The structure and regulation of myosin VI : a new paradigm for the myosin superfamily / Amber Lynn Wells.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Wells, Amber Lynn.

Contributor:

Sweeney, H. Lee, advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Cell and Molecular Biology.

Academic Dissertations as Topic.

Medical Subjects:

Cell and Molecular Biology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Physical Description:

xx, 224 pages : illustrations (some color) ; 29 cm

Production:

2002.

Summary:

The myosin superfamily is comprised of nearly twenty classes of actin-based motors. Prior to 1999, it was assumed that all of these motors would share a common mechanism for movement and would move toward the barbed (+) end of an actin filament. A likely candidate for a reverse direction myosin emerged from sequence comparisons, based on the hypothesis that a reversal of direction would require a repositioning of the myosin "lever arm." The only myosin class that has an unusual insertion preceding the lever arm is myosin VI. Accordingly, we cloned and expressed (baculovirus) a fragment of myosin VI and demonstrated that indeed, it moved in the opposite direction as compared to other myosin classes (i.e. toward the pointed (-) end of the actin filament). Next, the structural basis of myosin VI directionality was examined by construction of chimeras between class II and class VI myosins. Lastly, two putative mechanisms for myosin VI motor regulation were studied. Phosphorylation of myosin VI T406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate. In the absence of calcium, double-headed myosin VI constructs move in an in vitro motility assay at a velocity that is twice that of single-headed constructs. Increasing calcium above 10 muM slows both the rate of ADP release from actomyosin VI and motility, and also appears to decouple the two heads of myosin VI. Calmodulin mutants reveal that this is dependent on calcium binding to the N-terminal lobe of calmodulin. It is unclear what uncoupling of the heads, combined with a slowing of ADP release would be predicted to do to processivity in the native myosin VI. However, increasing calcium reduces the double-headed myosin VI step size and may increase the duty cycle of individual heads. Thus increasing calcium concentration has intriguing effects on myosin VI kinetics that likely alter the nature of single molecule movement. In collaborative experiments we demonstrated that myosin VI, as predicted from its kinetics, is a processive motor that likely moves via a unique mechanism. Thus myosin VI represents a new paradigm for the myosin superfamily.

Notes:

Supervisor: H. Lee Sweeney.

Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 2002.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 3043976.

OCLC:

244972054