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High-resolution atomic force microscopy (AFM) allows a new insight into the surface of mammalian cells. Using the human red blood cell (RBC) as a model, we have demonstrated an important correlation between the conformation of membrane proteins measured from the external face of the cell and the cell shape.
Parvalbumin is thought to act as a Ca2+ buffer in skeletal muscle fibers, but its physiological role in brain, kidney, and testis remains unclear. We have transfected parvalbumin cDNA into a human ovarian adenocarcinoma cell line, which normally does not express this protein. The induced expression of parvalbumin under the control of three different promoters causes: (1) changes in the morphology of the cells from epitheloid to fusiform, (2) an increase in motility of whole cell clusters, and (3) a decrease in the mitotic rate. Transfection with a mutated cDNA of rat parvalbumin incapable of binding Ca2+ had no effect on these three parameters. Our results indicate that ectopic expression of parvalbumin influences not only cell division [Rasmussen and Means (1989) Mol. Endocrinol. 3, 588-596], but also cell shape and motility by modulating intracellular Ca2+ handling. This may be a basic function of parvalbumin when it is intrinsically expressed in differentiated nonmuscle cells.
The fission yeast Schizosaccharomyces pombe has been used to identify Arabidopsis thaliana proteins that may play a role in cell shape maintenance or cell cycle regulation. An Arabidopsis thaliana cDNA library was constructed in pREP5N vector under the control of the inducible nmt1 promoter and transformed into S. pombe. Expression of the A. thaliana sequences was induced and clones showing severe morphological changes were identified and analysed. Comparison of the sequences of the inserts with the sequence data bases revealed that several cDNAs encode proteins known to play a role in function of the cytoskeleton, the cell cycle and establishment of cell polarity. These include alpha-1, alpha-2, alpha-6 and beta-6 tubulins, myosin heavy chain-like protein, ubiquitin conjugating enzymes UBC9 (E2), 26S protease subunits, Ranbinding protein, myb protein, PRL1 gene product and rho protein. Approximately 30% of the clones encode novel sequences. The results suggest that S. pombe phenotypic screening can be used to identify plant proteins involved in cell shape maintenance and regulation during cell cycle and development.
The migration of cells on structured surfaces is known to be affected by its surface topography. Although the effects of topography have been extensively investigated the crucial parameters determining the cell-surface reaction are largely unknown. The present study was performed to describe and to define the role of groove/elevation (ridge) dimensions at the micrometre scale on fibroblast cell migration by correlating cell shape, migration angle alpha, cell orientation beta and velocity with these dimensions. For this a quantitative method was developed. We could show that the surface structures significantly influenced migration direction alpha, cell orientation beta and mean velocity, as well as migration speed in the directions parallel and perpendicular to the grooves/elevations in a surface structure dependant way. Cell migration velocity parallel, respectively, perpendicular to the structures was significantly affected by the geometries and dimensions of the substratum. Surface structures were not able to significantly affect distribution patterns of cell shapes. Overall, it could be shown that differently structured surfaces influenced the cells but no crucial feature could be clearly identified, suggesting that the reaction of the surface structure might be far more complex than generally is assumed.
Fibroblasts are thought to require an intact system of cytoplasmic microtubules in order both to adopt and to maintain a polarized morphology in culture, but whether the same is true for epithelial cells is not clear. We therefore compared the effects of the microtubule-disrupting drugs, colcemid and nocodazole, on the morphology of isolated embryonic chick heart fibroblasts (HF), corneal epithelial (CE) and epidermal epithelial (EE) cells. Immunofluorescence observations showed that all three cell types contained abundant microtubules when cultured in control medium and that these were absent from cells cultured in medium containing either colcemid or nocodazole. Qualitative observations suggested that these drugs inhibited the polarization of spreading HF but had no effect on the morphology of either of the epithelial cell types. We confirmed this observation quantitatively using two measures of cell shape, elongation and dispersion, both of which increase with increasing polarization. Our measurements show that microtubule-disrupting drugs significantly reduce both the elongation and dispersion of spreading HF but do not have a significant effect on either of these measures for the epithelial cell types. We quantified in a similar way the effects of microtubule-disrupting drugs on the morphology of all three cell types that had previously spread in control medium. We found that transferring spread HF to colcemid-containing medium significantly reduced both the elongation and dispersion of these cells but that the same treatment had no effect on these measures for either of the epithelial cell types. Our observations suggest that, unlike the fibroblasts, the epithelial cell types we have studied do not require microtubules either to adopt or to maintain a polarized morphology.
