Basement membrane (BM), a sheet-like form of extracellular matrix, surrounds most tissues. During organogenesis, specific adhesions between adjoining tissues frequently occur; however, their molecular basis is unclear. Using live-cell imaging and electron microscopy, we identify an adhesion system that connects the uterine and gonadal tissues through their juxtaposed BMs at the site of anchor cell (AC) invasion in C. elegans. We find that the extracellular matrix component hemicentin (HIM-4), found between BMs, forms punctate accumulations under the AC and controls BM linkage to promote rapid invasion. Through targeted screening, we identify the integrin-binding cytolinker plakin (VAB-10A) and integrin (INA-1/PAT-3) as key BM-BM linkage regulators: VAB-10A localizes to the AC-BM interface and tethers hemicentin to the AC while integrin promotes hemicentin punctae formation. Together, plakin, integrin, and hemicentin are founding components of a cell-directed adhesion system, which we name a BM-LINKage (B-LINK), that connects adjacent tissues through adjoining BMs.

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.

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.

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins and their animal orthologs localized to BMs. Network analysis and screening in C. elegans and zebrafish uncovered BM regulators, including ADAMTS, ROBO, and TGFβ. More than 100 BM network genes associate with human phenotypes, and by screening 63,039 genomes from families with rare disorders, we found loss-of-function variants in LAMA5, MPZL2, and MATN2 and show that they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity of BMs and their impact on in human health.

Basement membrane (BM) matrices surround and separate most tissues. However, through poorly understood mechanisms, BMs of adjacent tissue can also stably link to support organ structure and function. Using endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging, we identified extracellular matrix proteins mediating a BM linkage (B-LINK) between the uterine utse and epidermal seam cell BMs in Caenorhabditis elegans that supports the uterus during egg-laying. We found that hemicentin is secreted by the utse and promotes fibulin-1 assembly to jointly initiate the B-LINK. During egg-laying, however, both proteins' levels decline and are not required for B-LINK maintenance. Instead, we discovered that hemicentin recruits ADAMTS9/20, which facilitates the assembly of high levels of type IV collagen that sustains the B-LINK during the mechanically active egg-laying period. This work reveals mechanisms underlying BM-BM linkage maturation and identifies a crucial function for hemicentin and fibulin-1 in initiating attachment and type IV collagen in strengthening this specialized form of tissue linkage.

Large gaps in basement membrane (BM) occur during organ remodelling and cancer cell invasion. Whether dividing cells, which temporarily reduce their attachment to BM, influence these breaches is unknown. Here we analyse uterine-vulval attachment during development across 21 species of rhabditid nematodes and find that the BM gap that forms between these organs is always bounded by a non-dividing vulval cell. Through cell cycle manipulation and live cell imaging in Caenorhabditis elegans, we show that actively dividing vulval cells facilitate enlargement of this breach by promoting BM movement. In contrast, targeted cell cycle arrest halts BM movement and limits gap opening. Further, we demonstrate that the BM component laminin accumulates at the BM gap edge and promotes increased integrin levels in non-dividing vulval cells, stabilizing gap position. Together, these studies reveal that cell division can be used as a mechanism to regulate BM breaches, thus controlling the exchange of cells between tissues.

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.

Transport of G protein-coupled receptors (GPCRs) to the cell surface membrane is critical in order for the receptors to recognize their ligands. However, mammalian GPCR odorant receptors (ORs), when heterologously expressed in cells, are poorly expressed on the cell surface. Here we show that the transmembrane proteins RTP1 and RTP2 promote functional cell surface expression of ORs expressed in HEK293T cells. Genes encoding these proteins are expressed specifically in olfactory neurons. These proteins are associated with OR proteins and enhance the OR responses to odorants. Similar although weaker effects were seen with a third protein, REEP1. These findings suggest that RTP1 and RTP2 in particular play significant roles in the translocation of ORs to the plasma membrane as well as in the functioning of ORs. We have used this approach to identify active odorant ligands for ORs, providing a platform for screening the chemical selectivity of the large OR family.

Ras GTPases regulate many developmental and physiological processes and mutations in Ras are associated with numerous human cancers. Here, we report the function, levels, and localization of an N-terminal knock-in of mNeonGreen (mNG) into C. elegans LET-60 /Ras. mNG:: LET-60 interferes with some but not all LET-60 /Ras functions. mNG:: LET-60 is broadly present in tissues, found at different levels in cells, and concentrates in distinct subcellular compartments, including the nucleolus, nucleus, intracellular region, and plasma membrane. These results suggest that mNG:: LET-60 can be a useful tool for determining LET-60 levels and localization once its functionality in a developmental or physiological process is established.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the Caenorhabditis elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell-specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor-2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

Olfactory systems have evolved the extraordinary capability to detect and discriminate volatile odorous molecules (odorants) in the environment. Fundamentally, this process relies on the interaction of odorants and their cognate olfactory receptors (ORs) encoded in the genome. Here, we conducted a cell-based screen using over 800 mouse ORs against seven odorants, resulting in the identification of a set of high-affinity and/or broadly-tuned ORs. We then test whether heterologously expressed ORs respond to odors presented in vapor phase by individually expressing 31 ORs to measure cAMP responses against vapor phase odor stimulation. Comparison of response profiles demonstrates this platform is capable of discriminating between structural analogs. Lastly, co-expression of carboxyl esterase Ces1d expressed in olfactory mucosa resulted in marked changes in activation of specific odorant-OR combinations. Altogether, these results establish a cell-based volatile odor detection and discrimination platform and form the basis for an OR-based volatile odor sensor.

