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PubMed2004

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts.

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.

Actins↗
PubMed1978

Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin- and tubulin-containing structures.

Cytoskeletons prepared by Triton X-100 treatment of tissue culture cells appear in stereo electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. Microfilament bundles and also tonofilament-like bundles are readily discerned when present in the cell type. In addition thinner fibers, some of which branch (smallest diameter 30--40 A), as well as fibers of larger diameter, some of which correspond to microtubules, can be seen. Since such cytoskeletons are an open, membrane-free system, individual fibrous organizations can be identified by specific antibodies. An indirect immunoferritin procedure using antibodies to tubulin or actin visualizes microtubules or actin-containing structures. Stereo electron microscopy of cytoskeletons decorated with actin antibody reveals, in addition to the F-actin-containing microfilament bundles, an extended fine actin lattice. This actin net is displayed throughout the cytoplasm not only between the microfilament bundles but also in those regions of the cytoskeleton that in the intact cell correspond to the submembraneous regions. Thus all actin-containing fibrous cytoplasmic structures may be interconnected in the living cell.

Actins↗
PubMed1998

Regulation of a calcium-dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet-derived growth factor. Dependence on calcium and the actin cytoskeleton.

A novel, p125FAK homologue, CADTK, has been detected in neural, epithelial, or hematopoietic cells but not in fibroblasts. We now demonstrate CADTK expression in a mesenchymal cell, rat aortic smooth muscle cells (RSMC). Angiotensin II (Ang II) or platelet-derived growth factor (PDGF-BB and PDGF-AA) markedly stimulated CADTK tyrosine phosphorylation in RSMC but did not affect p125FAK phosphorylation. The PDGF-depedent CADTK tyrosine phosphorylation was slower and more prolonged than that of Ang II, correlating well with the differential effects of these agonists on cytosolic calcium ([Ca2+]i) signaling. An intracellular calcium chelator inhibited both the rapid and sustained activation of CADTK by Ang II and PDGF. Extracellular calcium chelation inhibited the PDGF-stimulated increase in CADTK tyrosine phosphorylation as well as the sustained (but not the early) activation by Ang II. In contrast, p125FAK tyrosine phosphorylation was maximal in quiescent, adherent RSMC and was not affected by incubation with EGTA. Depletion of protein kinase C activity partially inhibited both the Ang II- and PDGF-induced CADTK tyrosine phosphorylation. Additional results confirm a relation between CADTK and the cytoskeleton. First, the tyrosine phosphorylation of paxilin correlated with activation of CADTK; this increase was inhibited by EGTA. Second, cytochalasin D blocked the PDGF- or Ang II-stimulated tyrosine phosphorylation of CADTK, suggesting a role for the cytoskeleton in agonist-dependent CADTK activation. Third, immunofluorescence analysis of CADTK localization demonstrated actin-like cytoskeleton staining extending into focal contacts. These results suggest that in mesenchymal cells, CADTK is localized to and activated by an actin cytoskeleton-dependent mechanism; a mechanism that is regulated in a calcium and protein kinase C-dependent manner independently of p125FAK.

Angiotensin II↗
PubMed1985

Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin.

Microvilli isolated from intestinal epithelial cells contain a cytoskeletal Mr 110,000 polypeptide complexed with calmodulin (110K-CM) that is believed to link the microfilament core bundle laterally to the plasma membrane. Previous work has shown that physiological levels of ATP can partially solubilize the 110K-CM complex from isolated microvillus cytoskeletons or isolated microvilli. However, once extracted, the 110K-CM complex has been found to be difficult to maintain stably soluble in aqueous buffers. This is due to the presence of an endogenous ATPase (approximately 100 nmol Pi/min per mg at 37 degrees C) in microvillus cytoskeletal preparations that depletes the ATP with subsequent precipitation of 110K-CM. Addition of ATP to such precipitates resolubilizes 110K-CM. Inclusion of an ATP regenerating system in the solubilization of 110K-CM from cytoskeletons, or membrane-bound brush borders, increases the amount of 110K-CM solubilized. Solubilization of 110K-CM from microvillus cytoskeletons was found to require a divalent cation (Mg2+, Mn2+, or Co2+, but not Zn2+) and a nucleoside triphosphate (ATP, GTP, CTP, or ITP). ADP did not solubilize 110K-CM, but could partially inhibit ATP-dependent solubilization. Solubilized 110K was phosphorylated during extraction of microvillus cores with [gamma-32P]ATP, but this was unrelated to the solubilization of 110K-CM as the endogenous kinase was specific for ATP, whereas the solubilization was not. The 110K-CM was purified using ATP extraction of brush border cytoskeletons in the presence of an ATP regenerating system, gel filtration of the solubilized extract, an ATP depletion step to specifically precipitate 110K-CM with F-actin, and resolubilization followed by phosphocellulose chromatography. The purified complex was stably soluble in aqueous buffers both in the presence and absence of ATP. It bound almost quantitatively to F-actin in the absence of ATP, and showed nucleotide solubilization characteristics from F-actin similar to that found for solubilization of 110K-CM from microvillus cores. At low ATP levels, the binding to F-actin was increased in the presence of ADP. These results suggest that the purified complex has been isolated in a native form. The data confirm and extend the studies of Howe and Mooseker (1983, J. Cell Biol., 97:974-985) using a partially purified preparation of 110K-CM and further emphasize that 110K-CM is a stably water soluble complex and not an integral membrane protein.