A 77-year-old white woman presented with 1 1/2-year history of progressively enlarging cutaneous papules, nodules, and plaques, some of which had spontaneously regressed. Her past medical history included untreated chronic lymphocytic leukemia for 10 years' duration. Multiple skin biopsy specimens revealed a diffuse superficial and deep dermal spindle-cell infiltrate accompanied by occasional foamy round cells and multinucleated giant cells. The spindle-shaped cells were focally arranged in a storiform pattern with prominent fibrous stroma. The spindle-shaped cells stained positively for numerous macrophage markers including CD45, factor XIIIa, Leu M5, HLA-DR, CD4, and Leu M3, consistent with dermal dendrocytes. They were also positive for nonspecific esterase and acid phosphatase, which is typical of tissue macrophages. The spindle-shaped cells were negative for CD-1, S-100, and ATPase activity, thus excluding a Langerhans cell immunophenotype. Combining the clinical features, light microscopy, immunohistochemistry, and enzymatic analysis, this patient appears to represent a novel cutaneous fibrohistiocytic proliferative disorder that features large numbers of dermal dendrocytes.
In Volvox carteri adults, reproductive cells called gonidia are enclosed within a spherical monolayer of biflagellate somatic cells. Embryos must "invert" (turn inside out) to achieve this configuration, however, because at the end of cleavage the gonidia are on the outside and the flagellar ends of all somatic cells point inward. Generation of a bend region adequate to turn the embryo inside out involves a dramatic change in cell shape, plus cell movements. Here, we cloned a gene called invA that is essential for inversion and found that it codes for a kinesin localized in the cytoplasmic bridges that link all cells to their neighbors. In invA null mutants, cells change shape normally, but are unable to move relative to the cytoplasmic bridges. A normal bend region cannot be formed and inversion stops. We conclude that the InvA kinesin provides the motile force that normally drives inversion to completion.
The growth and regression of capillary blood vessels during angiogenesis is greatly influenced by changes in the activity of matrix metalloproteinases (MMPs), which selectively degrade extracellular matrix (ECM) and thereby modulate capillary endothelial cell shape, growth and viability. However, changes in cell-ECM binding and cell spreading have also been reported to alter MMP secretion and activation. Studies were carried out to determine whether changes in integrin binding or cell shape feed back to alter MMP-2 processing in human capillary endothelial (HCE) cells. Catalytic processing of proMMP-2 to active MMP-2 progressively decreased when HCE cells were cultured on dishes coated with increasing densities of fibronectin (FN), which promote both integrin binding and cell spreading. Conversely, the highest levels of active MMP-2 were detected in round cells cultured on low FN. When measured 24 hours after plating, this increase in active MMP-2 was accompanied by a concomitant rise in mRNA and protein levels for the membrane-type 1 MMP (MT1-MMP), which catalyzes the cleavage of proMMP-2. To determine whether proMMP-2 processing was controlled directly by integrin binding or indirectly by associated changes in cell shape, round cells on low FN were allowed to bind to microbeads (4.5 microm diameter) coated with a synthetic RGD peptide or FN; these induce local integrin receptor clustering without altering cell shape. ProMMP-2 activation was significantly decreased within minutes after bead binding in these round cells, prior to any detectable changes in expression of MT1-MMP, whereas binding of beads coated with control ligands for other transmembrane receptors had no effect. This inhibitory effect was mimicked by microbeads coated with activating antibodies against alphaVbeta3 and beta1 integrins, suggesting a direct role for these cell-surface ECM receptors in modulating proMMP-2 activation. Similar inhibition of proMMP-2 processing by integrin binding, independent of cell spreading, was demonstrated in cells that were cultured on small, microfabricated adhesive islands that prevented cell spreading while presenting a high FN density directly beneath the cell. Interestingly, when spread cells were induced to round up from within by disrupting their actin cytoskeleton using cytochalasin D, proMMP-2 processing did not change at early times; however, increases in MT1-MMP mRNA levels and MMP-2 activation could be detected by 18 hours. Taken together, these results suggest the existence of two phases of MMP-2 regulation in HCE cells when they adhere to ECM: (1) a quick response, in which integrin clustering alone is sufficient to rapidly inhibit processing of proMMP-2 and (2) a slower response, in which subsequent cell spreading and changes in the actin cytoskeleton feed back to decrease expression of MT1-MMP mRNA and, thereby, further suppress cellular proteolytic activity.
