cells are stimulated by RE, they elongate dramatically into type III cells as described in [1], provided they are stimulated at the optimal cell density. We show that the transformation of one type to another is directly under the control of RE, and we demonstrate that the changes in cell morphology are accompanied by alterations in cytoplasmic actin filaments. Type I cells contain few microfilaments, while type II cells display actin-tropomyosin polygonal fibre networks that reform during conversion to type III cells and then to elongated stress fibres. The change from type I to type II cells is also accompanied by massive accumulation of surface-associated fibronectin. We conclude that factors obtained directly from the eye have a direct ability to control morphology and proliferation of ocular cells like lens cells perhaps by modulation of cellular adhesiveness mediated by surface fibronectin and reorganization of cytoplasmic actin-based filaments.

Actins↗

PubMed1998

Regulation of expression of A-kinase anchoring proteins in rat granulosa cells.

FSH action on granulosa cells involves the generation of cAMP and subsequent activation of the cAMP-dependent protein kinase (PKA). The PKA holoenzyme is targeted to specific subcellular sites through the interaction of the regulatory subunits with A-kinase anchoring proteins (AKAPs). We previously reported that FSH regulates expression of AKAPs. In this report we examine the relationship between AKAP expression and cell shape. Granulosa cells cultured in the absence of FSH tend to spread and flatten. Cell spreading is accompanied by an increased expression of a 140-kDa AKAP. This spreading/flattening phenotype is independent of the specific extracellular matrix proteins (fibronectin, polylysine, and gelatin) on which cells are plated. Addition of FSH prevents both cell spreading and induction of AKAP 140. Culturing cells on poly (2-hydroxyethyl methacrylate), a surface-coating agent that inhibits cell spreading and adhesion, also inhibits expression of AKAP 140. Addition of phorbol myristate acetate, an agent known to antagonize FSH actions, blocks FSH regulation of both cell shape and AKAP 140 expression. Addition of dexamethasone plus FSH causes a synergistic increase in progesterone levels but has no effect on cell shape or induction of AKAP 140. Dexamethasone produces a dose-dependent increase in AKAP 80 expression, which is blocked by FSH, suggesting cross talk between the glucocorticoid and FSH receptor signaling pathways. These data suggest that expression of AKAP 140 is linked to regulation of cell shape, and that changes in the expression of AKAPs are regulated by several different signaling pathways.

Animals↗

PubMed1999

Technical Advance: Confocal measurement of the three-dimensional size and shape of plant parenchyma cells in a developing fruit tissue.

Parenchyma cells from the inner mesocarp of a grape berry (Vitis vinifera L. cv. Chardonnay) were visualised in three-dimensions within a whole mount of cleared, stained tissue using confocal laser scanning microscopy and digital image reconstruction. The whole berry was fixed, bisected longitudinally, cleared in methyl salicylate, stained with safranin O and mounted in methyl salicylate. Optical slices were collected at 1.0 µm intervals to a depth of 150 µm. Neighbouring z-series were joined post-collection to double the field-of-view. Attenuation at depth of the fluorescent signal from cell walls was quantified and corrected. Axial distortion due to refractive index mismatch between the immersion and mounting media was calibrated using yellow-green fluorescent microspheres and corrected. Transmission electron microscopy was used to correct fluorescent measurements of cell wall thickness. Digital image reconstructions of wall-enclosed spaces enabled cells to be rendered as geometric solids of measurable surface area and volume. Cell volumes within the inner mesocarp tissue of a single grape berry exhibited a 14-fold range, with polysigmoidal distribution and groupings around specific size classes. Cell shape was irregular and the planes of contact were rarely flat or simple. Variability in cell shape was indicated by the range in surface area to volume ratios, from 0.080 to 0.198 µm-1. Structural detail at the internal surface of the cell wall was apparent. The technique is applicable to a wide range of morphometric analyses in plant cell biology, particularly developmental studies, and reveals details of cell size and shape that were previously unattainable.

Journal Article↗

PubMed1993

Differentiation of hematuria using a uniquely shaped red cell.

Although variously shaped urinary red cells have been reported in glomerulonephritic hematuria, no specific shapes with concrete definition have been proposed. This made morphological differentiation of hematuria vague and caused different results among different observers. To solve these problems and improve the diagnostic rate, we employed a uniquely shaped red cell, which only appeared in glomerulonephritic hematuria, as a probe for diagnosis. We studied 182 hematuria cases from 90 glomerulonephritic patients and 95 hematuria cases from 68 urological disease patients. Fresh urine was collected and observed by differential interference microscopy. The red cell, referred to as G1, has a distinctive doughnut-like shape with blebs and was highly specific for glomerulonephritic hematuria. Occurrence of G1 cells increased at lower pH an higher osmolality of urine. A presence of 5% or more G1 cells could be an indicator of glomerulonephritic hematuria. Specificity and sensitivity of this criterion were 100 and 73%. However, when only acidic concentrated urine (pH < or = 6.4, osmolality > or = 400 mosm/kg H2O) was used, the specificity and sensitivity increased to 100 and 99.2%, respectively. Glomerulonephritic and urological hematuria were correctly diagnosed by counting the urinary red cells with doughnut-like shape in acidic and concentrated urine. This method seems to be superior to others in diagnostic rate, simplicity and clarity.

Adolescent↗

PubMed1976

Stabilization of the shape of sickled cells by calcium and A23187.