Actins↗
PubMed1984

Rapid modulation of N-formyl chemotactic peptide receptors on the surface of human granulocytes: formation of high-affinity ligand-receptor complexes in transient association with cytoskeleton.

When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4 degrees C. These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively. After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min. Exposure at 15 degrees C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37 degrees C. At 4 degrees C, complex formation was approximately 10% of the maximum amount formed at 37 degrees C. Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation. Electron microscopic autoradiography indicated that after 1 min of incubation at 37 degrees C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton. After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution. We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton.

Cell Membrane↗
PubMed1995

Jasplakinolide's inhibition of the growth of prostate carcinoma cells in vitro with disruption of the actin cytoskeleton.

BACKGROUND: Jasplakinolide, a cyclodepsipeptide produced by an Indo-Pacific sponge, Jaspis johnstoni, has been reported to inhibit the growth of breast cancer cells. PURPOSE: The effects of jasplakinolide on the proliferation of three human immortalized prostate carcinoma cell lines (PC-3, LNCaP, and TSU-Pr1) were studied. The growth-inhibitory effect of jasplakinolide on the PC-3 cell line was studied in detail to elucidate its mechanism of action. METHODS: Cell counts were used to study growth inhibition. A protein-based microplate assay was used to assess the time of exposure needed to cause persistent growth inhibition and to study the effects of jasplakinolide analogues. Metabolic changes were assessed by following the incorporation of radiolabeled precursors. The effects of jasplakinolide on the cytoskeleton were studied by fluorescent microscopy, using rhodamine phalloidin (RP) and antibodies to cytoskeletal components. Changes in RP binding were quantified by extracting bound fluorescent material from fixed cells and measuring the amount of fluorescence in a spectrofluorometer. RESULTS: The growth of PC-3, LNCaP, and TSU-Pr1 cells was potently inhibited by exposure to jasplakinolide for 48 hours; doses of jasplakinolide that led to 50% growth inhibition were 65 nM for PC-3 cells, 41 nM for LNCaP cells, and 170 nM for TSU-Pr1 cells. In PC-3 cells, exposure to 160 nM for 48 hours led to total growth inhibition, which persisted for several days even after drug removal. Several jasplakinolide analogues also inhibited the growth of PC-3 cells, although analogues in which the rigidity of the macrolide ring was altered were ineffective. No early changes in the synthesis of DNA, RNA, or protein or in intracellular adenosine triphosphate levels were seen in the PC-3 cells after exposure to jasplakinolide. Growth inhibition by jasplakinolide was accompanied by striking morphologic changes. Exposure for several doublings led to multinucleated cells. Further investigation of these changes in the PC-3 cells revealed a dramatic and early disruption of the actin cytoskeleton and a statistically significant decrease in RP binding. The doses of jasplakinolide, the time of exposure, and the pattern of growth inhibition by structural analogues corresponded with the changes seen in actin distribution. CONCLUSIONS: Jasplakinolide represents a novel marine natural product with potent in vitro antiproliferative activity against human prostate carcinoma cell lines, and it appears to target the actin cytoskeleton. IMPLICATIONS: Jasplakinolide is a potential candidate for further preclinical development and a lead structure for a novel class of therapeutic agents that can disrupt the actin cytoskeleton in mammalian cells.

Actins↗
PubMed1998

The chondramides: cytostatic agents from myxobacteria acting on the actin cytoskeleton.