PURPOSE: To investigate the effects of cholesterol-lowering statin drugs on trabecular meshwork cellular properties and aqueous humor outflow. METHODS: Primary cell cultures of porcine trabecular meshwork (PTM) and ciliary body (PCB) were treated with either lovastatin or compactin, to determine the effects of statins on cell shape, actin cytoskeletal organization, and cell-extracellular matrix interactions (focal adhesions) by immunofluorescence staining. Changes in myosin light-chain (MLC) phosphorylation were evaluated by Western blot analysis. Changes in Rho GTPase content of membrane fractions from lovastatin-treated PTM cells were assessed by Western blot analysis. A constant-flow, organ-culture perfusion system was used to measure the effects of statins on aqueous humor outflow facility in the anterior segments of porcine eyes. RESULTS: PTM and PCB cells treated with lovastatin or compactin exhibited dramatic changes in cell shape and cytoskeletal organization within 24 hours, consisting of cell rounding, actin depolymerization, and decreased focal adhesions. These effects were found to be reversible on supplementation with geranylgeranyl pyrophosphate. Both lovastatin and compactin decreased MLC phosphorylation in PTM and PCB cells. PTM cells treated with lovastatin exhibited marked decreases in membrane-bound Rho GTPase. In addition, perfusion of organ-cultured porcine eye anterior segments with 100 microM lovastatin for 96 hours caused a significant increase in aqueous humor outflow facility (110%) compared with control eyes, in a reversible manner. CONCLUSIONS: This study demonstrates that the statin drugs lovastatin and compactin induce changes in cell shape and actin cytoskeletal organization and decrease MLC phosphorylation in PTM and PCB cells, all of which are events that are likely to lead to cellular and tissue relaxation. In addition, these effects of the statins appear to be mediated by inhibition of isoprenylation of the small GTP-binding proteins such as Rho GTPase. An important finding is that statins exert an ocular hypotensive response in an organ-culture perfusion model, indicating the potential for this class of drugs in glaucoma therapy.
The ability of cells to respond to their surrounding environment and relay signals to the cell interior is essential for numerous processes during the development and maintenance of tissues. Eph receptors and their membrane-bound ligands, the ephrins, are unique in the receptor tyrosine kinase family in that their signaling is bidirectional, through both the receptor and the ligand. Eph receptors and ephrins are essential for a variety of biological processes, and play a particularly important role in regulating cell shape and cell movement. Recent data have linked Eph receptor-ephrin signaling complexes to the Ras and Rho families of small molecular weight GTPases and also to heterotrimeric G proteins. Understanding the signaling networks involved is an important step to understand the molecular basis for normal and defective cell-cell communication through Eph receptors and ephrins.
Type II pneumocytes are the stem cells for the alveolar epithelium, proliferating and differentiating into type I cells during lung growth or after injury. The pattern of cytokeratin expression by type II cells in vitro has been linked to the state of differentiation of these cells. In particular, cytokeratin 19 expression has been associated with the type II cell phenotype. We now examine the roles of cell shape and cell-cell interactions in the regulation of cytokeratin expression by rat type II cells in vitro. Type II cell spreading and intercellular contacts were modulated by seeding cultures at high or low density (3.5 or 0.5 x 10(5) cells/cm2). When cultured at high density, cells demonstrated increased cytokeratin 19 protein synthesis and diminished cytokeratin 18 protein synthesis compared with highly spread cells at low density. This effect was a reflection of changes in the abundance of mRNAs for the individual cytokeratins. Alveolar epithelial cells at high density also formed extensive desmosomes between the cells. Desmosome formation was significantly decreased when cells were seeded at low density or in reduced (0.05 mM) calcium medium. Cytokeratin 19 mRNA and protein expression were also significantly decreased when desmosome formation was inhibited in reduced calcium medium, while calcium content of the medium had little effect on cytokeratin 18. These studies suggest that type II cell expression of cytokeratin 19, a differentiation-related cytokeratin, is regulated by factors influencing cell shape and intercellular contacts between epithelial cells. They further suggest that cell-cell interactions between epithelial cells may play a role in the modulation of epithelial cell phenotype.
