Reorganization Of Cytoskeletal Elements Research Articles

Measuring the phagocytic activity of cells.

Phagocytosis is a critical biological activity through which the host can protect itself from infectious and non-infectious environmental particles and remove unwanted host cells in order to maintain tissue homeostasis. Phagocytosis is an ancient, conserved process that is apparent in all multicellular organisms. The process of phagocytosis requires the recognition of ligands on particles by specific receptors expressed by the phagocyte that promote internalization via reorganization of cytoskeletal elements and directed formation of the phagosome. Subsequent phagosome-lysosome fusion delivers the contents for destruction and recycling in the acidic compartment. Significantly, receptor engagement and uptake can also trigger intracellular signaling pathways that initiate appropriate innate immune and pro-inflammatory or anti-inflammatory responses dependent upon the nature of the particle. The important benefits of phagocytosis to host survival are exemplified by the detrimental effects to health that occur when phagocytic efficiency is diminished. In an overview, we discuss the different experimental approaches or options that can be considered when investigating and determining the characteristics and quantification of phagocytic activity. These criteria will include choice of phagocytic cell type, selection, and method of labeling of particle for monitoring internalization, targeting of particles to specific receptors, and quantification of ingestion either at the single cell or at the population level. We provide two detailed examples of phagocytosis assays.

A Rho-Type GTPase, rho-4 , Is Required for Septation in Neurospora crassa

Proteins in the Rho family are small monomeric GTPases primarily involved in polarization, control of cell division, and reorganization of cytoskeletal elements. Phylogenetic analysis of predicted fungal Rho proteins suggests that a new Rho-type GTPase family, whose founding member is Rho4 from the archiascomycete Schizosaccharomyces pombe, is involved in septation. S. pombe rho4Delta mutants have multiple, abnormal septa. In contrast to S. pombe rho4Delta mutants, we show that strains containing rho-4 loss-of-function mutations in the filamentous fungus Neurospora crassa lead to a loss of septation. Epitope-tagged RHO-4 localized to septa and to the plasma membrane. In other fungi, the steps required for septation include formin, septin, and actin localization followed by cell wall synthesis and the completion of septation. rho-4 mutants were unable to form actin rings, showing that RHO-4 is required for actin ring formation. Characterization of strains containing activated alleles of rho-4 showed that RHO-4-GTP is likely to initiate new septum formation in N. crassa.

Sodium monochloroacetate causes cytotoxic effects, an increased lactate and pyruvate level and induces ultra structural and cytoskeletal alterations in cultured kidney and liver epithelial cells.

1. Monochloroacetic acid (MCAA) and its sodium salt, sodium monochloroacetate (SMCA) are widely used in chemical industries as intermediates in the synthesis of carboxymethylcellulose, phenoxyacetic acid, thioglycolic acid, glycine, indigoid dyes and others. Moreover, MCAA has been found as a by-product of the chlorination disinfection of drinking water and as an environmental contaminant of the atmosphere from the photodechlorination reactions of chlorinated hydrocarbons. Little is known about the mode of action of both compounds on the cellular level. From cases of accidental poisoning of man it is known that MCAA accumulates in liver and kidney. 2. In this study, the cytotoxicity of SMCA on cultured liver (Chang liver cells) and kidney epithelial cells of the proximal tubule (Opossum kidney cells) was investigated and its effect on metabolism, ultrastructure and organization of cytoskeleton was examined. 3. Independent from the growth state of the cells (proliferating or quiescent), the results clearly show that SMCA causes a dose-dependent decrease in cell viability after an exposure period of 24 h. In all experiments, proliferating cells were more sensitive than quiescent and confluent cells. Liver cells were less sensitive against SMCA treatment than kidney epithelial cells. In contrast to liver cells, kidney cells exhibited a dose-dependent decrease in cell volume. The decrease in cell viability was accompanied by an increase of lactate and pyruvate concentrations released into the culture medium. In the case of Opossum kidney cells, lactate and pyruvate levels increased 5 - 6-fold, whereas in the case of Chang liver cells the increase was approximately twofold. While the ultrastructure of liver cells remained unaltered after drug treatment, kidney cells exhibited cytoplasmic vacuolization, membraneous disruption and especially mitochondrial alterations. In accordance with the changes in the ultrastructure of Opossum cells, was the reorganization of cytoskeletal elements with an increased stress fiber network at the basolateral surface as well as a partial depolymerization of microtubules and vimentin filaments. A cytoskeletal reorganization was not observed for Chang liver cells after SMCA treatment. 4. The results demonstrate that SMCA causes a dose-dependent cytotoxicity which is accompanied by metabolic, mitochondrial and cytoskeletal alterations in the cells.

