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Published on: January 12, 2015
Recombinant expression of the brush border myosin I heavy chain
1Whitehead Institute, Massachusetts Institute of Technology, Cambridge, USA.
Insights
Myosin I motors, important for membrane motility, localize to cell membranes. This study found that chicken myosin I functions similarly in COS cells, indicating general cellular factors support its activity.
Area of Science:
- Cell Biology
- Molecular Motors
- Biochemistry
Background:
- Myosin I motors are implicated in membrane motility, but their specific functions and localization determinants remain unclear.
- Different myosin I isoforms exhibit varied cellular localizations, suggesting isoform-specific regulation.
- The brush border myosin I from chicken intestinal epithelium is a well-characterized mammalian myosin I.
Purpose of the Study:
- To investigate if the localization and biochemical activity of chicken brush border myosin I depend on epithelial cell-specific factors.
- To determine if non-epithelial cells can support the general membrane localization and biochemical functions of brush border myosin I.
Main Methods:
- Transfection of COS cells with the cDNA for the heavy chain of chicken intestinal epithelium brush border myosin I.
- Analysis of recombinant myosin I localization within transfected COS cells.
- Biochemical assays on purified recombinant myosin I to assess actin binding and ATPase activity.
Main Results:
- Recombinant brush border myosin I was successfully produced in COS cells.
- The recombinant myosin I localized to plasma membrane protrusions, particularly at spreading edges, and to cytoplasmic structures.
- Purified recombinant myosin I exhibited ATP-dependent actin binding and calcium-sensitive, actin-activated MgATPase activity, mirroring the native enzyme.
- High expression levels of recombinant myosin I correlated with an irregular cell surface.
Conclusions:
- COS cells possess the necessary cellular factors for the general membrane localization of brush border myosin I.
- The biochemical activity of brush border myosin I is supported by factors present in COS cells, similar to those in intestinal epithelium.
- These findings suggest that the fundamental mechanisms governing myosin I localization and function are conserved across different cell types.
Abstract:
Although the specific functions of myosin I motors are not known, their localization to membrane structures suggests a function in membrane motility. Different myosin I isoforms in the same cell or in different cells can possess different localizations. To determine if the localization and biochemical activity of the best-characterized mammalian myosin I, chicken intestinal epithelium brush border myosin I, was dependent on determinants of the membrane or actin cytoskeleton specific to epithelial cells, we transfected the cDNA for the heavy chain of this myosin into COS cells. Transient transfection of COS cells with the chicken brush border myosin heavy chain resulted in the production of recombinant myosin I. Recombinant brush border myosin I localized to protrusions of the plasma membrane, particularly at spreading edges, and also to unknown cytoplasmic structures. Some cells expressing particularly high levels of brush border myosin I possessed a highly irregular surface. Recombinant brush border myosin I purified from COS cells bound to actin filaments in an ATP-dependent manner and decorated actin filaments to form a characteristic appearance. The recombinant myosin also catalyzed calcium-sensitive, actin-activated MgATPase activity similar to that of the native enzyme. Thus, any cellular factor required for the general membrane localization or biochemical activity of brush border myosin I is present in COS cells as well as intestinal epithelium.