Voltage-dependent K+ currents in pear-shaped hair cells of the frog crista ampullaris were investigated in thin slice preparations using the whole-cell variant of the patch-clamp technique. Microscopy observation revealed that pear-shaped cells are located in intermediate and peripheral regions of the crista, whereas they are absent in the central region. Voltage-clamp recordings in cells from the peripheral regions revealed that the total outward K+ current could be separated pharmacologically into three distinct components: a A-type K+ current (IA); an inactivating calcium-activated K+ current (IK(Ca)) and a delayed rectifier K+ current (IK). IK and IK(Ca) exhibited similar magnitude and accounted for most of the membrane cell conductance. The same experimental protocol applied to cells from the intermediate regions showed the presence of a large and sustained IK(Ca) which represented 95% of the total outward current. In this region IA was absent. The present results demonstrated that pear-shaped hair cells located in two discrete regions of frog crista ampullaris exhibit a different complement of voltage-dependent conductances, suggesting that they can play a different role in processing the natural stimulus.
The WAVE complex is an essential regulator of actin-related protein (ARP) 2/3-dependent actin filament nucleation and cell shape change in migrating cells. Although the composition of the WAVE complex is well characterized, the cellular mechanisms that control its activity and localization are not well known. The 'distorted group' defines a set of Arabidopsis genes that are required to remodel the actin cytoskeleton and maintain the polarized elongation of branched, hair-like cells termed trichomes. Several loci within this group encode homologs of ARP2/3 subunits. In addition to trichome distortion, ARP2/3 subunit mutants have reduced shoot fresh weight and widespread defects in epidermal cell-cell adhesion. The precise cellular function of plant ARP2/3, and the means by which it is regulated, is not known. In this paper, we report that the 'distorted group' gene PIROGI encodes a homolog of the WAVE complex subunit SRA1. The similar cell shape and actin phenotypes of pir and ARP2/3 complex subunit mutants suggest that PIROGI positively regulates ARP2/3. PIROGI directly interacts with the small GTPase ATROP2 with isoform specificity and with selectivity for active forms of the protein. PIROGI shares only 30% amino acid identity with its human homolog. However, both WAVE subunit homologs are functionally interchangeable and display identical physical interactions with RHO family GTPases and the Arabidopsis homolog of the WAVE complex subunit NAP125. These results demonstrate the utility of the 'distorted group' mutants to study ARP2/3 complex functions from signaling input to cell shape output.
We report 19 Leydig cell tumors (LCTs) of the testis with adipose differentiation (n = 12) and/or spindle cell growth (n = 8) in patients 28-70 years of age; three tumors with adipose differentiation showed psammomatous calcifications, two of which also had foci of ossification. In eight tumors fat-like cells apparently derived from lipid accumulation within neoplastic Leydig cells and appeared as focal to prominent clusters in a background of vacuolated, neoplastic Leydig cells. The fat-like cells were usually immunoreactive for Leydig cell markers (inhibin-alpha, calretinin, and melan-A) but were typically strongly positive for the adipose tissue marker, S-100 protein, supporting a hybrid cell phenotype. Four tumors had fat of stromal derivation. In two of these there were intermixed mature adipocytes, but in two others only lipoblastic cells were present. These four tumors lacked vacuolated, neoplastic Leydig cells, and the fat cells in the single case studied were negative for inhibin-alpha and melan-A but positive for S-100. Three of the 12 LCTs with adipose differentiation were clinically malignant, and each had several of the established malignant features. Eight tumors with spindle cells occurred in men 34-70 years of age. Two tumors had ill-defined fascicles of spindle cells, and three showed prominent edematous to myxoid areas with spindle-shaped tumor cells. Two additional tumors had a fibroma-like spindled component that blended with islands of more plump, polygonal to spindle-shaped Leydig cells. Finally, one tumor had foci resembling an unclassified sarcoma that merged with conventional LCT; the spindle cell component in this case did not react for Leydig cell markers in contrast to the spindle cells in five of the six other cases in which immunostains were performed. Spindle cell differentiation, by itself, did not appear to have prognostic significance. Of the six patients with available follow-up, two developed metastases, but their tumors had malignant features apart from spindle cells; the remaining four patients were disease free at a mean of 3.6 years. Awareness of these unusual patterns in LCTs may prevent misinterpretation of fat admixed with neoplastic Leydig cells as evidence of extratesticular growth (a criterion for malignant LCT) may help avoid misdiagnosis of a LCT as a testicular "tumor" of the adrenogenital syndrome (which may contain fat) and may prevent misdiagnosis of a LCT with spindle cells as a sarcoma or unclassified sex cord-stromal tumor.
We studied the shape of L1210 leukaemia cells adhering in a protein-free medium to sulfonated (styrene/methyl methacrylate) copolymer surfaces of two sulfonic group densities, and thus of differing wettability. The use of our image analysis method and the mathematical procedure [Kowalczynska, H.M. et al, Colloids Surfaces B: Biointerfaces, 30 (2003) 193-206.] allowed us to calculate the values of the so-called shape parameter, which quantitatively determines the three-dimensional cell shape. Here, we show that the values of the shape parameter of the adhering cells and the F-actin concentration, in the region near the cell-substratum interface, depend on the density of sulfonic groups present on the substratum surface.
