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

Lauren L Lohmer,Elliott J Hagedorn,Matthew R Clay,David R Sherwood,Qiuyi Chi,Ranjay Jayadev,Kaleb M Naegeli,Jieun E Park,Joshua W Ziel,Andrew D Chisholm

doi:10.1371/journal.pgen.1005786

Abstract

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.

Highlights

Basement membrane (BM) is a dense, highly cross-linked extracellular matrix that surrounds most tissues and acts as a barrier to migrating cells [1]
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 GDP-dissociation inhibitor (GDI)-1 that promotes membrane addition at invadopodia that may allow invadopodia to extend through basement membranes
The netrin receptor UNC-40/DCC localizes to the BM breach site and directs formation of a large protrusion that widens the BM breach and shuts down additional invadopodia formation [22]

Summary

Introduction

Basement membrane (BM) is a dense, highly cross-linked extracellular matrix that surrounds most tissues and acts as a barrier to migrating cells [1]. Invadopodia are protrusive, F-actin rich, membrane associated structures that were identified over twenty years ago in vitro within transformed cells and highly metastatic cancer cell lines [7,8,9]. The formation and regulation of invadopodia have been examined extensively in cancer cell lines and tumor models because these structures are thought to facilitate tumor cell invasion across BM barriers [9,10,11,12,13]. Due to the difficulty of examining the dynamic interactions between invasive cells, BM, and tissue being invaded in native physiological settings, the mechanisms that control invadopodia formation and activity in vivo remain largely unknown [18,19,20]

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Discussion

Conclusion