BACKGROUND: Chondramides are cyclodepsipeptides produced by strains of the myxobacterium, Chondromyces crocatus. These peptides, which have been reported to inhibit yeast and mammalian cell proliferation, are related to jasplakinolide, which has been isolated from marine sponges of the genus Jaspis and has been shown to interfere with the actin cytoskeleton (a structural component of cells that helps maintain their shape and is involved in processes, such as cell division and cell locomotion). We studied the effects of the chondramides (A, B, C, and D) on tumor cell growth, on cytoskeletal structure, and on actin polymerization in vitro and compared these effects with those of cytochalasin D and jasplakinolide. METHODS: Cell proliferation was measured by means of tetrazolium salt reduction assays. Effects on the cytoskeleton were studied by use of fluorescence techniques, and actin polymerization in vitro was measured by means of viscosimetry. RESULTS: Proliferation of tested tumor cell lines was inhibited by the chondramides. Concentrations that inhibited proliferation by 50% (IC50 values) ranged from 3 to 85 nM and were of the same order of magnitude as those found for cytochalasin D and jasplakinolide. Fluorescence staining of potoroo cells incubated with chondramides A and B showed that organization of the actin cytoskeleton was disrupted; however, the microtubule system was not affected. Viscosimetric measurement showed that, depending on the experimental conditions, chondramide A induced or accelerated actin polymerization in vitro. CONCLUSION: The chondramides--unlike jasplakinolide--can be produced in large amounts by fermentation, and, similar to jasplakinolide, they appear to have antiproliferative activity against carcinoma cell lines by targeting the actin cytoskeleton.

Actins↗
PubMed1999

Impacts of aluminum on the cytoskeleton of the maize root apex. short-term effects on the distal part of the transition zone

Using monoclonal tubulin and actin antibodies, Al-mediated alterations to microtubules (MTs) and actin microfilaments (MFs) were shown to be most prominent in cells of the distal part of the transition zone (DTZ) of an Al-sensitive maize (Zea mays L.) cultivar. An early response to Al (1 h, 90 μM) was the depletion of MTs in cells of the DTZ, specifically in the outermost cortical cell file. However, no prominent changes to the MT cytoskeleton were found in elongating cells treated with Al for 1 h in spite of severe inhibition of root elongation. Al-induced early alterations to actin MFs were less dramatic and consisted of increased actin fluorescence of partially disintegrated MF arrays in cells of the DTZ. These tissue- and development-specific alterations to the cytoskeleton were preceded by and/or coincided with Al-induced depolarization of the plasma membrane and with callose formation, particularly in the outer cortex cells of the DTZ. Longer Al supplies (>6 h) led to progressive enhancements of lesions to the MT cytoskeleton in the epidermis and two to three outer cortex cell files. Our data show that the cytoskeleton in the cells of the DTZ is especially sensitive to Al, consistent with the recently proposed specific Al sensitivity of this unique, apical maize root zone.

Journal Article↗
PubMed2003

p38 MAP kinase mediates mechanically induced COX-2 and PG EP4 receptor expression in podocytes: implications for the actin cytoskeleton.

A dynamic cytoskeleton allows podocytes to withstand significant mechanical stress on elevation of intraglomerular capillary pressure (Pgc). However, vasoactive hormones, such as prostaglandin E2 (PGE2), may challenge the integrity of the actin cytoskeleton, alter podocyte morphology, and compromise glomerular permeability. PGE2 synthesis correlates with the onset of proteinuria and increased Pgc following reduced nephron mass. We investigated the interplay among mechanical stress, cyclooxygenase (COX), E-prostanoid (EP) receptor expression, and the actin cytoskeleton, using an in vitro model of cell stretch. Immortalized mouse podocytes grown on flexible silicone membranes were cyclically stretched (5% elongation, 0.5 Hz) for 2 h. EP4 and COX-2 mRNA increased three- and sevenfold above nonstretched controls, whereas EP1 and COX-1 levels were unchanged. Six hours of stretch resulted in a threefold increase in PGE2-stimulated cAMP accumulation, a measure of EP4 receptor function, and an increase in COX-2 protein. The stretch-induced effects on COX-2/EP4 expression and EP4-induced cAMP production were attributable to p38 MAP kinase, as blockade of this pathway, but not of ERK or JNK, abrogated the response. These stretch-induced changes in expression were transcriptionally dependent as they were actinomycin D sensitive. Finally, we investigated the influence of enhanced EP4 signaling on the actin cytoskeleton. Addition of PGE2 resulted in actin filament depolymerization observable only in stretched cells. Our results indicate that key components of the eicosanoid pathway are upregulated by mechanically stimulated p38 MAP kinase in podocytes. Enhanced EP4 receptor signaling may undermine podocyte cytoskeletal dynamics and thereby compromise filtration barrier function under conditions of increased Pgc.

Actins↗
PubMed1980

Cytoskeleton changes in fibroblast adhesion and detachment.

The organization of the cytoskeleton in several anchorage-dependent fibroblast types has been compared with the pattern of adhesions to a glass substratum which will support either their growth or just their spreading. Components were stained separately for immunofluorescence microscopy using specific antisera against actin, tubulin, and gizzard 10-nm filament protein, and the adhesions were visualized by interference-reflexion microscopy. Of the cytoskeleton features, only stress fibres could be related to the pattern of focal adhesions; as shown before, each focal adhesion lies directly beneath a stress fibre, often near the terminus. Cells spread on fibronectin-treated glass in serum-free medium to arrest the development of focal adhesions, show correspondingly underdeveloped stress fibres. Actin geodesic domes, microtubules, and 10-nm filaments showed no relations with the adhesion pattern. During cell rounding leading to detachment with either EGTA or trypsin, stress fibres begin to disperse in advance of shape change whereas microtubules and 10-nm filaments seem to alter their distribution as a consequence of shape change. We therefore confirm that stress fibres are the cytoskeleton features most directly related to focal adhesions and are cytoskeleton targets for 2 agents which cause rounding and hence detachment. The sequences of events in dispersal of stress fibres by EGTA and by trypsin showed significant differences in detail. With trypsin, fibres higher in the cell and terminating at the cell edge were more sensitive than most basal fibres and, during disintegration, all types of fibre went through an intermediate 'beaded' structure. With EGTA, all stress fibres seemed to be similarly susceptible and the beaded stage was not seen. The implications of these differences for our understanding of the mechanisms of dispersal of stress fibres are discussed.

Actins↗
PubMed2006

A hypothesis linking low folate intake to neural tube defects due to failure of post-translation methylations of the cytoskeleton.

Neural tube defects are serious congenital malformations which can be prevented by periconceptional folic acid supplementation. We hypothesize that folic acid provides the methyl group used for post-translational methylation of arginine and histidine in the highly conserved regulatory domains of the cytoskeleton and that these are required for neural tissue differentiation. Presumptive neural tissue has an unusually high need for folates due to the activity of phosphoethanolamine methyl transferase in producing neural tissue specific lipids at a time when the cytoskeleton is also competing for methylation. According to the cell state splitter hypothesis, the cytoskeleton is required to coordinate the spatial and temporal component of differentiation. When folate supply is low and the cytoskeleton is not methylated properly, the result is a neural tube defect due to failure of this coordination.

Animals↗
PubMed1984

Calmodulin-binding proteins in teleost retina, rod inner and outer segments, and rod cytoskeletons.

125I-calmodulin gel overlay techniques have been used to identify calmodulin-binding proteins in teleost retina, in a rod fragment preparation which contains rod inner and outer segments (RIS-ROS), and in RIS-ROS cytoskeletons. We have previously shown that teleost rods change length in response to changes in light conditions, that rod movement is mediated by the actin filaments in the rod inner segment, and that both Ca2+ and cAMP appear to be involved in regulating rod movement. We report here the development of a rod fragment preparation (RIS-ROS), which retains the movable part of the rod, for use in biochemical analysis of rod motility. Gel overlay studies indicate that isolated whole retinas have six prominent calmodulin-binding proteins, migrating at 240 K, 190 K, 150 K, 61 K and a doublet at 18/19 K. In contrast, detached RIS-ROS have three different prominent calmodulin-binding proteins, migrating at 330 K, 33 K, and 31 K. RIS-ROS cytoskeletons have been produced by extraction with Triton X-100; they contain both actin filament bundles and microtubules associated with the connecting cilium. RIS-ROS cytoskeletons have 3 prominent calmodulin-binding proteins migrating at 240 and 18/19 K. These proteins produce faint bands in gel overlays of intact RIS-ROS, but prominent bands in overlays of whole retina. The 240 K protein of RIS-ROS cytoskeletons co-migrates with the 240 K calmodulin-binding subunit of rat brain fodrin. We suggest that the rod 240 K calmodulin-binding protein may be a spectrin-like protein which participates in Ca2+- and calmodulin-regulation of rod motility.

