The Myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization.
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.
van den Boom
Frank
F
Institute for General Zoology and Genetics, Westfalian Wilhelms-University, 48149 Münster, Germany.
Düssmann
Heiko
H
Uhlenbrock
Katharina
K
Abouhamed
Marouan
M
Bähler
Martin
M
D000199:Actins / Q000378:metabolism*; D000818:Animals; D005453:Fluorescence; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007797:Laminin / Q000737:chemistry; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D038761:Photobleaching; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D014407:Tumor Cells, Cultured
D000199:Actins; D020690:GTPase-Activating Proteins; D007797:Laminin; C113469:myosin IXB; C085379:rho GTPase-activating protein; D049452:Green Fluorescent Proteins; D009218:Myosins