The transcription factor HLH-2/E/Daughterless regulates anchor cell invasion across basement membrane in C. elegans.
ABSTRACT: Cell invasion through basement membrane is a specialized cellular behavior critical for many developmental processes and leukocyte trafficking. Invasive cellular behavior is also inappropriately co-opted during cancer progression. Acquisition of an invasive phenotype is accompanied by changes in gene expression that are thought to coordinate the steps of invasion. The transcription factors responsible for these changes in gene expression, however, are largely unknown. C. elegans anchor cell (AC) invasion is a genetically tractable in vivo model of invasion through basement membrane. AC invasion requires the conserved transcription factor FOS-1A, but other transcription factors are thought to act in parallel to FOS-1A to control invasion. Here we identify the transcription factor HLH-2, the C. elegans ortholog of Drosophila Daughterless and vertebrate E proteins, as a regulator of AC invasion. Reduction of HLH-2 function by RNAi or with a hypomorphic allele causes defects in AC invasion. Genetic analysis indicates that HLH-2 has functions outside of the FOS-1A pathway. Using expression analysis, we identify three genes that are transcriptionally regulated by HLH-2: the protocadherin cdh-3, and two genes encoding secreted extracellular matrix proteins, mig-6/papilin and him-4/hemicentin. Further, we show that reduction of HLH-2 function causes defects in polarization of F-actin to the invasive cell membrane, a process required for the AC to generate protrusions that breach the basement membrane. This work identifies HLH-2 as a regulator of the invasive phenotype in the AC, adding to our understanding of the transcriptional networks that control cell invasion.
Project description:Cell invasion through basement membrane (BM) occurs in many physiological and pathological contexts. MIG-10, the Caenorhabditis elegans Lamellipodin (Lpd), regulates diverse biological processes. Its function and regulation in cell invasive behavior remain unclear. Using anchor cell (AC) invasion in C. elegans as an in vivo invasion model, we have previously found that mig-10's activity is largely outside of UNC-6 (netrin) signaling, a chemical cue directing AC invasion. We have shown that MIG-10 is a target of the transcription factor FOS-1A and facilitates BM breaching. Combining genetics and imaging analyses, we report that MIG-10 synergizes with UNC-6 to promote AC attachment to the BM, revealing a functional role for MIG-10 in stabilizing AC-BM adhesion. MIG-10 is also required for F-actin accumulation in the absence of UNC-6. Further, we identify mig-10 as a transcriptional target negatively regulated by EGL-43A (C. elegans Evi-1 proto-oncogene), a transcription factor positively controlled by FOS-1A. The revelation of this negative regulation unmasks an incoherent feedforward circuit existing among fos-1, egl-43 and mig-10. Moreover, our study suggests the functional importance of the negative regulation on mig-10 expression by showing that excessive MIG-10 impairs AC invasion. Thus, we provide new insight into MIG-10's function and its complex transcriptional regulation during cell invasive behavior.
Project description:To transmigrate basement membrane, cells must coordinate distinct signaling activities to breach and pass through this dense extracellular matrix barrier. Netrin expression and activity are strongly associated with invasion in developmental and pathological processes, but how netrin signaling is coordinated with other pathways during invasion is poorly understood. Using the model of anchor cell (AC) invasion in C. elegans, we have previously shown that the integrin receptor heterodimer INA-1/PAT-3 promotes netrin receptor UNC-40 (DCC) localization to the invasive cell membrane of the AC. UNC-6 (netrin)/UNC-40 interactions generate an invasive protrusion that crosses the basement membrane. To understand how UNC-40 signals during invasion, we have used genetic, site of action and live-cell imaging studies to examine the roles of known effectors of UNC-40 signaling in axon outgrowth during AC invasion. UNC-34 (Ena/VASP), the Rac GTPases MIG-2 and CED-10 and the actin binding protein UNC-115 (abLIM) are dedicated UNC-40 effectors that are recruited to the invasive membrane by UNC-40 and generate F-actin. MIG-10 (lamellipodin), an effector of UNC-40 in neurons, however, has independent functions from UNC-6/UNC-40. Furthermore, unlike other UNC-40 effectors, its expression is regulated by FOS-1A, a transcription factor that promotes basement membrane breaching. Similar to UNC-40, however, MIG-10 localization to the invasive cell membrane is also dependent on the integrin INA-1/PAT-3. These studies indicate that MIG-10 has distinct functions from UNC-40 signaling in cell invasion, and demonstrate that integrin coordinates invasion by localizing these molecules to the cell-basement membrane interface.
Project description:Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.
