Using molecular genetics as a tool in understanding crawling cell locomotion in myoblasts.
We have used digitally recorded interference microscopy with automatic phase shifting (DRIMAPS) to investigate the crawling locomotion of normal and mutant mouse myoblasts. Contraction forces that give rise to cell body movement, tail retraction and cell adhesion to the substrate in myoblasts and other locomoting tissue cells arise from the interactions of actin and non-muscle myosin II. The activity of non-muscle myosin II is regulated differently from that of skeletal myosin. Using DRIMAPS, we found that crawling locomotion was altered in myoblasts that heterologously expressed human beta-cardiac myosin heavy chain (MHC); the cells moved more slowly and had reduced rates of protrusion and retraction. Immunolocalization demonstrated that MHC and non-muscle myosin II were not co-localized, suggesting that MHC does not compete directly with myosin II, but interferes with cell locomotion by binding inappropriately to actin filaments and possibly cross-linking them. Myosin I may be involved in protrusion of the lamellipodia. However, using DRIMAPS, we found that crawling locomotion was unaltered in myoblasts that heterologously expressed a truncated myosin I which lacked the membrane-binding tail domain. This suggests that, if endogenous myosin I is important for cell locomotion, this mutant was unable to interfere with its action. We conclude that the effects on locomotion of expressing foreign or mutant proteins of the cytoskeleton in vertebrate cells can be subtle and can be swamped by the intrinsic variability of the cells. Their characterization requires automated methods of acquiring data, such as DRIMAPS, and careful statistical analysis in order to take account of other sources of variation.
D000199:Actins / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement / Q000235:genetics* / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology*; D009154:Mutation; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000502:physiology