Sperm motility in the horseshoe crab. IV. Extracellular ions and intracellular pH are not mediators of motility initiation

Upon dilution into sea water, Limulus spermatozoa undergo a brief flurry of motility (duration < 60 sec), after which they are nonmotile until encountering a sperm motility initiating peptide (SMI) that emanates from eggs. Utilizing highly purified SMI extracts and simplified seawater formulations (from which individual ions have been deleted), we found that no specific extracellular ion is required for either dilution-initiated or SMI-initiated motility. Indeed, deletion of one ion (Na⁺) produced dilution-initiated motility of very long duration (several hours). When motility is initiated by SMI (in normal seawater) there is an increase in intracellular pH (pH_i), as indicated by the fluorescent probe, 9-amino acridine; however, this pH, change is not a trigger for motility. As a more general method examining ion movements, the fluorescent probe diS-C₃-(5) was used to qualitatively measure changes in the membrane potential of spermatozoa. Although crude SMI extracts caused membrane depolarization, further purification resulted in an almost complete separation of this activity from SMI, thus showing that SMI activation is apparently an electroneutral event. (The membrane-depolarizing factor has a molecular weight > 30,000 and does not initiate acrosome reactions.) Experiments utilizing the ionophore A23187 and Ca⁺²-blocking agents (verapamil and TMB-8) provided tentative evidence that mobilization of intracellular Ca⁺² may be required for motility initiation. These results show that neither changes in pH_i nor the influx of specific extracellular ions are direct mediators of SMI-initiated motility; however, experiments with pharmacologic agents indicate a possible role for intracellular Ca⁺².     

Differences in red cell shape of two inbred chicken lines

Summary Comparison of two inbred chicken lines (F_x > 99.9%) revealed significant differences in shape of the red blood cells (RBC). The length-width index was lower for both sexes in IC-line (1.46) when compared to CB-line chickens (1.88). Phenotypic expression of this character in F₁ hybrids and both backcross groups corresponded to the common manifestations of the metric parameters. The index in F₁ hybrid chickens deviated from intermediate values with the dominant tendency to oval RBC. An analysis of the segregating first backcross generation chickens did not show any association between RBC shape and the genotype in the blood group systems B, C, I, and D and the IgG allotypes. The differences in RBC shape were probably not associated with the survival of RBC in the blood circulation.     

Nature and origin of patterns of changes in cell shape in embryos

Spatial patterns of the future elongation of cells exist in the early embroyo. In the newt, such a pattern of changers of cell shape contributes to the formation of the neural plate. Regardless of where neural plate. Regardless of where neural plte cells are transplanted, they change shape as prescribed by tge pattern. Embryonic induction has a role in establishing this pattern.     

Effects of membrane reference state on shape memory of a red blood cell

By using a three-dimensional continuum model, we simulate the shape memory of a red blood cell after the remove of external forces. The purpose of this study is to illustrate the effect of membrane reference state on cell behavior during the recovery process. The reference state of an elastic element is the geometry with zero stress. Since the cell membrane is composed of cytoskeleton and lipid bilayer, both the reference states of cytoskeleton (RSC) and lipid bilayer (RSL) are considered. Results show that a non-spherical RSC can result in shape memory. The energy barrier due to non-spherical RSC is determined by the ratio of the equator length to the meridian length of the RSC. Thus different RSCs can have similar energy barrier and leading to identical recovery response. A series of simulations of more intermediate RSCs show that the recovery time scale is inversely proportional to the energy barrier. Comparing to spherical RSL, a spheroid RSL contributes to the energy barrier and recovery time. Furthermore, we observe a folding recovery due to the biconcave RSL which is different from the tank treading recovery. These results may motivate novel numerical and experimental studies to determine the exact RSC and RSL.     

