Saturday, November 23, 2024
11/23/2024
The ‘actin cytoskeleton’ is not one structure but a number of distinct structures assembled and disassembled for different purposes. In mammalian cells, a few abundant and easily recognizable structures dominate our view of the actin cytoskeleton, including: stress fibers, lamellipodia and filopodia. However, a growing number of less abundant and/or highly transient actin-based structures have been revealed, controlling important cellular processes. Two such actin structures are the subject of this application: 1) CIA, calcium-induced actin; and 2) ADA, acute depolarization-induced actin. Though highly transient, both structures are extensive in the cytosol and affect important processes. In addition, both CIA and ADA impact the structure and function of mitochondria. CIA depends on calcium activation of the formin protein INF2, which stimulates actin polymerization on the endoplasmic reticulum and throughout the cytosol. Downstream effects of CIA include increased mitochondrial calcium and increased mitochondrial fission. The importance of CIA is illustrated by the fact that INF2 mutations link to two diseases, focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth disease (CMTD). ADA is triggered by mitochondrial depolarization (either pharmacologically-induced or hypoxia-induced), which activates two parallel pathways: 1) mitochondrial calcium release activates protein kinase C-, activating in turn Rac, WAVE complex, and Arp2/3 complex; and 2) decreased ATP activates AMP-dependent protein kinase (AMPK) through LKB1, activating in turn Cdc42 and FMNL formins. The ADA actin network is tightly associated with mitochondria. An exciting new result is that one immediate consequence of ADA is rapid stimulation of glycolysis. Additionally, ADA temporarily inhibits longer-term consequences of mitochondrial depolarization such as mitochondrial reorganization and recruitment of the mitophagy protein Parkin. The goals in this grant period are to elucidate both the mechanisms triggering CIA and ADA, as well as their downstream effects. These goals will be accomplished using a combination of cellular approaches (live-cell microscopy, proteomics, metabolic analysis) and biochemical approaches (cell-free reconstitution, analysis of purified proteins on model lipid membranes). The questions to be asked include the following. 1) How is INF2 activated by increased calcium? 2) How are INF2-polymerized filaments organized into a network by myosin II and fascin? 3) How does CIA interface with known mitochondrial fission proteins such as Mff and Drp1 to stimulate fission? 4) How do PKC and AMPK activate Rac and Cdc42, respectively, during ADA? 5) How do Arp2/3 complex and FMNL formins work together during ADA? 6) How does ADA stimulate glycolysis? These questions address fundamental mechanistic questions important to a wide range of mammalian cells, and occupy an exciting frontier between cytoskeletal biology, mitochondrial biology, and metabolism. 1
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