Project description:Despite their profound importance in the development of cancer, the extracellular cues that target cell invasion through basement membrane barriers remain poorly understood. A central obstacle has been the difficulty of studying the interactions between invading cells and basement membranes in vivo. Using the genetically and visually tractable model of Caenorhabditis elegans anchor cell (AC) invasion, we show that UNC-6 (netrin) signalling, a pathway not previously implicated in controlling cell invasion in vivo, is a key regulator of this process. Site of action studies reveal that before invasion, localized UNC-6 secretion directs its receptor, UNC-40, to the plasma membrane of the AC, in contact with the basement membrane. There, UNC-40 polarizes a specialized invasive membrane domain through the enrichment of actin regulators, F-actin and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Cell ablation experiments indicate that UNC-6 promotes the formation of invasive protrusions from the AC that break down the basement membrane in response to a subsequent vulval cue. Together, these results characterize an invasive membrane domain in vivo, and reveal a role for UNC-6 (netrin) in polarizing this domain towards its basement membrane target.
Project description:Cellular invasion is a key part of development, immunity and disease. Using an <i>in vivo</i> model of <i>Caenorhabditis elegans</i> anchor cell invasion, we characterize the gene regulatory network that promotes cell invasion. The anchor cell is initially specified in a stochastic cell fate decision mediated by Notch signaling. Previous research has identified four conserved transcription factors, <i>fos-1</i> (Fos), <i>egl-43</i> (EVI1/MEL), <i>hlh-2</i> (E/Daughterless) and <i>nhr-67</i> (NR2E1/TLX), that mediate anchor cell specification and/or invasive behavior. Connections between these transcription factors and the underlying cell biology that they regulate are poorly understood. Here, using genome editing and RNA interference, we examine transcription factor interactions before and after anchor cell specification. Initially, these transcription factors function independently of one another to regulate LIN-12 (Notch) activity. Following anchor cell specification, <i>egl-43</i>, <i>hlh-2</i> and <i>nhr-67</i> function largely parallel to <i>fos-1</i> in a type I coherent feed-forward loop with positive feedback to promote invasion. Together, these results demonstrate that the same transcription factors can function in cell fate specification and differentiated cell behavior, and that a gene regulatory network can be rapidly assembled to reinforce a post-mitotic, pro-invasive state.
Project description:Integrin expression and activity have been strongly correlated with developmental and pathological processes involving cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of anchor cell (AC) invasion in C. elegans, we have recently shown that netrin signaling orients a specialized invasive cell membrane domain toward the basement membrane. Here, we demonstrate that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part by targeting the netrin receptor UNC-40 (DCC) to the AC's plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2, and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain.
Project description:Though critical to normal development and cancer metastasis, how cells traverse basement membranes is poorly understood. A central impediment has been the challenge of visualizing invasive cell interactions with basement membrane in vivo. By developing live-cell imaging methods to follow anchor cell (AC) invasion in Caenorhabditis elegans, we identify F-actin-based invadopodia that breach basement membrane. When an invadopodium penetrates basement membrane, it rapidly transitions into a stable invasive process that expands the breach and crosses into the vulval tissue. We find that the netrin receptor UNC-40 (DCC) specifically enriches at the site of basement membrane breach and that activation by UNC-6 (netrin) directs focused F-actin formation, generating the invasive protrusion and the cessation of invadopodia. Using optical highlighting of basement membrane components, we further demonstrate that rather than relying solely on proteolytic dissolution, the AC's protrusion physically displaces basement membrane. These studies reveal an UNC-40-mediated morphogenetic transition at the cell-basement membrane interface that directs invading cells across basement membrane barriers.
Project description:Matrix metalloproteinases (MMPs) are associated with decreased patient prognosis but have failed as anti-invasive drug targets despite promoting cancer cell invasion. Through time-lapse imaging, optical highlighting, and combined genetic removal of the five MMPs expressed during anchor cell (AC) invasion in C. elegans, we find that MMPs hasten invasion by degrading basement membrane (BM). Though irregular and delayed, AC invasion persists in MMP- animals via adaptive enrichment of the Arp2/3 complex at the invasive cell membrane, which drives formation of an F-actin-rich protrusion that physically breaches and displaces BM. Using a large-scale RNAi synergistic screen and a genetically encoded ATP FRET sensor, we discover that mitochondria enrich within the protrusion and provide localized ATP that fuels F-actin network growth. Thus, without MMPs, an invasive cell can alter its BM-breaching tactics, suggesting that targeting adaptive mechanisms will be necessary to mitigate BM invasion in human pathologies.
Project description:During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.
Project description: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.