Role of Fyn in the rearrangement of tubulin cytoskeleton induced through TCR

The translocation of the microtubule-organizing center (MTOC), its associated signaling complex, and the secretory apparatus is the most characteristic early event that involves the tubulin cytoskeleton of T or NK cells after their interaction with APC or target cells. Our results show that Fyn kinase activity is essential for MTOC reorientation in an Ag-dependent system. Moreover, T cells from Fyn-deficient mice are unable to rearrange their tubulin cytoskeleton in response to anti-CD3-coated beads. Analysis of conjugates of T cells from transgenic OT-I mice with dendritic cells revealed that an antagonist peptide induces translocation of the MTOC, and that this process is impaired in T cells from Fyn(-/-) OT-I mice. In addition, Fyn deficiency significantly affects the MTOC relocation mediated by agonist peptide stimulation. These results reveal Fyn to be a key regulator of tubulin cytoskeleton reorganization in T cells.     

Persistent changes in gene expression induced by estrogen and progesterone in the rat mammary gland.

Epidemiological studies have consistently shown that an early full-term pregnancy is protective against breast cancer. We hypothesize that the hormonal milieu that is present during pregnancy results in persistent changes in the pattern of gene expression in the mammary gland, leading to permanent changes in cell fate that determine the subsequent proliferative response of the gland. To investigate this hypothesis, we have used suppression subtractive hybridization to identify genes that are persistently up-regulated in the glands of E- and progesterone (P)-treated Wistar-Furth rats 28 d after steroid hormone treatment compared with age-matched virgins. Using this approach, a number of genes displaying persistent altered expression in response to previous treatment with E and P were identified. Two markers have been characterized in greater detail: RbAp46 and a novel gene that specifies a noncoding RNA (designated G.B7). Both were persistently up-regulated in the lobules of the regressed gland and required previous treatment with both E and P for maximal persistent expression. RbAp46 has been implicated in a number of complexes involving chromatin remodeling, suggesting a mechanism whereby epigenetic factors responsible for persistent changes in gene expression may be related to the determination of cell fate. These results provide the first support at the molecular level for the hypothesis that hormone-induced persistent changes in gene expression are present in the involuted mammary gland.     

The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate

Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton.     

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.     

Clustering of cell surface laminin enhances its association with the cytoskeleton

In order to provide evidence for an association of cell surface laminin with the cytoskeleton, we have examined the detergent extractability of cell surface laminin on murine fibrosarcoma cells. We utilized indirect immunofluorescence with affinity-purified anti-laminin antibodies to determine the distribution, mobility and detergent extractability of laminin bound to the cell surface. We demonstrate that antibody induces clustering of cell surface laminin rendering it resistant to detergent extraction. At low receptor occupancy, approx. 80% of cell surface laminin is detergent-extractable. If cell surface laminin is induced to cluster with anti-laminin antibody, IB4 isolectin from Bandeiraea simplicifolia or by high receptor occupancy, then it is rendered resistant to detergent extraction. This process is temperature-sensitive and inhibited by cytochalasin D (CD). On the basis of these findings, we propose a model in which laminin anchored in the basement membrane in vivo affects the cellular cytoskeleton by facilitating the clustering of cell surface transmembrane laminin receptors which are able to interact with cellular actin.     

Association of heparan sulfate proteoglycan and laminin with the cytoskeleton in rat liver

Rats were injected with 35SO4 and after 2 h their livers were removed and used to prepare a detergent-insoluble cytoskeleton fraction. Spectrin, cytokeratins, and actin were major protein components of the isolated cytoskeletons. The cytoskeleton fraction accounted for approximately 14% of the total trichloroacetic acid-insoluble 35SO4 radioactivity incorporated into the liver. The cytoskeleton-associated radioactivity was present in a single species of macromolecule. This molecule was not present to a significant extent in the detergent-soluble fraction containing the cell supernatant and dissolved membrane proteins. Further characterization revealed the cytoskeleton-associated molecule was a heparan sulfate proteoglycan: it was eluted from a Sepharose CL-4B column under denaturing conditions at Kav = 0.4; following mild alkaline hydrolysis the radioactivity was eluted at a Kav = 0.7; when this material was subjected to nitrous acid hydrolysis all of the radioactivity was eluted near the column included volume. The isolated cytoskeletons contained attached nuclei. Pure nuclei isolated without associated cytoskeletal elements contained less than 1% of the total liver trichloroacetic acid-insoluble 35SO4 radioactivity and no detectable heparan sulfate proteoglycan. These results suggested that other matrix proteins might be associated with the liver cytoskeleton. When the subcellular distribution of laminin was monitored by immunostaining proteins transferred to nitrocellulose, laminin was detected exclusively in the cytoskeleton fraction. These results provide evidence for an association between extracellular connective tissue proteins and intracellular structural proteins.     

From laminin to lamin: regulation of tissue-specific gene expression by the ECM

Mammary epithelial cells need a laminin-rich extracellular matrix (ECM) to achieve a functionally differentiated phenotype that includes secretion of milk-specific proteins such as beta-casein. There is good evidence that ECM-induced expression of beta-casein involves an 'ECM-response element' in the promoter of the casein gene that is activated by integrin-mediated signalling. This article proposes that ECM-induced structural changes in the cytoskeleton, histone organization and the nuclear matrix contribute to this tissue-specific gene expression.     

Role of the cytoskeleton in choanoflagellate lorica assembly

ABSTRACT. Cell division in Acanthoeca spectabilis produces a "naked" motile daughter cell (juvenile) that settles onto a surface and deposits siliceous costal strips that are stored extracellularly in bundles. When complete, the bundles of strips are assembled in a single continuous movement to form a basket-like lorica. Assembly can be divided into four overlapping stages. Stage 1 entails the left-handed rotation of strips at the anterior end while the posterior end remains stationary. Stage 2 includes the posterior protrusion of the cell to form a stalk. Stage 3 involves the anterior extension of the spines, and Stage 4 the dilation of the lorica chamber and deposition of the organic investment. Scanning electron microscopic images reveal a one-to-one association between the moving bundles of strips and the anterior ring of lorica-assembling tentacles. Treatment with microtubule inhibitors produces "dwarf" cells that lack stalks, have their spines extended, and possess collars but lack flagella. Treatment with microfilament (actin) inhibitors prevents extension of the anterior spines. These experiments demonstrate that posterior cell extension is primarily mediated by microtubules whereas extension of the spines is controlled by the actin cytoskeleton. The processes of cytoskeletal rotation and extracellular costal strip movement are compared, respectively, with rotation of nuclei in animal embryos and movement of mammalian cells over surfaces.