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

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.

Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain

“…Together, these results indicate that UNC-40 directs the formation of a large protrusion that locally expands the size of the AC to open a single large gap in the BM. This is in marked contrast to the distinctly different action of invadopodia, which breach BM, are more numerous, smaller, short-lived, and not directed by UNC-40 (Hagedorn et al, 2013, 2014; Lohmer et al, 2016). …”

“…In addition to membrane addition, lysosome exocytosis might contribute cathepsin proteases that could promote BM removal (Kirkegaard and Jäättelä, 2009). However, there are at least 43 genes encoding cathepsins in C. elegans (Xu et al, 2014) and extensive genetic and RNAi screens have not identified a role for cathepsins during AC invasion (Lohmer et al, 2016; Matus et al, 2010). Thus, if cathepsins do promote invasion they may function redundantly.…”

“…The AC uses dynamic, rapidly forming and disassembling invadopodia to breach the BM separating the uterine tissue from the vulval tissue (Hagedorn et al, 2013, 2014; Lohmer et al, 2016). Approximately 10 invadopodia are found at any one time prior to invasion.…”

A new front in cell invasion: The invadopodial membrane

“…This includes well-studied actin regulators, matrix metalloproteinases (MMPs), signaling pathways, and integrins, as well as genes involved with glycolysis, metabolism, protein degradation, chaperone activity, and protein synthesis for which an exact role in invadopodia formation has not been determined (Attanasio et al, 2011; Hoshino et al, 2013; Lohmer et al, 2016). The breadth of gene families associated with invadopodia likely reflects the complexity and intricate regulation of invadopodia and suggests that many aspects of their function and control remain unknown.…”

“…The basement membrane in C. elegans is highly conserved and all major basement membrane components and receptors found in vertebrates are also present in C. elegans (Kramer, 2005). A suite of unique attributes of C. elegans as a model organism — including fluorescently tagged basement membrane components, anchor cell specific expression of fluorescently tagged proteins, the highly stereotyped nature of invasion, and genetic analysis—have allowed detailed experimental dissection of invadopodia in vivo (Hagedorn et al, 2014; Hagedorn et al, 2013; Lohmer et al, 2016; Lohmer et al, 2014). …”