A Role for βcyto- and γcyto Actin at the Sarco/endoplasmic Reticulum-Mitochondrial Interface
2017-05
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A Role for βcyto- and γcyto Actin at the Sarco/endoplasmic Reticulum-Mitochondrial Interface
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2017-05
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Skeletal muscle accounts for 40% of mass in adult humans. The primary function of skeletal muscle is to produce movement via contraction of the sarcomere. The sarcomere is the basic functional until of skeletal muscle and two of the key components in sarcomeric contraction are myosin and actin. Actin, though often referred to as a single entity, separates into two functional groups muscle (αsk-actin, αca-actin, αsm- and γsm-actin) and cytoplasmic (βcyto- or γcyto actin) actins. Though αsk-actin is the predominate isoform in adult skeletal muscle, specifically in the sarcomere, the ubiquitously expressed βcyto- and γcyto actin cytoplasmic actin isoforms are also present. As may be predicted by their ubiquitous expression patterns, βcyto- and γcyto actin, are enmeshed in a wide array of cellular functions including, migration, cell shape, cell division, vesicle trafficking, and organelle anchoring among others. Though collectively involved in many processes the unique and redundant role of both βcyto- and γcyto actin are not fully delineated. βcyto- and γcyto actin are highly conserved, with 99% homology of the coding sequence. The evolutionary pressure which has maintained both sequences suggests that they are both are necessary. The importance of having both βcyto- and γcyto actin was demonstrated through whole body knockouts. Whole body ablation of either βcyto- or γcyto actin proved lethal to the majority of animals during embryogenesis or within 24 hours after birth. The severity of the whole body phenotype while one cytoplasmic actin is still present illustrates the vital role each isoform has. The lethal nature of whole body knockouts of either cytoplasmic actin isoform made studies into tissue specific phenotypes difficult. One tissue of interest was skeletal muscle, because of the role cytoplasmic actins have in linking the sarcomere to the extracellular matrix. Previously, insight was gained into the functional significance of βcyto- and γcyto actin in skeletal muscle via muscle specific knockouts. Each knockout revealed the presence of a mild myopathy associated with muscle death and regeneration which worsened overtime. The focus of my thesis is to better understand how muscle specific ablation of either βcyto- or γcyto actin resulted in a progressive mild myopathy. Since, the link between either βcyto- and γcyto actin and the observed myopathy wasn’t readily apparent I investigated the known interactions of cytoplasmic actins in skeletal muscle. It was previously reported that cytoplasmic actin colocalizes with a host of structures in skeletal muscle including two which could affect cell viability: mitochondria and peri-z-disk region where the sarcoplasmic reticulum also localizes. My thesis advances understanding for how ablation of cytoplasmic actin isoforms leads to a mild myopathy. Firstly, I showed that both βcyto- and γcyto actin isoforms were present at the interface between mitochondria and sarcoplasmic reticulum. Secondly, I showed that βcyto- and γcyto actin knockout skeletal muscle had perturbations in sarcoplasmic reticulum and mitochondria morphology. Additionally, I showed that both cytoplasmic actin isoforms contribute to mitochondrial fission. Finally, I demonstrated that in skeletal muscle lacking βcyto- and γcyto actin, changes indicative of decreased sarcoplasmic reticulum function preceded the observed morphological changes. Though cytoplasmic actin isoforms have been localized to an array of structures in skeletal muscle, we chose to focus on two organelles which can affect cell viability. Further investigation into the source of the observed progressive myopathy revealed morphological aberrations in both the mitochondria and sarcoplasmic reticulum. The stable nature of skeletal muscle structure hindered further examination of the observed mitochondrial morphological phenotype so, we utilized a mouse embryonic fibroblast model. In mouse embryonic fibroblasts lacking either or both cytoplasmic actin isoforms, mitochondria are elongated and display a decrease in fission events. As mitochondria are a central regulator of cell health and because of the effect we observed βcyto- and γcyto actin have on mitochondrial dynamics, we initially hypothesized that mitochondria would be mildly functionally impaired. However, upon investigation of function it was not mitochondrial function which was impaired, but that an indicator of sarcoplasmic reticulum function was decreased. In sarcoplasmic reticulum a functional phenotype preceded the development of morphological changes supporting a potential role for sarcoplasmic reticulum dysfunction to act as a possible stressor linked to the observed mild progressive myopathy.
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University of Minnesota Ph.D. dissertation.May 2017. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: James Ervasti. 1 computer file (PDF); xi, 136 pages.
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O'Rourke, Allison. (2017). A Role for βcyto- and γcyto Actin at the Sarco/endoplasmic Reticulum-Mitochondrial Interface. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/199033.
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