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

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 genomewide 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 ACBM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC42 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. PLOS Genetics | DOI:10.1371/journal.pgen.1005786 January 14, 2016 1 / 29 OPEN ACCESS Citation: Lohmer LL, Clay MR, Naegeli KM, Chi Q, Ziel JW, Hagedorn EJ, et al. (2016) A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation. PLoS Genet 12(1): e1005786. doi:10.1371/journal.pgen.1005786 Editor: Andrew D. Chisholm, University of California San Diego, UNITED STATES Received: August 10, 2015 Accepted: December 12, 2015 Published: January 14, 2016 Copyright: © 2016 Lohmer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by the National Institutes of Health through grants GM100083 and GM079329 to DRS. Further, this work was supported by the Pew Scholars Program in Biomedical Sciences to DRS and the Damon Runyon Cancer Foundation grant DRG-2225-15 to MRC. The Caenorhabditis Genetics Center also provided strains, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection