A Genome-Wide RNAi Screen Identifies CDC-42 and GDI-1 as In Vivo Regulators of Invadopodia

Abstract

Basement membrane (BM) is a sheet-like extracellular matrix that underlies most tissues and acts as a barrier to invading cells. Many cell types, including immune cells, cells migrating during development and morphogenesis, and metastatic cancer cells utilize F-actin-based structures called invadopodia to breach BM as they leave one tissue to enter another. Despite extensive study and interest in understanding invasion for its clinical importance, the molecular mechanisms regulating invadopodia and BM breach in vivo remain unclear. During uterine-vulval attachment in C. elegans, the specialized uterine anchor cell (AC) uses invadopodia to mediate breach of the underlying BM in order to contact the underlying vulval epithelium. The AC offers several advantages as a model, including experimental, visual, and genetic tractability, the presence of endogenous extracellular environment, and availability of the tissue targeted for invasion. In Chapter 2, I describe development of a novel technique for spatiotemporal-specific knockdown of proteins that will facilitate investigation of proteins, particularly those that are essential or required in other tissues, in the regulation of invadopodia and AC invasion. Using a sensitized genome-wide RNAi screen, classical genetics, and timelapse imaging of invadopodia at the AC-BM interface, Chapter 3 presents two in vivo invadopodia regulators that function by distinct mechanisms. This is the first in vivo evidence that the RhoGTPase CDC-42 regulates invadopodia formation through WSP-1. RabGTPase GDI-1 is a novel regulator of the unique membrane compartment required for invadopodia formation. CDC-42 and GDI-1 both function downstream of an unknown cue secreted by the cells targeted by the AC for invasion, illustrating that extracellular cues can play key roles in mediating cell invasion. The characterization of CDC-42 and GDI-1 as in vivo regulators of invadopodia is an important first step to understanding the mechanisms of this critical cellular process and we expect the AC will be an excellent model for future identification of novel regulators of BM breach.