Animals↗
PubMed1984

Regional differences in the neuronal cytoskeleton.

Regional differences in the neuronal cytoskeleton were investigated in the giant neurons of Aplysia. Using SDS-PAGE, we have compared the proteins which comprise the cytoskeletons of cell bodies and axons. Separate populations of cell bodies and axons were collected and the proteins stained by the Coomassie brilliant blue method. Individual identified cell bodies, with long segments of their axons attached, were isolated, and the proteins were labeled with the [125I]Bolton-Hunter reagent. The proteins which are stably associated with the cytoskeleton were obtained by extracting the neuronal material in a physiological buffer containing Triton X-100. As a correlative measure to the biochemical analyses, electron microscopy was performed on the cell body and axonal fractions. Our results demonstrate that the composition and biochemical properties of the cytoskeletal proteins in the neuron cell bodies differ from those associated with axons. Specifically, the amount of neurofilament proteins, designated NF60 and NF65 , is 5 times more abundant in the axon than in the cell body. The relative amounts of actin and tubulin are comparable in these two regions of the neuron. In addition, the ratio of NF60 and NF65 is different in the cell body and axon. The cell bodies contain proportionally more NF60 than the axons. However, the physical properties of the tubulin in the cell body, as measured by relative solubility, differ from that of the axon. The substantial differences between the composition of the cytoskeleton of the cell bodies and axons of Aplysia suggests that at least two distinct cytoskeletal networks exist in these neurons, one specific for the cell body and the other specific for the axon.

Animals↗
PubMed1981

The construction of the red cell cytoskeleton.

The state of actin in the erythrocyte membrane cytoskeleton has been examined. The presence of ADP, rather than ATP, as the predominant nucleotide species reinforces the view that actin occurs in the polymerized form. Redeterminations of the amounts of the three cytoskeletal proteins, spectrin, actin and 4.1, present in the cell allow evaluation of some of the stoichiometric constraints on the construction of the cytoskeleton. The available evidence is compatible with a network consisting of spectrin tetramers as the structural members, attached at both ends to junctions consisting of 4.1 and short filaments (or 'protofilaments') of actin. Electron micrographs of isolated cytoskeletons support such a picture. The dynamic state of the actin has been studied, using the DNAase assay method. The results indicate that whereas native monomeric actin is present in the cell, only a small proportion of the subunits of the protofilament can enter into an equilibrium with this pool. A considerable proportion of the cytoskeletal actin is not liberated under conditions that dissociate F-actin and is evidently tightly associated with spectrin and 4.1. Attention is drawn to the possible consequences of ATP depletion on the state of the actin and thus of the cytoskeleton.

Actins↗
PubMedDate not supplied

Cytoskeleton protein composition upon platelet stimulation with thrombin in essential thrombocythemia.

BACKGROUND: Structural and functional platelet alterations are present in essential thrombocythemia (E.T.). Platelet aggregation patterns are highly variable; moreover, structural, biochemical and metabolic platelet defects have also been shown. In this study, data on function and structure of platelets from a group of patients affected with E.T. are reported. Since cytoskeleton proteins are involved in activation and aggregation mechanisms, we investigated the cytoskeleton protein composition of resting, activated and aggregated platelets using electrophoretic analysis. PATIENTS AND METHODS: Studies were performed on 13 patients affected with E.T. On the basis of their functional behavior in response to thrombin, patients were divided into two groups: T1 (5 patients) and T2 (8 patients). A group of 11 healthy subjects was examined as control. Washed platelets were stimulated with 0.05 or 0.25 U/mL thrombin in the presence or the absence of EGTA in order to obtain activated or aggregated platelets. Triton-insoluble residues of activated and aggregated platelets were electrophoresed on 4.5-15% linear gradient SDS polyacrylamide gel, visualized by Coomassie blue stain and scanned with a densitometer. RESULTS: On the basis of the response to thrombin, group T1 was composed of subjects showing completely normal functional behaviour, while group T2 included patients who were more active than controls in response to low thrombin concentrations. By comparing the densitometric data of the two groups of patients it is possible to show that cytoskeleton structures in the T2 group have a significantly higher protein content than normals and than group T1. Platelet treatment with high thrombin concentrations cancels every difference between the two groups of patients with the exceptions of a 33-Kd protein and actin, which remain higher in group T2. CONCLUSION: Abnormal platelet function and quantitative cytoskeleton protein content can be correlated in patients affected with E.T. In the platelets of those patients who display higher responsiveness to thrombin, either altered calcium homeostasis or changes in membrane viscosity leading to altered thrombin receptor expression, or both these events, could be present.