Regulation of cell substrate adhesion: effects of small galactosaminoglycan-containing proteoglycans.

Cell adhesion is a process which is initiated by the attachment of cells to specific sites in adhesive matrix proteins via cell surface receptors of the integrin family. This is followed by a reorganization of cytoskeletal elements which results in cell spreading and the formation of focal adhesion plaques. We have examined the effects of a class of small galactosaminoglycan-containing proteoglycans on the various stages of cell adhesion to fibronectin-coated substrates. Our results indicate that dermatan sulfate proteoglycans (DSPGs) derived from cartilage, as well as other related small proteoglycans, inhibit the initial attachment of CHO cells and rat embryo fibroblasts to substrates composed of the 105-kD cell-binding fibronectin fragment, but do not affect cell attachment to intact fibronectin. Although this effect involves binding of DSPGs to the substrate via the protein core, the intact proteoglycan is necessary for the observed activity. Isolated core proteins are inactive. The structural composition of the galactosaminoglycan chain does not appear to be functionally significant since both chondroitin sulfate and various dermatan sulfate proteoglycans of this family inhibit cell attachment to the fibronectin fragment. Neither the percentage of cells spread nor the mean area of spread cells adhering to substrates of intact fibronectin was significantly affected by the DSPGs. However, significantly fewer cells formed focal adhesions in the presence of DSPGs as compared with untreated control cells. These results suggest that the binding of small galactosaminoglycan-containing proteoglycans to a fibronectin substrate may affect several stages in the cell adhesion process.

Rapid and transient reorganization of the cytoskeleton in GH3B6 cells during short-term exposure to thyroliberin

The cytoskeletal organization of the rat pituitary tumor cell line GH3B6 was analysed using immunofluorescence, in basal conditions and after stimulation by thyroliberin (TRH). Under basal conditions, a dense and entangled cytoplasmic microtubule network, a perinuclear cage of cytokeratin fibers, and a diffuse distribution of F-actin were revealed. Short-term stimulation of these cells by TRH induces a first early phase of PRL release (0-2 min), concomitant with a rarefaction of cytoplasmic PRL-containing granules, followed by a second plateau phase (5-30 min), concomitant with modifications of the Golgi zone. We show that TRH induced early and transient modifications in the cytoskeletal distribution during these short periods of stimulation. First, after 2 min of stimulation, small fluorescent tubulin blebs appeared under the plasma membrane. Then, after 5 min they disappeared, and a thin actin network, accentuated by thicker fibers, organized transiently in the cytoplasm. After 30 min, the microtubules and cytokeratin networks had extended throughout the cytoplasm and the actin distribution was diffuse again. So, in this study, we have shown the existence of a parallelism between the redistribution of intracellular PRL compartments and the reorganization of cytoskeletal elements, during exposure to TRH. We could not clearly correlate these modifications with transduction mechanisms involved in TRH action.

An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion.

Thrombin, in addition to its central role in hemostasis, possesses diverse cellular bioregulatory functions implicated in wound healing, inflammation, and atherosclerosis. In the present study we demonstrate that thrombin molecules modified either at the procoagulant or catalytic sites induce endothelial cell (EC) adhesion, spreading, and cytoskeletal reorganization. The most potent adhesive thrombin analogue (NO2-alpha-thrombin) was obtained by nitration of tyrosine residues. The cell adhesion promoting activity of NO2-alpha-thrombin was blocked upon the formation of thrombin-antithrombin III (ATIII) complexes and by antiprothrombin antibodies, but was unaffected by hirudin. Arg-Gly-Asp-containing peptides, fully inhibited EC adhesion to NO2-alpha-thrombin, while synthetic peptides corresponding to thrombin "Loop B" mitogenic site and the thrombin-derived chemotactic fragment "CB67-129", were uneffective. Immunofluorescence studies indicated that EC adhesion to NO2-alpha-thrombin was followed by cell spreading, actin microfilament assembly, and formation of focal contacts. By the use of specific antibodies, the vitronectin (vn) receptor (alpha v beta 3) was found to be localized in clusters upon cell adhesion to NO2-alpha-thrombin. An anti alpha v beta 3 antibody blocked EC adhesion and spreading while antifibronectin (fn) receptor (alpha 5 beta 1) antibodies were uneffective. While native thrombin exhibited a very low cell attachment activity, thrombin that was incubated at 37 degrees C before coating of plastic surfaces induced EC attachment and spreading. We propose that under certain conditions the naturally hindered RGD domain within thrombin is exposed for interaction with alpha v beta 3 on EC. This in turn promotes cell adhesion, spreading, and reorganization of cytoskeletal elements, which may altogether contribute to repair mechanisms in the disturbed vessel wall. This study defines a new biological role of thrombin and characterizes a new recognition mechanism on EC for this molecule.

