模拟微重力诱导的细胞微丝变化影响COL1A1启动子活性

细胞骨架系统是细胞内的重力感受系统。已知微重力导致的细胞形态、功能、信号传导等多种变化均与细胞骨架系统变化有关,但微重力对相关基因调控的影响知之甚少。本研究以构建的基因工程细胞株（EGFP-ROS）为对象,以回转器模拟微重力效应,利用增强型绿色荧光蛋白（enhanced green fluorescence protein,EGFP）荧光半定量和细胞微丝荧光染色分析技术,探讨回转模拟微重力条件下,细胞微丝系统对Ⅰ型胶原α1链基因（collagen type Ialpha chain 1 gene,COL1A1）启动子活性的影响。空间飞行和回转模拟微重力后,细胞微丝解聚、张力纤维减少,表明微重力可降低细胞微丝结构的有序性,诱导细胞骨架重排。适合剂量的细胞松弛素B处理EGFP-ROS细胞诱导微丝骨架解聚,同时导致COL1A1启动子活性增加,细胞荧光强度增强,并呈现剂量依赖性。因此,一定程度的细胞微丝系统破坏将导致COL1A1启动子活性的增强,证明细胞微丝骨架系统参与了微重力对COL1A1启动子活性调节,且在微重力信号传导中起重要作用。     

Visualization of the same PtK2 cytoskeletons by both immunofluorescence and low power electron microscopy.

A procedure is described which allows the examination of the cytoskeleton of a single PtK2 cell first by immunofluorescence and then by electron microscopy after staining with uranyl acetate. The immunofluorescent patterns of these detergent resistant cytoskeletons elicited with various monospecific antibodies closely resemble the patterns found in whole cells. Comparison of the immunofluorescence and electron micrographs directly supports the previous assignments of actin, myosin, filamin, α-actinin and tropomyosin as proteins associated with microfilament bundles in non-muscle cells. Actin is also found associated with a fine lattice-like structure present both in the ruffles and lying above the microfilament bundles in the cell body. The tonofilament bundles present in PtK2 cytoskeletons are not decorated by antibodies directed against the proteins associated with microfilament bundles. Antibodies directed against tonofilaments decorate specifically this system and not the microfilament bundles.     

Role of fibronectin in the organisation of the cytoskeleton during the spreading of rat mammary epithelial cells.

The correlation between the extracellular deposition of fibronectin and the development of the actin-containing cytoskeleton was studied during the attachment and spreading of the rat mammary epithelial cell line Rama 25. During the initial phase of cell spreading, actin is localised in peripheral microfilament bundles. As cell spreading increases, the peripheral ring is displaced towards the perinuclear region. Fibronectin, deposited beneath the basal surface, co-localises with the actin-containing peripheral ring. The peripheral ring subsequently disappears and is replaced by a system of radial microfilaments that extend from the perinuclear region to the cell periphery. At this stage, there is no correlation between the distribution of fibronectin and actin. As cells form colonies, radial microfilament bundles are replaced by peripheral microfilament bundles which do not co-localise with fibronectin. Cells at the edges of colonies extend lamellae that contain microfilament stress fibres. In these structures there is co-localisation of actin, fibronectin and the a5 beta 1-integrin fibronectin receptor.     

Dimethylsulphoxide affects the organisation of microfilaments in the mouse oocyte

The effect of dimethylsulphoxide (DMSO) on microfilament organisation has been studied in the mouse oocyte after staining with (NBD)-phallacidin. The cortical actin meshwork was disrupted by exposure of oocytes to 1.5 M DMSO at 37 degrees C, and this disruption was associated with changes in the cell surface, especially microvilli length and distribution, as observed by scanning electron microscopy. The irregular distribution of actin filaments observed also appears to lead to an irregular expansion of the cell after DMSO removal. However, when exposure to DMSO was combined with cooling, the effects on the microfilament system were much reduced. The reversibility of DMSO action is considered and the potential implications of microfilament disruption on the viability and functions of the oocyte discussed.     

