2012 — 2013 |
Ryder, Pearl Victoria |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
Regulation of Ap-3-Dependent Axonal Targeting by Hect Ubiquitin Ligases
DESCRIPTION (provided by applicant): Numerous ubiquitin ligases and potential substrates are present at multiple locations within the cell, yet ubiquitination occurs only at specific organelles. While the subcellular location where a substrate is ubiquitinated has functional consequences for the fate of the substrate, the mechanisms that specify this location are incompletely understood. In neurons, ubiquitination regulates synapse assembly and function by a host of selectively targeted pre- and post-synaptic ubiquitin ligases. The mechanisms that target these ligases to their subcellular locations are poorly understood and constitute the focus of this proposal. Our interest in these mechanisms stems from the role of the ubiquitin machinery in sporadic and familial forms of neurodegenerative diseases, such as Parkinson's disease. Exciting new data in our laboratory demonstrate that a member of the HECT (homologous to E6-AP carboxy terminus) superfamily of ubiquitin ligases, Nedd4, interacts with the adaptor protein complex-3 (AP-3). While Nedd4 acts at the late endosome, the mechanisms for targeting it to this compartment are unknown. Since the AP-3 complex acts as a vesiculation scaffold that brings together multiple proteins at endocytic organelles, we postulate that Nedd4 is recruited to endocytic compartments by interaction with AP-3. Our overall hypothesis is that HECT family ubiquitin ligases recognize specific membranous organelles by binding to organelle-restricted docking factors, such as AP-3. We will test our hypothesis through the following specific aims: (1) to determine if the AP-3 complex plays a role in subcellular localization of the Nedd4 ubiquitin ligase and (2) to determine if AP-3 regulates Nedd-4-dependent substrate recognition and post-translational modification. We will use subcellular fractionation and quantitative high-resolution immunofluorescence microscopy of fixed primary cultured neurons from wild-type and Nedd4- deficient mice and neuronal model cell lines in order to test our hypothesis. Our long-term goal is to define subcellular compartmentalization as a novel biological regulatory principle of ubiquitin machineries. Given the importance of the ubiquitin machinery to the normal and pathological synapse, our work will contribute new mechanistic understanding of the processes involved in the establishment and maintenance of synapses. PUBLIC HEALTH RELEVANCE: Relevance to human health and disease: The health of the brain depends on the maintenance of the connections between brain cells. Dysregulation of a protein modification machinery, the ubiquitination machinery, can lead to neurodegenerative disease such as Parkinson's disease. Our work will contribute details of the regulation of this machinery in both health and neurodegenerative diseases.
|
1 |
2019 |
Ryder, Pearl Victoria |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Regulation of Centrosomes by Localized Rna
PROJECT SUMMARY/ABSTRACT Aberrant centrosome function underlies pathologies with profound significance for human health, including growth deficiency syndromes and cancer. Centrosomes function as microtubule organizing centers, build the mitotic spindle, form the basal body to template cilia in ciliated cells, and serve as platforms for signaling cascades including cell cycle signaling. The centrosome consists of a pair of centrioles surrounded by a protein matrix termed pericentriolar material (PCM). The composition and quantity of PCM determines the microtubule nucleating activity of the centrosome and changes rapidly in cycling cells. The mechanisms responsible for rapid changes to centrosome composition and structure are incompletely understood. Intriguingly, a screen for localized mRNA in Drosophila early embryos identified multiple mRNA transcripts enriched at spindle poles. Mutations to several of these genes disrupt centrosome function and/or the mitotic spindle, suggesting that mRNA enrichment and local translation at the centrosome may be an unexplored mechanism to modulate centrosome composition. This proposal aims to fill this gap in knowledge by leveraging the genetically tractable Drosophila melanogaster early embryo, an ideal system to visualize hundreds of active MTOC centrosomes. My central hypothesis is that specific RNAs are actively localized to the centrosome by RNA binding proteins to regulate its structure and function. I will test this hypothesis with two complementary aims. In Specific Aim 1, I will define the role of centrosome mRNAs in centrosome composition and function using single molecule FISH, spinning disk confocal microscopy, super-resolution microscopy, quantitative image analysis, biochemical isolation of centrosomes, and 3?UTR swapping experiments in well-characterized Drosophila centrosome mutants and controls. In Specific Aim 2, I will identify mechanisms of centrosome mRNA localization by testing the hypothesis that Orb, a Drosophila cytoplasmic element binding protein ortholog and RNA binding protein, targets mRNAs to centrosomes. I will test this hypothesis using single molecule FISH, RNA immunoprecipitation, and chimeric reporters with mutated Orb consensus binding sequences. In Aim 2, I will also take an unbiased approach to identify other RNA binding proteins that contribute to centrosome mRNA localization via an RNA interference screen, which will form the basis for my research program as an independent investigator. This proposal exploits the Drosophila early embryo system to provide insight into the rapid changes in centrosome composition during the cell cycle, a biological process with implications for cancer pathogenesis and growth deficiency syndromes.
|
1 |