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© The Rockefeller University Press, 0021-9525/1997//633 $5.00
The Journal of Cell Biology, Volume 136, Number 3, , 1997 633-647

Article

Myosin I Overexpression Impairs Cell Migration



Kristine D. Novak and Margaret A. Titus

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

Dictyostelium myoB, a member of the myosin I family of motor proteins, is important for controlling the formation and retraction of membrane projections by the cell's actin cortex (Novak, K.D., M.D. Peterson, M.C. Reedy, and M.A. Titus. 1995. J. Cell Biol. 131:1205–1221). Mutants that express a three- to sevenfold excess of myoB (myoB+ cells) were generated to further analyze the role of myosin I in these processes. The myoB+ cells move with an instantaneous velocity that is 35% of the wild-type rate and exhibit a 6–8-h delay in initiation of aggregation when placed under starvation conditions. The myoB+ cells complete the developmental cycle after an extended period of time, but they form fewer fruiting bodies that appear to be small and abnormal. The myoB+ cells are also deficient in their ability both to form distinct F-actin filled projections such as crowns and to become elongate and polarized. This defect can be attributed to the presence of at least threefold more myoB at the cortex of the myoB+ cells. In contrast, threefold overexpression of a truncated myoB that lacks the src homology 3 (SH3) domain (myoB/SH3– cells) or myoB in which the consensus heavy chain phosphorylation site was mutated to an alanine (S332A-myoB) does not disturb normal cellular function. However, there is an increased concentration of myoB in the cortex of the myoB/SH3– and S332A-myoB cells comparable to that found in the myoB+ cells. These results suggest that excess full-length cortical myoB prevents the formation of the actin-filled extensions required for locomotion by increasing the tension of the F-actin cytoskeleton and/ or retracting projections before they can fully extend. They also demonstrate a role for the phosphorylation site and SH3 domain in mediating the in vivo activity of myosin I.

Abbreviations used in this paper: MIHCK, myosin I heavy chain kinase; nt's, nucleotides; SH3, src homology 3 domain.

Thanks to Dr. Dan Kiehart for many helpful discussions and for critically reading the manuscript. We would like to thank Jon Robertson of the DCMB Group DNA Synthesis Facility for the speedy production of oligos, Dr. Bruce Patterson (University of Arizona, Tucson, AZ) for providing the pBIG, pLittle, and pSmall vectors, and Dr. Angelika Noegel (Max Planck Institute für Biochemie, Martinsried, Germany) for the plasmid pDRH. We would also like to thank Dr. Mike Sheetz for his continuing support and encouragement.

The initial portions of this work were supported by grants from the American Cancer Society (CB-90A and JFRA no. 378), and subsequently by the March of Dimes (no. 1-FY95-0754) and the National Science Foundation (MCB-9507376). M.A. Titus is a member of the Duke Comprehensive Cancer Center.

Address all correspondence to Margaret Titus, Department of Cell Biology, Duke University Medical Center, Durham, NC 22710. Tel.: (919) 681-8859. Fax: (919) 684-5481. E-mail: m.titus{at}cellbio.duke.edu


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