It has been puzzled that in spite of its single-headed structure, myosin-IX shows the typical character of processive motor in multi-molecule in vitro motility assay, because this cannot be explained by hand-over-hand mechanism of the two-headed processive myosins. Here, we show direct evidence of the processive movement of myosin-IX using two different single molecule techniques. Using optical trap nanometry, we found that myosin-IX takes several large ( approximately 20nm) steps before detaching from an actin filament. Furthermore, we directly visualized the single myosin-IX molecules moving on actin filaments for several hundred nanometers without dissociating from actin filament. Since myosin-IX processively moves without anchoring the neck domain, the result suggests that the neck tilting is not involved for the processive movement of myosin-IX. We propose that the myosin-IX head moves processively along an actin filament like an inchworm via a unique long and positively charged insertion in the loop 2 region of the head.
"This movement is considered as " caterpillar " mechaa nism (Nishikawa et al., 2006). Myosin molecules lack ing tailldomain move along actin fibrils to the minuss end, whereas fulllsize molecules move to the plussend (O'Connell and Mooseker, 2003). "
[Show abstract][Hide abstract] ABSTRACT: The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.
"We have proposed previously that class IX myosins could represent actin-based motorized signaling molecules (Bähler, 2000). Although the mammalian class IX myosin Myo9b (formerly also called myr 5) is a singleheaded myosin, in vitro actin-gliding assays revealed that it takes many successive steps along actin filaments without dissociating, implying that it is a processive motor molecule (Inoue et al., 2002; Post et al., 2002; Nishikawa et al., 2006). However, kinetic analysis of Myo9b demonstrated that it spends during its ATPase cycle considerable time in the ATP-bound state, a state in which other myosins typically are not associated with actin filaments (Nalavadi et al., 2005; Kambara and Ikebe, 2006). "
[Show abstract][Hide abstract] ABSTRACT: Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.
Molecular Biology of the Cell 05/2007; 18(4):1507-18. DOI:10.1091/mbc.E06-08-0771 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Class IX myosins are found in animals from invertebrates to vertebrates. Invertebrates contain a single myosin IX gene, whereas
vertebrates contain two myosin IX genes, MYO9A and MYO9B. Mammalian Myo9b, the only class IX myosin studied so far, has unique
motor properties. It is the first myosin for which ATP hydrolysis is the rate-limiting step in the chemical cycle and although
it is a single-headed myosin, it can take multiple steps along F-actin before dissociating. Class IX myosins are motorized
signaling molecules that contain in their tail domain a Rho GTPase-activating protein (GAP) activity. Mammalian members of
myosin class IX negatively regulate the monomeric GTP-binding proteins RhoA-C. In cells, Myo9b accumulates in regions of active
actin polymerization such as in extending lamellipodia. In these regions Myo9b might locally down regulate contractility and
adhesion that are controlled by Rho activity and thereby contribute to sustained lamellipodial extension and cell polarity.
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