Possible involvement of myosin-X in intercellular adhesion: importance of serial pleckstrin homology regions for intracellular localization.
Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)-immunoblot analysis using antibodies against two different tail regions of mouse myosin-X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane-rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin-X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin-X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin-like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS-1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell-cell adhesion in a Rac- and Cdc42-dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42-dependent manner. Expression of green fluorescent protein (GFP)-MyTH4 fusion protein resulted in formation of phalloidin-positive granules, while GFP-FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin-X is involved in cell-cell adhesion-associated signaling-linked membrane and/or cytoskeleton reorganization.
Yonezawa
Satoshi
S
Institute for Developmental Research, Aichi Human Service Center, Kamiya-cho, Kasugai 480-0392, Japan. yonez@inst-hsc.pref.aichi.jp
Yoshizaki
Norio
N
Sano
Mamoru
M
Hanai
Atsuko
A
Masaki
Shigeo
S
Takizawa
Takenori
T
Kageyama
Takashi
T
Moriyama
Akihiko
A
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D003599:Cytoskeleton / Q000502:physiology; D005819:Genetic Markers; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D007672:Kidney Cortex / Q000166:cytology / Q000502:physiology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D008970:Molecular Weight; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
D005819:Genetic Markers; D008164:Luminescent Proteins; C409064:Myo10 protein, mouse; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D009218:Myosins