Association of African swine fever virus with the cytoskeleton

The association of African swine fever virus (ASFV) with the cytoskeleton was investigated. Immunofluorescent studies of ASFV infected cells with anti-ASFV serum showed a temporal and spatial development of viral inclusions which moved from a peripheral to a perinuclear location and fused to give a single large perinuclear factory. The migration and fusion of viral inclusions was inhibited by colchicine suggesting a function for microtubules in assembly site organization not previously described. Accumulation of virions outside the inclusions and inhibition of viral release was also observed in colchicine treated cells. Viral antigens and structural elements were retained on the cytoskeleton fraction of Triton X-100 extracted cells. Reorganization of cytoskeletal elements around the assembly sites was demonstrated by transmission electronmicroscopy and by immunofluorescent studies using monoclonal antibodies against actin, tubulin and vimentin. Intermediate filaments accumulated around the viral factories, microtubules were greatly decreased in number and microfilaments were reorganized in association with the plasma membrane. Bundles of 15 nm tubules of unknown origin were also observed around the assembly sites. The distribution of viral proteins in soluble, cytoskeleton and detergent insoluble nuclear fractions was studied by pulse-chase experiments with [ 35S]methionine. SDS-PAGE analysis showed the presence in the cytoskeletal and nuclear fractions of 150, 72, 38, 28, 19 and 15 kDa virus structural proteins which increased after a 5 h chase. Our results indicate a close association of ASFV replication with the cytoskeleton similar to events described during FV3 replication but which differ from those occurring in poxvirus-infected cells.

Structural features of cultured epithelial cells from the adult rat epididymis.

Epithelial cells isolated from the caput epididymidis of adult rats were placed in primary culture and examined daily for ten days for changes in external anatomy, reorganization of cytoskeletal components, maintenance of characteristic cytoplasmic features, and response to media formulated to minimize nonepithelial cell proliferation. Significant cell attachment to the substrate began after the first 24 hours of culture. After attachment, the cells underwent a progressive flattening and became closely applied to the substrate. This was accompanied by a redistribution of microvilli on the cell surface and a reorganization of cytoskeletal elements within the cell. After flattening, the cultured cells displayed an extensive array of 10-nm filaments which were associated with the desmosomes attaching adjacent cells. Immunofluorescence studies demonstrated that these were keratin-containing intermediate filaments and 2-D gel electrophoresis of intact cells and cell cytoskeletons revealed that a family of "keratin-like" polypeptides were major components of the cells. Epithelial cell attachment, morphology, and maintenance in the primary culture were unaffected by D-valine, cytosine arabinoside, or both; however, these agents, either individually or in combination, reduced significantly the number of cells incorporating 3H-thymidine. These data show that isolated epithelial cells retain some differentiated structural features that characterize the intact cell and that enriched cultures of epithelial cells can be maintained under conditions where fibroblast proliferation is inhibited.

Architecture and polypeptide composition of HeLa cytoskeletons: Modification of cytoarchitectural polypeptides during mitosis

Substrate-attached asynchronous HeLa cells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules, intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of α-actinin and 30% of β-tubulin). Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000 to 70,000 M r region as determined by staining with Coomassie blue and labelling with a mixture of 14C-labelled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerising protein from slime moulds, seven abundant acidic IEF ‡ ‡ IEF, isoelectric focusing. polypeptides were shown to be present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 M rmr) and six polypeptides (IEF 12, 68,000 M r; IEF 24, 56,000 M r; IEF 31, 50,000 M r; IEF 35, 49,000 M r; IEF 36, 48,500 M r and IEF 46, 43,500 M r) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis. Labelling of mitotic and interphase cells with [ 35S]methionine followed by one-dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.

Reorganization of cytoskeletal elements during transition of human monocytes into adherent macrophage-like cells

Reorganization of cytoskeletal elements during transition of human monocytes into adherent macrophage-like cells