Adult↗
PubMed1995

Evidence for thrombin-induced human platelet secretion regulated by the cytoskeleton.

Human blood platelets store an abundance of growth factors, adhensive proteins, coagulation factors, platelet-specific proteins, calcium, serotonin, and adenine nucleotides in their secretory organelles. After exposure to stimuli (thrombin, collagen, ADP, etc.), the platelets undergo a rapid series of morphological change from characteristic disks to spheres with several filopodia, adhension to the inner surface of blood vessels, and aggregation among the platelets. Most of these changes are usually accompanied by secretion or release of dense bodies (release I) and alpha-granules (release II). The secreted products play essential parts in a variety of important physiological and/or pathological events, such as hematosis, thrombosis, blood coagulation, inflammation, and atherosclerosis. Little is known, however, for regulatory mechanism of platelet secretion. Platelets are a distinct kind of cells with many special organelles, but without a nucleus. It also contains a large amount of cytoskeleton. The numerous investigators have suggested that the platelet responses to stimuli are intimately linked to events involving cytoskeleton within the platelets. During platelet activation, as revealed by electron microscopy, the secretory organelles become centralized and enveloped by circular microtubules and a filamentous network. It is implied a possible relationship between cytoskeleton and platelet secretion. However, the precise role of cytoskeleton in platelet secretion remains uncertain. By using exposed GMP-140 (a platelet granule-membrane protein with MW of 140 kDa) as a specific signal of secretion, the present study was designed to investigate the effects of microtubular and microfilamental inhibitors on thrombin-induced platelet secretion.

Adult↗
PubMed1991

Fine structure of the human synovial lining cell in osteoarthritis: its prominent cytoskeleton.

The cytoskeleton of the human osteoarthritic synovial lining cell (SLC) consists of an extensive number of vimentin intermediate filaments (IFs) in addition to microfilaments and microtubules. The IFs are especially prevalent in the SLC processes, but are commonly seen in a paranuclear arrangement. Processes, ending in numerous microvilli and blebs, project into the joint space. Scanning electron microscopy (SEM) further reveals the processes that may parallel the synovium surface for a short distance. IFs extend to the termination of such Numerous pinocytotic vesicles and extensive rough endoplasmic reticulum (rER) are characteristic of the type B cells. Lysosomes and long microvilli identify the type A cell. Punctate adherens, gap junctions, and cilia are the cell membrane specializations of the osteoarthritis (OA) synovium. A comparison with synovium from rheumatoid arthritis (RA) patients is made in order to assess the effect o this inflammatory disease on the SLC cytoskeleton, cell type relationship, and cell arrangement. The prominent cytoskeleton appears to play an important role in the architecture of the synovium. Our findings are further presented in the form of a drawing which in some aspects could describe the morphology of the normal synovium.

Actin Cytoskeleton↗
PubMed1989

Protein kinase C inhibitor H-7 alters the actin cytoskeleton of cultured cells.

The effects of the protein kinase C inhibitor H-7 on the actin cytoskeleton of cultured cells (Swiss 3T3 and PTK2) are described. As documented by fluorescence microscopy and the higher-resolution technique of photoelectron microscopy, the effects are rapid and dramatic; exposure to 30 microM H-7 in culture medium for less than 6 min is sufficient to induce a significant reduction in the numbers and thickness of actin microfilament bundles and alterations in the morphology of cell-cell boundaries in PTK2 cells. One-hour exposure to 30 microM H-7 results in nearly complete depletion of normal actin microfilament bundles from all of the cell types examined, without dramatic changes in overall cell shape. The intermediate filament and microtubule cytoskeletal networks did not appear to be affected to any extent over the times and doses examined. Forty-five minutes of exposure of Swiss 3T3 cells to 200 microM of either HA1004 (which is comparable to H-7 with respect to inhibition of cyclic nucleotide dependent kinases) or to the protein kinase C inhibitor sangivamycin did not induce the actin alterations characteristic of H-7. In addition, depletion of protein kinase C from Swiss 3T3 cells by means of phorbol ester-induced down-regulation did not prevent the effects of H-7 on the actin cytoskeleton. These results demonstrate that the protein kinase C inhibitor H-7 has a specific and rapid effect on the actin cytoskeleton, and furthermore H-7 may have biochemical effects beyond those mediated by inhibition of protein kinase C or the cyclic nucleotide dependent kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