Evaluation of the role of calcium in irreversible sickling has been approached by treating sickled cells with calcium and the ionophore A23187. A calcium-dependent stabilization of the sickled cell shape was observed after reoxygenation of cells in the presence of ionophore. At low calcium concentrations, this retention of sickled shape was maintained for periods up to 1 hr. However, the morphology of the oxygen-stable sickled cells was like that of deoxygenated sickle cells and significantly different from the characteristic morphology of native irreversibly sickled cells (ISCs). Because the stabilized cells did not fulfill the morphological criterion for ISCs, the shape-stabilizing effect of calcium in this system did not provide additional support for the hypothesis that calcium accumulation was the determining factor in ISC generation.

Anemia, Sickle Cell↗

PubMed2004

Traction in smooth muscle cells varies with cell spreading.

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

Adhesiveness↗

PubMed2002

[Primary culture of strial marginal cells of guinea pig cochlea].

OBJECTIVE: For further study of the function of marginal cells, primary cultures of marginal cells of guinea pig were established. METHOD: Tissue of stria vascularis was transferred from the cochlea to a cell-cultured dish using the explants culture technique. Explants were cultivated at 37 degrees C in a 5% CO2 incubator and examined daily by interted microscopy. The culture medium was changed twice a week. Antibodies of cytokeratin and vimentin were used to examine the cultured cells by immunohistochemical methods. The ultrastructure of the cultured cells was observed by making the electron microscopy specimens. RESULT: Explants were successfully cultivated for 4 weeks. Two different cells, hexagonal-shaped cells and spindle-shaped cells were observed. Cytokeratin and vimentin staining occurred in cytoplasm of hexagonal-shaped cells, whereas spindle-shaped cells gave a negative cytokeratin staining but a positive vimentin staining. Electron microscopy of the explants revealed that hexagonal-shaped cells have the epithelial morphologic characteristics such as tight junctions and desmosomes. CONCLUSION: Primary culture of marginal cells by explants culture technique provides an optimal model for investigation of the function of the marginal cells.

Animals↗

PubMed1986

Alignment of fibroblasts on grooved surfaces described by a simple geometric transformation.

The response of chick heart fibroblasts to grooved substrata was studied using microfabricated grooves and new measures of shape and alignment derived from the moments of cell shapes. Cell shape and alignment were measured on 23 different sets of regular, parallel grooves, which ranged in width from 1.65 to 8.96 micron, and in repeat spacing from 3.0 to 32.0 micron. The grooves were of constant depth, 0.69 micron. Digitized video images were analysed to extract the zero-, first- and second-order moments of the cell shapes, from which were calculated three measures of cell shape, and three measures of cell alignment. Regression analyses of the measures against parameters of the substratum such as groove width, repeat spacing and the ridge width between grooves show that ridge width is the main parameter affecting cell alignment (alignment being inversely proportional to ridge width), although groove width has a small additional effect. All the differences in cell shape between the different grooves can be summarized to a very good approximation as simple geometrical stretch transformations of the shapes of cells on planar surfaces. Our principal measure of cell alignment, paraxial elongation, is a measure of the necessary transformation. This finding has the interesting biological implication that the shape and orientation adopted by cells, in response to the grooves, are not governed by independent cellular mechanisms.

Animals↗

PubMed1984

Analysis of cell movement.

The methods of statistical physics have been applied to the analysis of cell movement. Human leukocytes (granulocytes) were observed using time-lapse photography. The center of gravity of a cell, variations of cell shape, and cell orientation were investigated. This analytical description leads to a better understanding of cell movement. Stationary motion of a cell is described by the anisotropy of the cell shape. The cell displacement can be characterized by three different types of movement: The persistent mode where the cell moves away from an arbitrary chosen origin with its track velocity. The diffusion mode where the cells become dispersed in space by a random walk process. The drift mode where the cell moves with a drift velocity, v parallel, in a concentration gradient of chemoattractant molecules. The chemokinetic response is described by the diffusion constant D (= 240 microns2/min) and the track velocity vc (= 30 microns/min). The chemotactic response is described by the degree of orientation P1 (= 0.8), which is identical with the McCutcheon index and the chemotropism index. Cell movement can be described by elementary moving states, and the life time of such a moving state is 0.5 min. The survival probability of the moving state is determined by an internal program. It is not described by a stochastic process. The angular change in moving direction is also programmed, as the square root of the mean square angular change is +/- 50 degrees. The plus and minus direction are equally probable in a chemokinetic response. However, in a chemotactic assay the plus and minus directions are not equally probably. We found that the information transfer from the chemotactic gradient to the migrating cell is 1 bit per change in moving direction. A disturbance in this information transfer leads to an order-disorder transition. Furthermore, we found that the migrating cell exhibits a directional memory of 75 s.

Biophysical Phenomena↗

PubMed1989

Autoradiographic investigations on cell shape-mediated growth regulation of lens epithelial cells in culture.

An autoradiographic method is described which is well suited for the determination of the labelling index in flattened as well as rounded cells. Using this method DNA synthesis of lens epithelial cells in culture was found to be dependent on cell attachment, cell flattening and intact microfilaments. Thus previous results on cell shape mediated growth regulation could be confirmed. Moreover, considering the labelling index it was possible to conclude that cell rounding or a disintegration of microfilaments did not impair ongoing DNA synthesis but did prevent cells from entering the S-phase of the cycle.

Animals↗