A procedure is described for the dispersion, partial purification, and identification of epithelial and nonepithelial cells from the rat ventral prostate, the latter based in part upon the ability of cells to bind androgen. Since initial efforts to mechanically disrupt prostatic tissue at 2 C lysed many cells, minced rat prostates were exposed to collagenase at 37 C and further dispersed by repetitive pipetting and passage through a tissue sieve. Washed cells in culture medium were centrifuged through an isokinetic Ficoll gradient from which three visible fraction (1, 2, and 4) and a less definite fraction 3 were harvested. Characterically, fraction 1 contained cellular debris; fraction 2 contained round nucleated cells, fewer elliptically shaped cells, red blood cells, and rare free cell nuclei; fraction 3 contained somewhat larger elliptical and round cells; and fraction 4 contained larger round and elliptically shaped cells. These were epithelial cells, as judged by electron microscopy. Isolated prostate cells from rats castrated for 24 h were incubated with [3H]testosterone; 80-90% of the retained radioactivity, the majority of which was dihydrotestosterone, was associated with washed cells from fraction 4. Similar results were obtained after in vivo administration of labeled androgen and subsequent analysis of radioactivity in cells from these fractions. Histochemically and enzymatically demonstrable formalin-insensitive acid phosphatase was increased in bands 3 and 4, which were enriched in epithelial cells. Many cells in fractions 2-4 were viable before and after exposure to Ficoll, as estimated by their ability to exclude trypan blue, incorporate radioactive uridine into RNA, generate cell monolayers during 1-4 weeks of culture, and actively metabolize androgens. Compared to fraction 4, cells from fraction 2, considered to be enriched in nonepithelial cells, actively metabolized but bound much less [3H]testosterone and its metabolites. A number of epithelial cells in fraction 4 isolated from prostates dissociated at 2 C were associated with typical-C-type RNA viruses.
Human fibroblast cultures derived from normal donors of different ages were examined for the proportion of spindle-shaped, sail-shaped, and large pleomorphic cells which constitute the cultures, and the capacities of these cells for unscheduled DNA synthesis (UDS). The large pleomorphic cells were most abundant in all cultures, followed in abundance by the sail-shaped and spindle-shaped cells. Major changes in the occurrence of the cells of different shape with increased donor age were not apparent, although the abundance of spindle-shaped cells decreased slightly with donor age. The large pleomorphic and sail-shaped cells exhibited similar UDS which was consistently greater than that of the spindle-shaped cells. No major changes in UDS were observed for either type of cell with increased donor age.
This in vitro study was undertaken to determine the changes in Ca2+ kinetics and cell shape of cultured putative glomerular mesangial cells in the rat in response to angiotensin II (ANG II). Intracellular Ca2+ ([Ca2+]i) was measured using quin 2. ANG II-stimulated Ca2+ efflux was also determined. ANG II induced rapid concentration-dependent increases in [Ca2+]i and Ca2+ efflux. ANG II also induced contraction of mesangial cells as assessed by alterations in cell shape. Even in Ca2+-free medium, ANG II increased [Ca2+]i and Ca2+ efflux, but to a lesser extent. Under this condition, contraction of mesangial cells induced by ANG II was also observed. Readdition of extracellular Ca2+ after the ANG II-induced increase in [Ca2+]i caused a second and slower [Ca2+]i increase. High potassium (50 mM) induced a change of [Ca2+]i, but to a lesser extent compared with the ANG II-induced change. The Ca2+ channel blocker verapamil (5 x 10(-5) M) partially inhibited ANG II-induced Ca2+ influx but totally blocked the increase in [Ca2+]i induced by high potassium. Verapamil did not inhibit ANG II-stimulated Ca2+ efflux or the change in cell shape. Dantrolene (10(-4) M), a blocker of Ca2+ release from endoplasmic reticulum, inhibited ANG II-stimulated Ca2+ efflux and change in cell shape. These results indicate that ANG II rapidly increases [Ca2+]i in cultured rat mesangial cells, in part by mobilizing Ca2+ from dantrolene-sensitive intracellular pools and in part through activation of receptor-operated and voltage-dependent Ca2+ channels. The [Ca2+]i mobilization, however, seems to be the primary modulator of initial glomerular mesangial cell contraction.