Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.

Interplay of Endocytosis and Growth Factor Receptor Signalling.

Growth factor receptors play a variety of roles during embryonic development and in adult homeostasis. These receptors are activated repeatedly in different cellular contexts and with different cellular outcomes. This begs the question as to how cells in a particular developmental, spatial and temporal context, or in adult tissue, interpret signalling by growth factor receptors in order to deliver qualitatively different signalling outputs. One mechanism by which this could occur is via endocytic regulation. The original paradigm for the role of endocytosis in growth factor receptor signalling was that receptor uptake has a quantitative role in signalling by reducing the number of cell surface receptors available for activation and targeting activated receptors for degradation. However, a range of studies over the last several years, in many different experimental systems, has demonstrated an additional qualitative role for endocytic trafficking in receptor signalling, with specific outcomes depending on the location of the signalling complex. Confinement of receptors within endosomes can spatially regulate signalling, facilitating specific protein interactions or post-translational modifications that alter throughout the trafficking process. Therefore, endocytosis does not simply regulate cell surface expression, but tightly controls protein interactions and function to produce distinct outcomes.