Ameboid cell motility: a model and inverse problem, with an application to live cell imaging data

In this article a mathematical model for ameboid cell movement is developed using a spring-dashpot system with Newtonian dynamics. The model is based on the facts that the cytoskeleton plays a primary role for cell motility and that the cytoplasm is viscoelastic. Based on the model, the inverse problem can be posed: if a structure like a spring-dashpot system is embedded into the living cell, what kind of characteristic properties must the structure have in order to reproduce a given movement of the cell? This inverse problem is the primary topic of this paper. On one side the model mimics some features of the movement, and on the other side, the solution to the inverse problem provides model parameters that give some insight, principally into the mechanical aspect, but also, through qualitative reasoning, into chemical and biophysical aspects of the cell. Moreover, this analysis can be done locally or globally and in different media by using the simplest possible information: positions of the cell and nuclear membranes. It is shown that the model and solution to the inverse problem for simulated data sets are highly accurate. An application to a set of live cell imaging data obtained from random movements of a human brain tumor cell (U87-MG human glioblastoma cell line) then provides an example of the efficiency of the model, through the solution of its inverse problem, as a way of understanding experimental data.     

A genome-wide resource of cell cycle and cell shape genes of fission yeast

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li⁺>Na⁺=K⁺>Rb⁺. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na⁺ or K⁺ incubation, whereas Li⁺ leads to flabby, flattened cells with a certain tendency to crenation, and Rb⁺ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li⁺. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

Auxin-dependent control of cytoskeleton and cell shape regulates division orientation in the Arabidopsis embryo

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Septins regulate border cell surface geometry,shape, and motility downstream of Rho in Drosophila

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Bilayer balance and regulation of red cell shape changes

Discocytic human red cells undergo discocyte-echinocyte and discocytestomatocyte transformations under the action of a wide variety of lipid-soluble anionic and cationic agents respectively. These shape transformations are explained by the bilayer couple hypothesis of Sheetz and Singer to be the result of preferential distribution of the anionic agents in the outer half of the bilayer and the cationic agents in the inner half of the bilayer. We demonstrate that echinocytogenic effects indeed occur when the naturally occurring phospholipid lysophosphatidylcholine (LPC) is localized in the outer half of the bilayer, and stomatocytogenic effects occur when LPC is in the inner half. However, in contrast to the bilayer couple hypothesis, our results show that simple equivalent membrane surface area expansion on each layer is insufficient to maintain the discocytic shape and there exists a differential concentration effect of LPC on the two halves of the bilayer.     

The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro

Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.     

Extracellular matrix- and cytoskeleton-dependent changes in cell shape and stiffness

Cell spreading is correlated with changes in important cell functions including DNA synthesis, motility, and differentiation. Spreading is accompanied by a complex reorganization of the cytoskeleton that can be related to changes in cell stiffness. While cytoskeletal organization and the resulting cell stiffness have been studied in motile cells such as fibroblasts, less is known of these events in nonmigratory, epithelial cells. Hence, we examined the relationship between cell function, spreading, and stiffness, as measured by atomic force microscopy. Cell stiffness increased with spreading on a high density of fibronectin (1000 ng/cm(2)) but remained low in cells that stayed rounded on a low fibronectin density (1 ng/cm(2)). Disrupting actin or myosin had the same effect of inhibiting spreading, but had different effects on stiffness. Disrupting f-actin assembly lowered both stiffness and spreading, while inhibiting myosin light chain kinase inhibited spreading but increased cell stiffness. However, disrupting either actin or myosin inhibited DNA synthesis. These results demonstrate the relationship between cell stiffness and spreading in hepatocytes. They specifically show that normal actin and myosin function is required for hepatocyte spreading and DNA synthesis and demonstrate opposing effects on cell stiffness upon disruption of actin and myosin.     

Loss of O-antigen increases cell shape abnormalities in penicillin-binding protein mutants of Escherichia coli

Escherichia coli mutants lacking multiple penicillin-binding proteins (PBPs) produce aberrantly shaped cells. However, most of these experiments have been performed in E. coli K12 strains, which do not attach a complete O-antigen to their outer membrane lipopolysaccharide. We constructed mutants in different genetic backgrounds and found that the frequency of morphological deformities was higher in strains lacking the O-antigen. Also, complementing O-negative mutants with a heterologous O-antigen from Klebsiella returned a substantial fraction of misshapen cells to a normal morphology. Thus, the O-antigen contributes to cell shape in E. coli, perhaps by reducing the number of ectopic poles, which may be the proximal cause of shape abnormalities.     

