Showing papers by "Toshimasa Ishizaki published in 2001"

Focal Contacts as Mechanosensors Externally Applied Local Mechanical Force Induces Growth of Focal Contacts by an Mdia1-Dependent and Rock-Independent Mechanism

Abstract: The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

Coordination of microtubules and the actin cytoskeleton by the Rho effector mDia1.

Abstract: Coordination of microtubules and the actin cytoskeleton is important in several types of cell movement. mDia1 is a member of the formin-homology family of proteins and an effector of the small GTPase Rho. It contains the Rho-binding domain in its amino terminus and two distinct regions of formin homology, FH1 in the middle and FH2 in the carboxy terminus. Here we show that expression of mDia1(DeltaN3), an active mDia1 mutant containing the FH1 and FH2 regions without the Rho-binding domain, induces bipolar elongation of HeLa cells and aligns microtubules in parallel to F-actin bundles along the long axis of the cell. The cell elongation and microtubule alignment caused by this mutant is abolished by co-expression of an FH2-region fragment, and expression of mDia1(DeltaN3) containing point mutations in the FH2 region causes an increase in the amount of disorganized F-actin without cell elongation and microtubule alignment. These results indicate that mDia1 may coordinate microtubules and F-actin through its FH2 and FH1 regions, respectively.

Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells.

Abstract: mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu(434) and Leu(455) in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.