2017 — 2018 |
Higuchi-Sanabria, Ryo |
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. |
The Cytoskeletal Stress Response: a Novel Facet to Protecting Cell Integrity During Aging @ University of California Berkeley
Project Summary/Abstract This grant proposal seeks to identify the first known cytoskeleton-specific stress response and the potential regulators of this pathway. Previous work in yeast has identified an age-associated decline in cytoskeletal function and its implications in lifespan, while recent work in C. elegans have identified HSF-1 in mediating a protective role in cytoskeletal integrity. Marrying these findings, we propose to characterize both systemic and tissue-specific cytoskeletal decline as a function of age in the multicellular model organism, C. elegans. Next, we would like to identify the key players working both in synergy or independently of HSF-1 in protecting cytoskeletal integrity under stress and aging. Finally, we can determine whether the cytoskeletal stress response itself declines over age, as many stress response pathways do ? including mitochondrial, endoplasmic reticulum, and cytoplasmic ? and whether ectopic activation can rescue cytoskeletal function in advanced age. We will employ both biochemical and imaging methods to test our hypotheses. We propose to study cytoskeletal function by imaging of actin using LifeAct. In addition, we will purify actin proteins in a tissue- specific manner and study their function in worms at various stages of adulthood. Finally, we propose to perform a candidate screen of 400 transcription factors to identify novel regulators of cytoskeletal function. Here, endocytosis and organization of muscle fibers will be used as a readout for cytoskeletal integrity and function. At the culmination of this study, we will have characterized the cytoskeletal stress response as a function of age. Moving forward, this will open exciting avenues of research in continuing to map out the mechanistic pathway of the cytoskeletal stress response, as well as identifying the conservation of this mechanism in mammals and disease models.
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2020 — 2021 |
Higuchi-Sanabria, Ryo |
K99Activity Code Description: To support the initial phase of a Career/Research Transition award program that provides 1-2 years of mentored support for highly motivated, advanced postdoctoral research scientists. R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
More Than Just a Load Control: Cytoskeletal Form and Function During Aging @ University of California Berkeley
Project Summary. Although the cytoskeleton has historically been understood as the structural framework of the cell, the proper function of actin is also required for a diverse array of cellular pathways. The collapse of these cellular processes manifests during aging and exposure to a myriad of stresses, which is in part due to the breakdown of the cytoskeleton under these conditions. Interestingly, the breakdown of the cytoskeleton throughout age has been adopted as common knowledge in the field of aging biology, despite the lack of clear and direct evidence. A major contributor to the lack of these essential studies is the lack of tools available for in vivo, live-cell imaging of the actin cytoskeleton in multi-cellular organisms. Early in my postdoctoral career, I developed a system for robust, tissue-specific, live-cell imaging of the cytoskeleton in the muscle, intestine, and hypodermis of C. elegans, utilizing LifeAct fused to a fluorescent molecule. LifeAct-mRuby reliably binds to F-actin, allowing visualization of functional, filamentous actin in the cells it is expressed. Using this system, I performed an exhaustive characterization of the decline of actin cytoskeletal integrity during aging. This work laid the foundation of my currently ongoing work in identification of novel regulators of the actin cytoskeleton. Having set up a system to interrogate cytoskeletal quality, I can now interrogate novel genes in their potential role for actin regulation. Using this and other platforms, I performed a multi-pronged screening approach to identify novel genetic regulators of actin. These studies combined in vivo live cell imaging of actin filaments, synthetic lethality screening with known regulators of the actin cytoskeleton, and both transcriptome analysis and whole genome CRISPR-Cas9 screening of organisms experiencing actin stress. Cross-referencing these rich datasets has revealed two critical nodes of genes: 1) modifiers of chromatin state and their downstream transcriptional regulators and 2) genes involved in lipid storage and global lipid homeostasis. In Aim 1.1, I hypothesize that a general chromatin state exists to promote a healthy transcriptome for proper cytoskeletal form and function, and that this breaks down as a function of age. Moreover, a healthy metabolic state can work either upstream of ? or independent of ? chromatin remodeling to also promote cytoskeletal health. In Aim 1.2, I propose to study whether any of the identified processes can function in a tissue- specific manner and a cell non-autonomous manner, by answering two questions: 1) is overexpression of chromatin remodeling or lipid homeostasis factors in a single tissue sufficient to preserve organismal lifespan? and 2) does overexpression of these genes in neurons drive protection of the actin cytoskeleton in peripheral tissue? Aim 2 uses 2 biochemical approaches to assess cytoskeletal function. First, proximity labeling will be used to characterize novel protein interactors of actin important for proper form and function. Second, we are building a tool for a biochemical approach for quantifying actin function with single cell resolution. This study will open exciting avenues of research in understanding the role of cytoskeletal function on physiological aging.
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