Graduate Thesis Or Dissertation

 

Initial in vitro and in vivo Characterization of the Membrane Trafficking Protein Fer1L6 Public Deposited

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  • A fundamental difference between prokaryotic and eukaryotic cells is the presence of membrane bound organelles in eukaryotes. The dynamics of membrane trafficking within the cell are responsible for everything from intercellular communication and cell homeostasis, to mitosis, cell migration, and differentiation. These processes require exocytosis and compensatory endocytosis, often working in concert to create vesicle cycling. While constitutive exo‐ and endocytosis occurs independently of extracellular stimulus, , regulated membrane trafficking events are quiescent until triggered by a messenger, such as calcium. Exocytosis is primarily carried out by SNARE proteins, while endocytosis is carried out by either clatherin‐related proteins or clatherin‐independent proteins, such as Eps 15 homology domain proteins (EHDs). However, none of these proteins are directly calcium sensitive. Thus, calcium regulated exocytosis requires an additional calcium‐regulated protein. In neuronal exocytosis the synaptotagmin family confers calcium sensitivity to the process, and members are typically composed of a membrane anchor and two C2 domains. Another family of proteins involved in calcium sensitive exocytosis is the ferlin family. Composed of 5 to 7 C2 domains, these membrane proteins have been implicated in various membrane trafficking diseases, ranging from muscular dystrophy to non‐syndromic deafness, and have been implicated in multiple cancer types. The goal of this dissertation is to characterize the sixth mammalian ferlin protein, Fer1L6, which has not been previously studied. Currently, Fer1L6 has no known disease state links. Nevertheless, characterization of both tissue specificity and functional roles of the Fer1L6 protein will be highly valuable to furthering our understanding of the ferlin protein family, and strengthen our understanding of membrane dynamics inside eukaryotic cells. To date little is known about the function or expression of Fer1L6, despite the fact that it is a predicted protein coding gene in a wide range of vertebrate genomes. Reverse genetic techniques were used to elucidate a possible functional role of Fer1L6 in the model organism D. rerio (zebrafish). A Fer1L6 morpholino knockdown resulted in abnormal skeletal muscle development, in which the sarcoplasmic reticulum and t‐tubules do not form properly. Additionally, the myofibrils and myosepta are disorganized and irregularly shaped. Heart rate quantitation and microscopy of cardiac muscle shows underdevelopment of the heart chambers with decreased heart rate. However, initial studies with a first generation Fer1L6 mutant zebrafish line do not exhibit any of the same gross phenotypes that were observed with the morpholino knockdown. Further investigation of the mutant line is required to fully characterize the effects of the mutation. The muscle related phenotypes seen in the morpholino studies initiated an investigation into Fer1L6 in the C2C12 myoblast cell line. Using both q‐PCR and western blots, no changes in Fer1L6 expression was detected during myoblast differentiation into myotubes. Immunostaining of undifferentiated myoblasts showed Fer1L6 localization to the perinuclear region with Fer1L6 puncta also emanating outwards towards the plasma membrane. Additionally, there were small levels of accumulation at the plasma membrane. These results are consistent with previously proposed models in which Fer1L6 is involved in trans‐Golgi to plasma membrane endocytic recycling events with Rab 11. The results of this dissertation begin the process of determining a functional role for Fer1L6, and demonstrate the feasibility for both zebrafish and C2C12 cells as a model for future studies on the endogenous protein. The results validate high‐throughput proteomic studies, and lend support to recently literature with over expression in cell culture. Overall, we find that Fer1L6 is a widely expressed protein that may be involved in both embryonic development and maintaining homeostasis of tissues through endosomal recycling pathways.
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  • 2017-08-16 to 2018-02-13

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