We have a long-standing interest in actin polymerization mechanisms, which has led us to investigate quantitatively minor populations of filaments with important cellular roles. One such actin population functions in mitochondrial fission. Mitochondrial fission is required for proper mitochondrial distribution, mitophagy, oxidative stress response, and adaptation to varying metabolic substrates. Defects in mitochondrial fission are linked to the pathology of major neurodegenerative diseases, including Alzheimer's, Huntington's, Parkinson's, and ALS. The dynamin family GTPase Drp1 is a central player in mitochondrial fission, oligomerizing at fission sites and promoting membrane constriction. Still, the mechanisms that trigger mitochondrial fission are murky. We have discovered that actin polymerization at fission sites plays a major role in Drp1 recruitment and mitochondrial fission in mammals. This finding came from our focus on an endoplasmic reticulum-bound formin, INF2, which assembles this filament population. Through these studies, we have developed live-cell systems for imaging mitochondrial fission at high spatial and temporal resolution, which have allowed us to define the order of events leading to Drp1 oligomerization on mitochondria. We have also established refined biochemical systems to study interaction of actin with Drp1, INF2 and other components of the fission process, which will enable eventual cell-free reconstitution of fission. These discoveries have fundamentally changed our view of mitochondrial fission. Our goal in the next five years is to define one ?type? of mammalian mitochondrial fission in detail (stimulated by calcium ionophore), and subsequently to use this knowledge to define fission mechanisms induced by other stimuli. We have two longer-term goals: to reconstitute actin-mediated mitochondrial fission using purified components (which would indicate full mechanistic understanding), and to define the signaling in-puts that activate fission in specific physiological situations. Mutations in INF2 are causally linked to two human diseases: focal and segmental glomerulosclerosis (a kidney disease) and Charcot-Marie-Tooth disease (a peripheral neuropathy). Thus, our work impacts both fundamental cell biology and disease- based research. A second focus of the laboratory is filopodia assembly by the formin FMNL3. While not discussed in this Research Strategy, we will continue our filopodia work in this MIRA. Similar to our INF2 studies, years of careful cellular and biochemical work are leading to surprising discoveries, including 1) links between filopodia and both cell-cell and cell-substratum adhesion, and 2) a role for FMNL3 in endosomal dynamics. Our overall vision is that there are undiscovered populations of actin filaments, transient and of low abundance, which mediate key cellular functions. The combined studies in my laboratory are revealing these actin filament populations.

Public Health Relevance

Project Description/Narrative We are discovering novel functions for actin cytoskeleton in organelle dynamics, with one focus being mitochondrial fission. This work has direct impact on two human diseases: focal segmental glomerulosclerosis (kidney) and Charcot-Marie-Tooth disease (a neuropathy). It also impacts major neurodegenerative diseases (Alzheimer?s, Parkinson?s and others).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZGM1)
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Flicker, Paula F
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Dartmouth College
Schools of Medicine
United States
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Young, Lorna E; Higgs, Henry N (2018) Focal Adhesions Undergo Longitudinal Splitting into Fixed-Width Units. Curr Biol 28:2033-2045.e5
Young, Lorna E; Latario, Casey J; Higgs, Henry N (2018) Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly. J Cell Sci 131:
Chakrabarti, Rajarshi; Ji, Wei-Ke; Stan, Radu V et al. (2018) INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division. J Cell Biol 217:251-268
Ji, Wei-Ke; Chakrabarti, Rajarshi; Fan, Xintao et al. (2017) Receptor-mediated Drp1 oligomerization on endoplasmic reticulum. J Cell Biol 216:4123-4139