Regulation of tropomyosin expression in transformed granulosa cell lines with steroidogenic ability

The expression of the different tropomyosin isoforms was analyzed in primary granulosa cell cultures and in established granulosa cell lines cotransfected with SV40 and Ha-ras DNA which retain a high steroidogenic response to cAMP stimulation. In contrast to normal cells which greatly reduce the expression of all tropomyosin isoforms during development of steroidogenic ability, in the doubly transformed cells only the synthesis of the high molecular weight isoforms nos 2 and 3 was decreased. The expression of isoforms 1 and 5 was elevated in the cotransfected lines and that of tropomyosin 1 was further enhanced by cAMP stimulation. The increased synthesis of tropomyosins 1 and 5 is unique to SV40 transformation, since it was observed also in cells transfected with SV40 DNA alone. These cells displayed a well organized microfilament system, but have lost the ability to differentiate. The reduced expression of tropomyosins 2 and 3 and a poorly organized microfilament system appear to be a dominant feature of both the highly differentiated normal- and transformed-granulosa cells. It is suggested that the switches in tropomyosin isoform expression during development of the steroidogenic phenotype and in cell transformation may account for necessary changes in microfilament organization which accompany these cellular processes.     

pp60v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status

The mechanism by which Rous sarcoma virus (RSV) induces a reorganization of actin and its associated proteins and a reduction in microfilament bundles is at present poorly understood. To examine the relationship between the organization of the microfilament system and the polymerization state of actin after transformation, we have investigated these changes in a Rat-1 cell line transformed by LA29, a temperature-sensitive (ts) mutant of RSV. Parallel immunofluorescence and biochemical analysis demonstrated that LA29 pp60v-src was ts for tyrosine kinase activity and cytoskeletal association. Changes in the distribution and organization of actin, alpha-actinin and vinculin were dependent on the association of a kinase-active pp60v-src molecule with the detergent-insoluble cytoskeleton. Whilst there was a transformation-dependent loss of microfilament bundles, biochemical quantitation demonstrated that the polymerization state of the actin in both detergent-soluble and insoluble fractions of these cells grown at temperatures either permissive or restrictive for transformation was quantitatively unchanged. These results indicate that the loss of microfilament bundles after transformation is not due to a net depolymerization of filamentous actin but rather to a reorganization of polymeric actin from microfilament bundles and stress fibers to other polymeric forms within the cell. The polymeric nature of the actin in these cells was confirmed by electron microscopy of cytoskeletons and substrate-adherent membranes.     

Controlling the cortical actin motor

Actin is the essential force-generating component of the microfilament system, which powers numerous motile processes in eukaryotic cells and undergoes dynamic remodeling in response to different internal and external signaling. The ability of actin to polymerize into asymmetric filaments is the inherent property behind the site-directed force-generating capacity that operates during various intracellular movements and in surface protrusions. Not surprisingly, a broad variety of signaling pathways and components are involved in controlling and coordinating the activities of the actin microfilament system in a myriad of different interactions. The characterization of these processes has stimulated cell biologists for decades and has, as a consequence, resulted in a huge body of data. The purpose here is to present a cellular perspective on recent advances in our understanding of the microfilament system with respect to actin polymerization, filament structure and specific folding requirements.     

Studies on microplasmodia ofPhysarum polycephalum

Summary The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 m) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (m long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.     

Mechanisms of cellular adhesion : II. The interplay between adhesion, the cytoskeleton and morphology in substrate-attached cells

cAMP/theophylline exaggerates cell shape—whether the fibroblastic morphology of controls or the epithelioid shape of colchicine-treated cells. The ultrastructural basis is that cAMP/theophylline increases the number and linearity of microtubules and microfilament bundles, although where also treated with colchicine, the cells adopt a well-spread shape maintained by microfilament bundles alone. Since interference reflection microscopy shows that colchicine promotes the marked alignment of focal contacts (which terminate microfilament bundles) it is concluded that microtubules encourage angular cell form and modify the pattern of adhesions by influencing the directionality of microfilament bundle formation although they are inessential for the maintenance of the spread form or adhesion per se.     

Sensitivity of Fibroblasts and Their Cytoskeletons to Substratum Topographies: Topographic Guidance and Topographic Compensation by Micromachined Grooves of Different Dimensions

Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-μm-wide and 0.5-μm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.     

Human immunodeficiency virus (HIV)-1 proteins and cytoskeleton: partners in viral life and host cell death

Cytoskeletal components play a major role in the human immunodeficiency virus-1 (HIV-1) infection. A wide variety of molecules belonging to the microfilament system, including actin filaments and actin binding proteins, as well as microtubules have a key role in regulating both cell life and death. Cell shape maintenance, cell polarity and cell movements as well as cytoplasmic trafficking of molecules determining cell fate, including apoptosis, are in fact instructed by the cytoskeleton components. HIV infection and viral particle production seem to be controlled by cytoskeleton as well. Furthermore, HIV-associated apoptosis failure can also be regulated by the actin network function. In fact, HIV protein gp120 is able to induce cytoskeleton-driven polarization, thus sensitizing T cells to CD95/Fas-mediated apoptosis. The microfilament system seems thus to be a sort of cytoplasmic supervisor of the viral particle, the host cell and the bystander cell's very fate.     

