TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. TRIOBP has three isoforms, named TRIOBP-1, TRIOBP-4, and TRIOBP-5. In vitro, TRIOBP isoform 4 (TRI-OBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRI-OBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. Recent our study demonstrated that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific. Structural analysis of TRIOBP by amino acid sequence showed ID proteins. Thus the second structure of TRIOBP may not have. To investigate the structural property of TRIOBP-4, we analyzed the structure of TRIOBP-4 by using circular dichroism, dynamic light scattering, and fluorescence correlation spectroscopy. Our analysis show that TRIOBP has a beta-sheet, but not alpha-helix. To investigate the structure of tight F-actin bundle structure with TRIOBP, we analyzed 3-D structure of the bundle by using 3-D image analysis from transmitted electron microscope images. Eukaryotic cells rely on their cytoskeleton to carry out coordinated motion. To adapt to the changing requirements, the cell's cytoskeleton constantly remodels through the action of myosin II motors that interact with numerous actin filaments simultaneously. Here we study the various roles of myosin II clusters in the formation and evolution of in-vitro actomyosin networks. In our experiments the motor clusters can vary in size between 14 and 144 myosin II molecules and apply forces ranging between several to tens of pico Newtons. During the initial process of network formation the motor clusters become embedded within the network structure, where they act as internal active cross-linkers. Myosin II clusters enhance the nucleation of actin network in a concentration dependent manner, in the presence of passive crosslinkers, thus functioning as a 'network co-nucleator'. As network formation is achieved, myosin II turns into a 'network reorganizer', where it takes part in the remodeling and coarsening of the overall network structure. As a result of the strong confinement (the motor clusters within the network bundles exhibit high processivity with a fraction of attached motors p att R0.15), their effect in the nucleation and reorganization of the actin network is enhanced, rendering even small clusters of 14 myosin II molecules efficient. The stresses building-up in the networks lead to complex dynamics and can drive their contraction and rupture, depending on motor concentration and cluster size. Above a certain concentration, the severing and disassembly properties of the motors dominate, and they function as 'network disassembly agents'. Myosin II motors are shown to be unique motors that function as complex machines that per...
ATPase cycle (see Figure). The reduced run lengths with increasing ATP, ADP, and Pi suggest that runs terminate from two distinct states; one with both heads weakly-bound (state 3) another with ADP in the trailing head while the leading head has yet to undergo its powerstroke (state 1). In addition, to strain dependent accelerated ADP-release from the trailing head (State 4), the model also predicts that strain accelerates ATP binding (state 5) two-fold. These data and model analysis suggest that myosin Va processivity involves a complex branched kinetic pathway, providing the motor versatility when meeting the physical challenges presented by the intracellular environment.
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