Myosin regulatory proteins mediate hypertrophic cardiomyopathy and mitochondrial homeostasis
- The eukaryotic actin and microtubule cytoskeleton is extremely dynamic and complex. Actin and microtubule filaments, together with their accessory proteins, perform structural roles and provide tracks for molecular motors to move along. Converting chemical energy released upon nucleotide hydrolysis into mechanical work, molecular motors provide the driving force behind most non-Brownian movement within a eukaryotic cell. Myosins are a type of molecular motor that undergo conformational changes upon the hydrolysis of ATP and product release that enables them to translate along actin filaments. Myosin motors are responsible for the sliding of actin filaments during muscle contraction, as well as transport and anchoring of organelles, vesicles, and other intracellular components (Hartman et al., 2011). Identified for its role in muscle contraction, muscle myosin II (MII) hydrolyzes ATP to generate force and drive the sliding of actin filaments in the sarcomere (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954). MII functions in both skeletal and cardiac muscle, and is responsible for the muscle contraction, isometric force generation, and the contraction force associated with each heartbeat. In myocytes, MII is highly organized with about a dozen other proteins into sarcomeric units that form a near crystalline structure and comprise about 50% of myocyte volume. Availability of MII binding sites along the actin filament is regulated by MII accessory proteins in a Ca2+-dependent manner. Thus, MII-generated force and muscle contraction is dependent upon intracellular Ca2+ concentration and cooperation of accessory proteins. Human mutations in MII or its accessory proteins can yield a variety of disease states, depending on the mutated protein and the tissue in which it is expressed. In this work, we characterize four different mutations in the MII accessory protein troponin I that cause hypertrophic cardiomyopathy (HCM). A condition affecting approximately 1 in 500 individuals in the United States, the underlying mechanisms of HCM remain elusive (Maron et al., 1995a). Using a six-component system, we reconstituted the sarcomere in vitro and examined biochemical properties of the system with wild type and mutant troponin I protein. We found that three of the four mutations significantly increase the affinity of regulated actin ("regulated thin filament") for Ca2+. The fourth mutation we examined did not affect the affinity of the system for Ca2+, but rather affected the mechanochemical cycle of the MII motor. Because no therapeutics currently exist to treat the underlying causes of HCM, these studies highlight the complexities underlying HCM and the importance of understanding the complete biochemical and biophysical nature of the mutations that cause it. We next explore the role of myosin Va (MVa) and its accessory proteins in neuronal function. Mice expressing mutant MVa have marked neurological defects and die prematurely (Mercer et al., 1991). While new biological roles of MVa are emerging, it is likely that there are many that are yet to be discovered. To identify novel MVa functions, we chose to determine the proteins to which it binds in the cell. Affinity chromatography was used to ascertain candidate MVa binding partners from brain extract. Each candidate was tested for direct binding, and the protein spire1 was found to be a direct binder to MVa. Spire1 is an actin nucleator that was originally identified in Drosophila, and few mammalian functional studies had been performed at the start of this work. We serendipitously amplified a novel isoform of spire1 from mouse brain cDNA that targeted to mitochondria. Additional work with MVa-null mice showed that this mitochondrial localization was independent of MVa, and any potential role spire1 may play in mediating mitochondrial dynamics or homeostasis is also MVa-independent. Detailed analysis of mammalian spire1 isoforms showed that at least 3 exons are differentially-spliced to form spire1 of various compositions. Interested in the potential functional differences among isoforms, we created spire1 constructs lacking each of the 3 alternate exons, and found alternate exon C to be necessary and sufficient for mitochondrial localization of spire1. After confirming the expression of exon C-containing spire1 in multiple mouse tissues, we probed functional roles for spire1 by expressing different truncations in mammalian cells. We identified a striking mitochondrial phenotype that strongly suggests spire1 mediates mitochondrial fission and/or fusion in an actin-dependent manner. Finally, we studied the biochemical link between spire1 and mitochondria. We found alternate exon C to bind directly and specifically to cardiolipin, a mitochondrial-specific lipid in eukaryotes. We performed affinity chromatography to look for potential proteins that interact with exon C and tether it to mitochondria, but the purified spire1 protein with which we were working was not tractable for these experiments. This work highlights the similarities and differences between two different myosins, their accessory proteins, and their diverse biological functions.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Ingle, Sadie Rae Bartholomew
|Stanford University, Department of Biochemistry.
|Spudich, James A
|Spudich, James A
|Straight, Aaron, 1966-
|Straight, Aaron, 1966-
|Statement of responsibility
|Sadie Rae (Bartholomew) Ingle.
|Submitted to the Department of Biochemistry.
|Thesis (Ph.D.)--Stanford University, 2013.
- © 2013 by Sadie Rae Ingle
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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