Maintaining Cell Morphology Research Articles

Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis

The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

Localization of caspase 12 in masseter muscle of mdx mice during regeneration

The mdx mouse, a model of muscular dystrophy, lacks dystrophin, a cell membrane protein. It is known that this lack of dystrophin results in muscle fiber necrosis from 2 weeks after birth, and the majority of necrotic fibers are replaced by regenerated fibers by 4 weeks of age. Recent studies reported the detection of mitochondrial and endoplasmic reticulum stress proteins during muscle fiber necrosis in mdx mice, but did not histologically localize them to determine the timing of their expression during the process from cell necrosis to regeneration. Therefore, in this study, we investigated histological localization and gene-level expression in the mdx mouse masseter muscle of caspase-12 protein (among the caspases, which are cell stress-related genes) involved in the endoplasmic reticulum stress pathway. We observed caspase-12 expression in muscle cells that seemed to be in the process of necrosis in the mdx mouse masseter muscle at 2 weeks after birth, but not in regenerated muscle cells with centrally located nuclei observed at 3 to 4 weeks of age. These results suggest that due to the lack of dystrophin, it becomes difficult for muscle cells to maintain their morphology, and endoplasmic reticulum stress occurs to maintain cell morphology during the process of cell necrosis.

Activation of caspase 3, 9, 12, and Bax in masseter muscle of mdx mice during necrosis

The mdx mouse, a model of muscular dystrophy, lacks dystrophin, a cell membrane protein. It is known that the lack of dystrophin causes muscle fiber necrosis from 2 weeks after birth, and the majority of necrotic muscle fibers are replaced by regenerated muscle fibers by 4 weeks after birth. A recent study indicated the possibility that mitochondria-mediated intracellular stress, a phenomenon similar to apoptosis, may be produced during muscle fiber necrosis, but did not analyze endoplasmic reticulum-mediated intracellular stress. Therefore, we examined the expression of the caspase-12 gene involved in the endoplasmic reticulum stress pathway and the Bax, caspase-9, and caspase-3 genes involved in the mitochondrial stress pathway in the mdx masseter muscle. We found over-expression of caspase-12 in cells at 2-3 weeks after birth when muscle fiber necrosis was not prominent. This suggests that stress occurs in the endoplasmic reticulum to maintain cell morphology in the absence of dystrophin. In addition, Bax was abundantly expressed in the mdx masseter muscle at 3 weeks after birth, and the expression of caspase-9 and -3 was prominent at 3-4 weeks after birth when necrosis and regeneration were marked. These results indicate that endoplasmic reticulum and mitochondrial stresses are produced during necrosis of the mdx masseter muscle, and suggest that these events are a phenomenon similar to apoptosis.

Increased Association of Dynamin II with Myosin II in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin II has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin II and myosin II. The dynamin II expression level was higher when co-expressed with myosin II in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin II interacted with myosin II. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin II and myosin II gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

Opposing Roles of Zyxin/LPP ACTA Repeats and the LIM Domain Region in Cell-Cell Adhesion

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cell-cell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherin-actin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cell-cell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane‐bound complex

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.

Microfilament actin remodeling as a potential target for cancer drug development.

Actin was first identified in non-muscle cells only about three decades ago, and at about the same time, it was found that actin filaments were disrupted in the malignant transformed cells. The actin network is a rather complex, yet important structural and functional system of all eukaryotic cells. Actin filaments provide the basic infrastructure for maintaining cell morphology and functions such as adhesion, motility, exocytosis, endocytosis, and cell division. Growing evidence from this laboratory and others shows that alterations of actin polymerization, or actin remodeling, plays a pivotal role in regulating the morphologic and phenotypic events of a malignant cell. Actin remodeling is the result of activation of oncogenic actin signaling pathways (e.g., Ras and Src), or inactivation of several important actin-binding proteins that have tumor suppressor functions (e.g., gelsolin). Distinctive protein expression patterns of some of these genes in cancer and progressive carcinogenic processes have been observed. It has become evident that actin dynamics are regulated by a complex interplay of the small GTPase proteins of Ras superfamily Rac, Rho, and Cdc42, and efforts to develop specific inhibitors for these small G proteins as anticancer drug are underway. In this review we will discuss how actin remodeling is altered in the malignant transformation process, the functional significance of actin alteration in association with malignant phenotypes, and the approaches of targeting actin remodeling for chemopreventive and chemotherapeutic drug development. Approaches including using nature products directly modulating actin polymerization, using inhibitors of actin pathway small G proteins, and using gene-augmentation for actin binding proteins will be discussed. In addition, the concept of using F/G-actin ratio as a surrogate marker for actin-pathway based therapy will also be introduced.

Assessment of microtubule stabilizers by semiautomated in vitro microtubule protein polymerization and mitotic block assays.

Paclitaxel (Taxol) a clinically active anticancer agent, exerts its cytotoxicity by inducing tubulin polymerization, leading to cellular mitotic block. In contrast, other antimitotic drugs, such as colchicine, podophyllotoxin, and vinblastine, act by depolymerizing microtubules. We report here (a) a semiautomated assay which measures the tubulin-polymerizing activity of paclitaxel analogs and (b) a cellular assay to measure the potential of these compounds to block cells in mitosis. The microtubule-polymerizing assay measured the turbidity of bovine brain microtubule protein (MTP) polymerized by the test compound in a 96-well plate. We maximized the sensitivity of this assay by conducting the polymerization reaction at 20 degrees C, at which temperature the baseline reaction, i.e. the basic ability of the untreated MTP control to polymerize, was minimal. At 20 degrees C, the effect of 0.05 microg/ml of paclitaxel on MTP could be detected, whereas at 37 degrees C, > 1 microg/ml of paclitaxel was required to detect a significant effect relative to untreated MTP. We describe the analysis of the complex curves of MTP polymerization with varying concentrations of test compounds. The polymerization of microtubules leads to cells being blocked in mitosis. This mitotic blocking effect in intact cells was determined using a cell settling chamber which allowed eight samples to be deposited on a slide. This method required a smaller number of cells (10(3) - 10[5]), maintained cell morphology, and allowed for rapid screening of samples. The activity of several new paclitaxel analogs is reported.

The fission yeast sts5+ gene is required for maintenance of growth polarity and functionally interacts with protein kinase C and an osmosensing MAP-kinase pathway.

Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division. Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed. Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth. The sts5 mutant is not defective in cell wall integrity. Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal. Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation. Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation. The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis. The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants. Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed. Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting. A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure. These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.