A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation

摘要

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C . elegans . Confirming the specificity of this screen, we identified the Rho GTPase cdc-42 , which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue. Author Summary During animal development specialized cells acquire the ability move and invade into other tissues to form complex organs and structures. Understanding this cellular behavior is important medically, as cancer cells can hijack the developmental program of invasion to metastasize throughout the body. One of the most formidable barriers invasive cells face is basement membrane–-a thin, dense, sheet-like assembly of proteins and carbohydrates that surrounds most tissues. Cells deploy small, protrusive, membrane associated structures called invadopodia (invasive feet) to breach basement membranes. How invadopodia are formed and controlled during invasion has been challenging to understand, as it is difficult to examine these dynamic structures in live animals. Using the nematode worm Caenorhabditis elegans , we have conducted the first large-scale screen to isolate genes that control invadopodia in live animals. Our screen isolated 13 genes and we confirmed two are key invadopodia regulators: the Rho GTPase CDC-42 that promotes F-actin polymerization at invadopodia to generate the force to breach basement membranes, and the Rab GDI-1 that promotes membrane addition at invadopodia that may allow invadopodia to extend through basement membranes. This work provides new insights into invadopodia construction and identifies potential novel targets for anti-metastasis therapies.