Intracellular localization of sirtuin and cell length analysis of Lactobacillus paracasei suggest possible role of sirtuin in cell division and cell shape regulation

Sirtuin has been associated in prolonging lifespan of different model organisms. It has been shown to have an enzymatic activity of NAD⁺-dependent protein deacetylation targeting acetylated proteins. To determine targets and possible roles of sirtuin (LpSirA) in the Lactobacillus paracasei BL23, deletion (ΔsirA), sirtuin overexpressor (highsirA) and GFP fusion (highsirA-Venus) strains were generated, and microscopic localization and cell length analysis were done. Microscopic analysis revealed localization of LpSirA at cell division plates, at cell poles and all throughout the cell length in a spiral manner. Cell length analysis revealed that 46.9% of the ΔsirA cells were observed to be shorter (<2 μm), whereas 12.6% of the highsirA cells were observed to be longer (>4 μm) in comparison with the wild-type with only 17.1% short cells and 5.3% long cells. Our results suggest that sirtuin may have an essential role in cell division and cell shape regulation.     

Modulation of adrenal cell functions by cadmium salts. 4. Ca2+-dependent sites affected by CdCl2 during basal and ACTH-stimulated steroid synthesis

In previous studies, nonlethal CdCl₂ concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl₂ inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl₂. Since CdCl₂ competed at metabolic steps requiring Ca²⁺ in other tissues, experiments were designed to examine Cd²⁺ competition with Ca²⁺ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl₂ were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl₂ in the presence or absence of EGTA, a relatively specific Ca²⁺, but not Cd²⁺, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl₂, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca²⁺ transfer across plasma membranes. Besides determining Ca²⁺ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca²⁺ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mol/L fura-2, cells remained untreated or medium was infused with CdCl₂, ACTH, ACTH/CdCl₂ or ACTH followed after 50 s by CdCl₂. Using Ca²⁺-supplemented media, we observed that Cd²⁺ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca²⁺-containing medium supplemented with Ca²⁺ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd²⁺-treated cells was only reduced by EGTA, when Ca²⁺ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd²⁺-treated cells, suggesting that extracellular Ca²⁺ resources may compete against Cd²⁺ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca²⁺ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca²⁺ concentrations before returning to basal, steady-state levels. Cd²⁺ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd²⁺ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl₂-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd²⁺ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd²⁺-induced hyperbolic rise in intracellular Cd²⁺. These fluorescence measurements suggested that cytoplasmic Ca²⁺ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca²⁺ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd²⁺ freely enters the cell under basal conditions and Cd²⁺ entry is accelerated by ACTH stimulation. Data were consistent with Ca²⁺ being required for optimal stimulated steroid production and Cd²⁺ probably competing with Ca²⁺ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation.     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Ultrastructural observations on changes in cell shape in chromatophores of the sea urchin Centrostephanus longispinus

Summary Alterations in cell shape of the light-sensitive chromatophores of the sea urchin Centrostephanus longispinus were studied by scanning- and transmission electron microscopy. Transition of the aggregated to the dispersed state is accompanied by incorporation of vesicles into the membrane of the pigment cell. During dispersion a system of microtubules originating from centriole-like structures is established throughout the stellate cell. Within restricted areas of the cell, cytoplasmic differentiation and condensation is found. The possible functional significance of the findings is briefly discussed.     

Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport

The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin alpha5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin alpha5, whereas Rad associates with importins alpha3, alpha5 and beta through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex 'in situ' as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function.     

Relationship between the shape and the membrane potential of human red blood cells

Summary Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, orE _m , is set in the range –85 to +10 mV with valinomycin at varied external K⁺, or K _o .E _m was measured with the fluorescent potentiometric indicator, diS-C₃(5), as calibrated by a pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated toE _m . The effects of increased intracellular Ca²⁺, or Ca _o , on echinocytosis and onE _m are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Ca _o than that which induces the half-maximal hyperpolarization associated with Ca-induced K⁺ conductance (Gardos effect). Thus, cells hyperpolarized by increased Ca _o remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Ca _o , extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low K _o , a new observation consistent with increased DIDS-sensitive uptake of⁴⁵Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Ca.