We have begun work investigating the function of myosin 18A (M18A). To this end, we have generated M18A conditional knockout mice that will be used to remove M18A from specific tissues. We have also generated M18A-specific antibodies and fluorescently-tagged M18A plasmids that are being used to investigate the subcellular localization of M18A in generic cells. Using various imaging techniques, we have localized M18A to acto-myosin rich regions, including internal stress fibers and lamellar protrusions. We have begun developing siRNA and shRNA technologies to suppress M18A expression in these cells to help reveal its cellular function. We will continue to investigate the role of M18A in both the whole animal and in cultured cells. To aid in our understanding of these acto-myosin rich regions, we have begun further investigation into non-muscle myosin II (NM II). NM II powers a variety of developmental and cellular processes, including adhesion, migration, and division. A single monomer of NM II is a hexamer consisting of two myosin heavy chains (MHC), two essential light chains, and two regulatory light chains. Three distinct genes encode MHC proteins, resulting in three distinct monomer populations, termed NM IIA, NM IIB and NM IIC. Knockout and rescue experiments in mice have demonstrated both unique and redundant functions for individual isoforms, which possess distinct biophysical properties. To exert their function, NM II monomers polymerize into anti-parallel filaments that engage the actin cytoskeleton. It is unclear whether or not the three NMII isoforms co-polymerize to produce mixed filaments or if filaments consist entirely of a single isoform. Clarifying this issue is critical for understanding and interpreting experiments investigating the roles of individual NM II isoforms. Molecular biological approaches have produced conflicting results, in part because pull-downs and IPs can yield false positives due to multiple filaments being connected through the actin cytoskeleton. While in vitro experiments have provided insights into the distinct biophysical characteristics of the NM II isoforms, they do not reproduce the physiological conditions experienced by the proteins in living cells. Finally, while it is clear that NM II isoforms display overlapping localization in some areas of the cell, this does not prove co-polymerization, as this imaging is typically well below the resolution required to distinguish a single filament from multiple filaments. NM II filaments are typically 300 nm in length, similar to the resolution limit of standard light microscopy. Therefore, to determine if NM II isoforms can co-polymerize in live cells, we used fluorophore-tagged MHC isoforms in conjunction with two-color super-resolution TIRF structured illumination microscopy (TIRF-SIM). This technique provides a lateral resolution of approximately 100 nm, nearly 2.5x higher than the resolution achieved with standard light microscopy. Using TIRF-SIM and MHC constructs with a fluorophore on either the head or the tail, we can unequivocally identify individual NM II filaments, along with the orientation of each filament. Using these techniques, we demonstrate that exogenously expressed NM IIA and NM IIB co-polymerize in live cells to produce heterogeneous filaments. Surprisingly, even in areas where the isoforms are thought to be segregated, such as at the leading edge of a polarized cell where NM IIA is thought to be uniquely localized, we still see evidence of co-polymerization in nearly every filament. We have confirmed these results with endogenous proteins using isoform-specific antibodies. These results argue that NM II isoforms are performing both their isoform-specific and redundant roles while co-polymerized with other isoforms, and it suggests that it is the ratio of each isoform within the filament that provides it with its biophysical properties. These results should be considered whenever interpreting experiments on individual NM II isoforms.

Project Start
Project End
Budget Start
Budget End
Support Year
30
Fiscal Year
2013
Total Cost
$628,082
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
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