Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.
Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.
Cytokinesis in many eukaryotes requires a contractile ring of actin and myosin that cleaves the cell in two. Little is known about how actin filaments and other components assemble into this ring structure and generate force. Here we show that the contractile ring in the fission yeast Schizosaccharomyces pombe is an active site of actin assembly. This actin polymerization activity requires Arp3, the formin Cdc12, profilin and WASP, but not myosin II or IQGAP proteins. Both newly polymerized actin filaments and pre-existing actin cables can contribute to the initial assembly of the ring. Once formed, the ring remains a dynamic structure in which actin and other ring components continuously assemble and disassemble from the ring every minute. The rate of actin polymerization can influence the rate of cleavage. Thus, actin polymerization driven by the Arp2/3 complex and formins is a central process in cytokinesis. Our studies show that cytokinesis is a more dynamic process than previously thought and provide a perspective on the mechanism of cell division.
Focal adhesions (FAs) are clustered integrins and associated proteins that mediate cell adhesion and signaling. A green fluorescent protein-beta1 integrin chimera was used to label FAs in living cells. In stationary cells, FAs were highly motile, moving linearly for several plaque lengths toward the cell center. FA motility was independent of cell density and resulted from contraction of associated actin fibers. In migrating cells, FAs were stationary and only moved in the tail. FA motility in stationary cells suggests that cell movement may be regulated by a clutch-like mechanism by which the affinity of integrins to substrate may be altered in response to migratory cues.
We have developed a new approach to detect mechanical forces exerted by locomoting fibroblasts on the substrate. Cells were cultured on elastic, collagen-coated polyacrylamide sheets embedded with 0. 2-micrometer fluorescent beads. Forces exerted by the cell cause deformation of the substrate and displacement of the beads. By recording the position of beads during cell locomotion and after cell removal, we discovered that most forces were radially distributed, switching direction in the anterior region. Deformations near the leading edge were strong, transient, and variable in magnitude, consistent with active local contractions, whereas those in the posterior region were weaker, more stable, and more uniform, consistent with passive resistance. Treatment of cells with cytochalasin D or myosin II inhibitors caused relaxation of the forces, suggesting that they are generated primarily via actin-myosin II interactions; treatment with nocodazole caused no immediate effect on forces. Immunofluorescence indicated that the frontal region of strong deformation contained many vinculin plaques but no apparent concentration of actin or myosin II filaments. Strong mechanical forces in the anterior region, generated by locally activated myosin II and transmitted through vinculin-rich structures, likely play a major role in cell locomotion and in mechanical signaling with the surrounding environment.
Factors that are involved in actin polymerization, such as the Arp2/3 complex, have been found to be packaged into discrete, motile, actin-rich foci. Here we investigate the mechanism of actin-patch motility in S. pombe using a fusion of green fluorescent protein (GFP) to a coronin homologue, Crn1p. Actin patches are associated with cables and move with rates of 0.32 microm s(-1) primarily in an undirected manner at cell tips and also in a directed manner along actin cables, often away from cell tips. Patches move more slowly or stop when actin polymerization is attenuated by Latrunculin A or in arp3 and cdc3 (profilin) mutants. In a cdc8 (tropomyosin) mutant, actin cables are absent, and patches move with similar speed but in a non-directed manner. Patches are sites of Arp3-dependent F-actin polymerization in vitro. Rapid F-actin turnover rates in vivo indicate that patches and cables are maintained continuously by actin polymerization. Our studies give rise to a model in which actin patches are centres for actin polymerization that drive their own movement on actin cables using Arp2/3-based actin polymerization.
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