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

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.

Basement Membranes in the Worm: A Dynamic Scaffolding that Instructs Cellular Behaviors and Shapes Tissues.

The nematode worm Caenorhabditis elegans has all the major basement membrane proteins found in vertebrates, usually with a smaller gene family encoding each component. With its powerful forward genetics, optical clarity, simple tissue organization, and the capability to functionally tag most basement membrane components with fluorescent proteins, C. elegans has facilitated novel insights into the assembly and function of basement membranes. Although basement membranes are generally thought of as static structures, studies in C. elegans have revealed their active properties and essential functions in tissue formation and maintenance. Here, we review discoveries from C. elegans development that highlight dynamic aspects of basement membrane assembly, function, and regulation during organ growth, tissue polarity, cell migration, cell invasion, and tissue attachment. These studies have helped transform our view of basement membranes from static support structures to dynamic scaffoldings that play broad roles in regulating tissue organization and cellular behavior that are essential for development and have important implications in human diseases.

Cadherin 6 promotes neural crest cell detachment via F-actin regulation and influences active Rho distribution during epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) is a complex change in cell phenotype that is important for cell migration, morphogenesis and carcinoma metastasis. Loss of epithelial cell adhesion and tight regulation of cadherin adhesion proteins are crucial for EMT. Cells undergoing EMT often display cadherin switching, where they downregulate one cadherin and induce expression of another. However, the functions of the upregulated cadherins and their effects on cell motility are poorly understood. Neural crest cells (NCCs), which undergo EMT during development, lose N-cadherin and upregulate Cadherin 6 (Cdh6) prior to EMT. Cdh6 has been suggested to suppress EMT via cell adhesion, but also to promote EMT by mediating pro-EMT signals. Here, we determine novel roles for Cdh6 in generating cell motility during EMT. We use live imaging of NCC behavior in vivo to show that Cdh6 promotes detachment of apical NCC tails, an important early step of EMT. Furthermore, we show that Cdh6 affects spatiotemporal dynamics of F-actin and active Rho GTPase, and that Cdh6 is required for accumulation of F-actin in apical NCC tails during detachment. Moreover, Cdh6 knockdown alters the subcellular distribution of active Rho, which is known to promote localized actomyosin contraction that is crucial for apical NCC detachment. Together, these data suggest that Cdh6 is an important determinant of where subcellular actomyosin forces are generated during EMT. Our results also identify mechanisms by which an upregulated cadherin can generate cell motility during EMT.

Rho activation is apically restricted by Arhgap1 in neural crest cells and drives epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transitions (EMTs) are crucial for morphogenesis and carcinoma metastasis, yet mechanisms controlling the underlying cell behaviors are poorly understood. RhoGTPase signaling has been implicated in EMT; however, previous studies have yielded conflicting results regarding Rho function, and its role in EMT remains poorly understood. Elucidation of precise Rho functions has been challenging because Rho signaling is highly context dependent and its activity is tightly regulated spatiotemporally within the cell. To date, few studies have examined how Rho affects cell motility in intact organisms, and the pattern of Rho activity during motile cell behaviors of EMT has not been determined in any system. Here, we image endogenous active Rho during EMT in vivo, and analyze effects of Rho and Rho-kinase (ROCK) manipulation on cell motility in vivo. We show that Rho is activated in a discrete apical region of premigratory neural crest cells during EMT, and Rho-ROCK signaling is essential for apical detachment and generation of motility within the neuroepithelium, a process that has been poorly understood. Furthermore, we find that Arhgap1 restricts Rho activation to apical areas, and this restriction is necessary for detachment. Our results provide new insight into mechanisms controlling local Rho activation and how it affects dynamic cell behaviors and actomyosin contraction during key steps of EMT in an intact living organism.

Head and neck cancer stem cells: the side population.

