Defining cytoskeletal mechanisms driving cell motility in Naegleria

PROJECT SUMMARY/ABSTRACT Although actin is highly conserved throughout eukarya, the mechanisms used to regulate its assembly and disassembly vary across phyla. Precisely timed and placed actin assembly orchestrates nearly every cellular process, including cell migration. While actin-driven cell migration has been defined in some detail in animal cells, it is unknown if diverse eukaryotic pathogens operate using the same set of rules. This proposal will address the hypothesis that there are conserved principles of cell migration by investigating Naegleria, which diverged from the “yeast-to-human” lineage over a billion years ago. Specifically, this work will define the contributions of the cytoskeleton to cell crawling in the “brain-eating amoeba” Naegleria fowleri: a pathogen that crawls into and within the brain, causing a deadly form of meningitis for which there are no reliable treatments. Dr. Velle’s initial postdoctoral research using the commonly-used, non-pathogenic model system Naegleria gruberi highlights the potential for universal rules of motility; N. gruberi crawls on flat surfaces using thin, actin- based protrusions assembled by proteins called the Arp2/3 complex. This actin and Arp2/3 based mechanism is also how animal cells crawl on flat surfaces. However, outside of laboratory conditions, cells rarely—if ever—crawl on flat, uniform surfaces. Animal cells are well-known to switch to a different mode of motility when crawling through complex, restrictive environments, but this has not been tested in Naegleria. Because N. fowleri crawl through narrow channels in the skull and into the brain, despite no known chemotactic signals, dissecting cell migration in restrictive environments is essential for understanding disease. Therefore, Aim 1 will determine mechanisms of cell crawling under confinement at the level of cell behavior. Aim 2 will focus on the actin networks in cells; while the protrusions driving N. gruberi migration on flat surfaces look similar to those of animal cells, defining the actin architecture using Platinum Replica Electron Microscopy (PREM) will reveal if the ultrastructure is conserved. This aim will also provide critical training to complement Dr. Velle’s background in light microscopy. The world expert in PREM, Dr. Svitkina, will provide this training as a member of the scientific advisory committee. Aim 3 will use biochemistry—a technique the applicant has no prior training in—to examine the upstream mechanisms of Arp2/3 complex activation. Dr. Velle has recruited Dr. Bruce Goode, an expert actin biochemist, for this training. Because the leading labs in the field of cell migration frequently employ both microscopy and in vitro actin biochemistry, the proposed training in PREM and biochemistry will ensure the applicant is skilled in the techniques required for success. Dr. Velle has also recruited Dr. Matt Welch, an actin expert, Dr. Meg Titus, who has expertise in actin and amoebae, and Dr. Jim Morris, an expert in N. fowleri, to her scientific advisory committee to provide scientific and career mentoring. Collectively, the proposed work will provide the technical training, and career mentorship required to launch Dr. Velle’s career as an independent investigator with a research program focused on Naegleria’s migration.