MICROFILAMENTS AND CELL LOCOMOTION

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro. Such cells are found to contain two types of microfilament systems: First, a sheath of 50–70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes. Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell. The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network. Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited. Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis. Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity. The microfilament network is, therefore, indispensable for locomotion.     

Microfilament assembly is involved in B-cell apoptosis.

Crosslinking of the B-cell antigen receptor (BCR) initiates a chain of reactions which culminate in a number of biologic responses, including entry into the cell cycle or cell death. The signals and processes which lead to cell death are slowly being unraveled. Based on the dramatic changes in cell shape which occur during progression of the apoptotic response, activation of cytoskeletal assembly may be critical as this appears to be essential to the mitogenic response. In this study, we demonstrate that crosslinking of the human BCR with anti-IgM antibodies results in the rapid assembly of actin. Our data also suggest that this conversion of G- to F-actin may be a prerequisite for the apoptosis response, as prevention of this conversion by botulinum C2 toxin or cytochalasin D results in rescue of the cells from apoptosis. Prevention of tyrosine kinase activation, disruption of microfilament assembly, and rescue of B lymphocytes from apoptosis imply that tyrosine phosphorylation is needed for both microfilament assembly and apoptosis. In each instance where microfilament assembly is inhibited, anti-IgM-induced activation of the protease CPP32 (caspase) is also inhibited. Taken together, these results suggest that the microfilament system is actively involved in delivering signals for apoptosis.     

Destruction of microfilament bundles in mouse embryo fibroblasts treated with inhibitors of energy metabolism

Immunofluorescence with an antiactin antibody and electron microscopy were used to study the distribution of actin in cultured mouse fibroblasts during treatment with inhibitors of energy metabolism. The inhibitors induce gradual disorganization of actin-containing microfilament bundles. At the first stage of the process the bundles degrade into separate fragments; later only small patches of actin can be found in the inhibitor-treated cells. This transformation takes about 90 min and is fully reversible as microfilament bundles are recovered after incubation of the cells in the inhibitor-free growth medium. The inhibitors do not alter actin distribution in the presence of glucose. This shows that their action is due to a reduction of the ATP level in the cells. A 90 min incubation with the inhibitors does not markedly alter either the cell shape or the microtubule system. Inhibitors of the energy metabolism prevent cytochalasin action on cells. Cytochalasin B (CB) or cytochalasin D (CD) rapidly disorganize the microfilament bundles and cause cell arborization. However, microfilament bundle destruction in the cells incubated in the mixture of cytochalasin and any of the inhibitors requires 90 min and is not accompanied by dramatic changes in the cell morphology, so the process is indistinguishable from microfilament bundle destruction in the presence of the inhibitors alone.     

Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts

The distribution of microfilament bundles in cells was examined using antibodies to fibroblast myosin and indirect immunofluorescence microscopy. There is no correlation between the presence of bundles of microfilaments and normal growth control. A normal cell line (Balb/c 3T3) cultured on a poorly adhesive substratum showed no microfilament bundles. Similarly, a mutant cell line (AD6) with normal growth, but a rounded shape due to defective adhesiveness to substratum, showed no bundle formation. On the other hand, two transformed cell lines with a flat morphology (Swiss SV3T3 and Balb MSV-85) showed extensive bundle formation. When a transformed cell line with poor adhesiveness (MC5-5) was treated with CSP (a major surface glycoprotein of normal cells) which increases adhesiveness to substratum, the cells formed extensive microfilament bundles without any decrease in growth. We conclude that the distribution of microfilament bundles is related to adhesiveness to substratum and cell shape but not to growth properties.     