OBJECTIVES/HYPOTHESIS: The cancer stem cell (CSC) theory concludes that a subpopulation of cancer cells, the cancer stem cells, can self-renew and are responsible for tumor growth. Previous studies have identified cells able to efflux Hoechst 33342 dye as the side population (SP). SP cells and CSCs share many characteristics, suggesting the SP isolated from malignant tumors contains CSCs. STUDY DESIGN: Experimental Study. METHODS: The SP was isolated from a head and neck cancer cell line and analyzed for CSC-like characteristics. RESULTS: The SP demonstrated the ability to reproduce both SP and non-side population (NSP) cells from as few as one cell. The SP had lower expression of active ß-catenin and more resistance to 5-fluorouracil; the SP also demonstrated greater expression of Bmi-1 (4.3-fold) and ABCG2 (1.4-fold). SP cells were able to produce tumors in an animal model, whereas NSP were not. SPs were identified in two primary human tumors. CONCLUSIONS: This work adds to the evidence that the SP in head and neck cancer represents cells with CSC properties and provides a method by which CSCs can be isolated and studied.

Regulation of cell adhesions and motility during initiation of neural crest migration.

Accurate neural crest cell (NCC) migration requires tight control of cell adhesions, cytoskeletal dynamics and cell motility. Cadherins and RhoGTPases are critical molecular players that regulate adhesions and motility during initial delamination of NCCs from the neuroepithelium. Recent studies have revealed multiple functions for these molecules and suggest that a precise balance of their activity is crucial. RhoGTPase appears to regulate both cell adhesions and protrusive forces during NCC delamination. Increasing evidence shows that cadherins are multi-functional proteins with novel, adhesion-independent signaling functions that control NCC motility during both delamination and migration. These functions are often regulated by specific proteolytic cleavage of cadherins. After NCC delamination, planar cell polarity signaling acts via RhoGTPases to control NCC protrusions and migration direction.

Control of neural crest cell behavior and migration: Insights from live imaging.

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system, and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.

Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase.

BACKGROUND: In accord with the cancer stem cell (CSC) theory, only a small subset of cancer cells are capable of forming tumors. We previously reported that CD44 isolates tumorigenic cells from head and neck squamous cell cancer (HNSCC). Recent studies indicate that aldehyde dehydrogenase (ALDH) activity may represent a more specific marker of CSCs. METHODS: Six primary HNSCCs were collected. Cells with high and low ALDH activity (ALDH(high)/ALDH(low)) were isolated. ALDH(high) and ALDH(low) populations were implanted into NOD/SCID mice and monitored for tumor development. RESULTS: ALDH(high) cells represented a small percentage of the tumor cells (1% to 7.8%). ALDH(high) cells formed tumors from as few as 500 cells in 24/45 implantations, whereas only 3/37 implantations of ALDH(low) cells formed tumors. CONCLUSIONS: ALDH(high) cells comprise a subpopulation cells in HNSCCs that are tumorigenic and capable of producing tumors at very low numbers. This finding indicates that ALDH activity on its own is a highly selective marker for CSCs in HNSCC.

Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.

The induction and migration of neural crest cells (NCCs) are essential to the development of craniofacial structures and the peripheral nervous system. A critical step in the development of NCCs is the epithelial to mesenchymal transition (EMT) that they undergo in order to initiate migration. Several transcription factors are important for the NCC EMT. However, less is known about the effectors regulating changes in cell adhesion, the cytoskeleton, and cell motility associated with the EMT or about specific changes in the behavior of cells undergoing EMT in vivo. We used time-lapse imaging of NCCs in the zebrafish hindbrain to show that NCCs undergo a stereotypical series of behaviors during EMT. We find that loss of cell adhesion and membrane blebbing precede filopodial extension and the onset of migration. Live imaging of actin dynamics shows that actin localizes differently in blebs and filopodia. Moreover, we find that disruption of myosin II or Rho-kinase (ROCK) activity inhibits NCC blebbing and causes reduced NCC EMT. These data reveal roles for myosin II and ROCK in NCC EMT in vivo, and provide a detailed characterization of NCC behavior during EMT that will form a basis for further mechanistic studies.