Distribution of microfilament bundles during rotation of the nucleus in 3T3 cells treated with monensin

Cytoskeletal aspects of monensin-treated 3T3 cells with rotating nuclei were studied by immunofluorescence. The pattern of intermediate filaments and microtubules appeared unchanged when compared with control cells having a stationary nucleus. In contrast, the actin microfilament bundles appeared to have a consistent distribution in cells with rotating nuclei. Typically, we did not find long microfilament bundles that traverse the length of the cytoplasm of cells that were fixed at the time of nuclear rotation. Instead, there was a local distribution of short microfilament bundles situated ventrally to the nucleus and oriented at various angles to one another and to the predominant distribution of microfilament bundles in the cell. The observations suggest that the actin cytoskeleton is reorganized locally before or during rotation of the nucleus.     

Curcumin inhibits cell motility and alters microfilament organization and function in prostate cancer cells

Curcumin is a dietary phytochemical associated with anti-tumorigenic effects, but the mechanisms by which it inhibits cancer cell growth and metastasis are not completely understood. For example, little information is available regarding the effects of curcumin on cytoskeletal organization and function. In this study, time-lapse video and immunofluorescence labeling methods were used to demonstrate that curcumin significantly alters microfilament organization and cell motility in PC-3 and LNCaP human prostate cancer cells in vitro. Curcumin rapidly arrests cell movements and subsequently alters cell shape in the highly motile PC-3 cell line, but has a less noticeable effect on the relatively immobile LNCaP cell line. Stress fibers are augmented, and the overall quantity of f-actin appears to increase in both types of cells following curcumin treatment. Cytochalasin B (CB) disrupts microfilament organization in both cell lines, and causes vigorous membrane blebbing in PC-3 cells, but not LNCaP cells. Pre-treatment of cells with curcumin suppresses changes in microfilament organization caused by CB, and blocks PC-3 membrane blebbing. At least some of the effects of curcumin appear to be mediated by protein kinase C (PKC), as treatment with the PKC inhibitor bisindolylmaleimide inhibits the ability of curcumin to block CB-induced membrane blebbing. These findings demonstrate that curcumin exerts significant effects on the actin cytoskeleton in prostate cancer cells, including altering microfilament organization and function. This is a novel observation that may represent an important mechanism by which curcumin functions as a chemopreventative agent, and as an inhibitor of angiogenesis and metastasis.     

Deep-etch visualization of the Sertoli cell (blood-testis) barrier in the boar.

The Sertoli cell (blood-testis) barrier in the boar was visualized by the freeze-fracture, deep-etch, rotary-replication technique. Three kinds of cross-bridging structures were clearly recognized in the following three ectoplasmic specialization (ES) regions; (1) cross-bridges in the intercellular space between adjacent Sertoli cell membranes; (2) cross-bridges in the space between the Sertoli cell membrane and microfilament bundles; and (3) cross-bridges in the space between microfilament bundles and subsurface cisternae. Results from immunolocalization, vinculin and alpha-actinin were recognized in the Sertoli cell barrier. Our findings show that these structural elements of the Sertoli cell barrier are held together by these cross-bridging structures, and provide important morphological evidence that implicates the ES in the dynamic function of the microfilament bundles of the Sertoli cell barrier.     

Cell locomotion, nerve elongation, and microfilaments

A basic difference in locomotion between migratory cells and nerves correlates with a difference in distribution of certain microfilament systems. Lattice filaments are present where extension and movement of cell surface occur in both cell types. Bundles of sheath filaments which bind heavy meromyosin, are present in migratory cells, where displacement of the cell soma over the substratum occurs, but absent from nerves, where the cell body and axon remain fixed upon the substratum and “locomotion” is restricted to the axonal tip. It is proposed that the microfilament lattice is involved in the extension phase of locomotion, and the microfilament sheath in the contractile phase.     

Cytoskeleton of mouse embryo fibroblasts. Electron microscopy of platinum replicas

We have studied the distribution of cytoskeletal elements in detergent-extracted mouse embryo fibroblasts using the platinum replica technique. It was shown that lamelloplasm can be subdivided into three zones: 1) the ruffle edge with dense microfilament meshwork; 2) the sparse zone adjacent to the ruffle edge and containing relatively few cytoskeletal elements; 3) the lamella proper occupied with a three-dimensional network of microfilaments, microtubules, intermediate filaments; this zone contained adhesion plaques corresponding to cell-substrate focal contacts and associated with the microfilament bundle ends. The cytoskeleton structure of the central (endoplasm) region of the cell was markedly different from that of the lamelloplasm. Its main feature was a dense microfilament sheath at the dorsal cell surface. Sites of microfilament bundle convergence can be visualized near the nucleus after partial removal of the sheath by more complete detergent extraction.