Epithelial cells and their underlying basement membranes (BMs) slide along each other to renew epithelia, shape organs, and enlarge BM openings. How BM sliding is controlled, however, is poorly understood. Using genetic and live cell imaging approaches during uterine-vulval attachment in C. elegans, we have discovered that the invasive uterine anchor cell activates Notch signaling in neighboring uterine cells at the boundary of the BM gap through which it invades to promote BM sliding. Through an RNAi screen, we found that Notch activation upregulates expression of ctg-1, which encodes a Sec14-GOLD protein, a member of the Sec14 phosphatidylinositol-transfer protein superfamily that is implicated in vesicle trafficking. Through photobleaching, targeted knockdown, and cell-specific rescue, our results suggest that CTG-1 restricts BM adhesion receptor DGN-1 (dystroglycan) trafficking to the cell-BM interface, which promotes BM sliding. Together, these studies reveal a new morphogenetic signaling pathway that controls BM sliding to remodel tissues.

Invadopodia are protrusive, F-actin-driven membrane structures that are thought to mediate basement membrane transmigration during development and tumor dissemination. An understanding of the mechanisms regulating invadopodia has been hindered by the difficulty of examining these dynamic structures in native environments. Using an RNAi screen and live-cell imaging of anchor cell (AC) invasion in Caenorhabditis elegans, we have identified UNC-60A (ADF/cofilin) as an essential regulator of invadopodia. UNC-60A localizes to AC invadopodia, and its loss resulted in a dramatic slowing of F-actin dynamics and an inability to breach basement membrane. Optical highlighting indicated that UNC-60A disassembles actin filaments at invadopodia. Surprisingly, loss of unc-60a led to the accumulation of invadopodial membrane and associated components within the endolysosomal compartment. Photobleaching experiments revealed that during normal invasion the invadopodial membrane undergoes rapid recycling through the endolysosome. Together, these results identify the invadopodial membrane as a specialized compartment whose recycling to form dynamic, functional invadopodia is dependent on localized F-actin disassembly by ADF/cofilin.

Many stem cell niches contain support cells that increase contact with stem cells by enwrapping them in cellular processes. One example is the germ stem cell niche in C. elegans, which is composed of a single niche cell termed the distal tip cell (DTC) that extends cellular processes, constructing an elaborate plexus that enwraps germ stem cells. To identify genes required for plexus formation and to explore the function of this specialized enwrapping behavior, a series of targeted and tissue-specific RNAi screens were performed. Here we identify genes that promote stem cell enwrapment by the DTC plexus, including a set that specifically functions within the DTC, such as the chromatin modifier lin-40/MTA1, and others that act within the germline, such as the 14-3-3 signaling protein par-5. Analysis of genes that function within the germline to mediate plexus development reveal that they are required for expansion of the germ progenitor zone, supporting the emerging idea that germ stem cells signal to the niche to stimulate enwrapping behavior. Examination of wild-type animals with asymmetric plexus formation and animals with reduced DTC plexus elaboration via loss of two candidates including lin-40 indicate that cellular enwrapment promotes GLP-1/Notch signaling and germ stem cell fate. Together, our work identifies novel regulators of cellular enwrapment and suggests that reciprocal signaling between the DTC niche and the germ stem cells promotes enwrapment behavior and stem cell fate.

Basement membranes (BMs) are supramolecular matrices built on laminin and type IV collagen networks that provide structural and signaling support to tissues. BM complexity, however, has hindered an understanding of its formation, dynamics, and regulation. Using genome editing, we tagged 29 BM matrix components and receptors in C. elegans with mNeonGreen. Here, we report a common template that initiates BM formation, which rapidly diversifies during tissue differentiation. Through photobleaching studies, we show that BMs are not static-surprisingly, many matrix proteins move within the laminin and collagen scaffoldings. Finally, quantitative imaging, conditional knockdown, and optical highlighting indicate that papilin, a poorly studied glycoprotein, is the most abundant component in the gonadal BM, where it facilitates type IV collagen removal during BM expansion and tissue growth. Together, this work introduces methods for holistic investigation of BM regulation and reveals that BMs are highly dynamic and capable of rapid change to support tissues.

Invasive cells use transient, energy-consuming protrusions to breach basement membrane (BM) barriers. Using the ATP sensor PercevalHR during anchor cell (AC) invasion in Caenorhabditis elegans, we show that BM invasion is accompanied by an ATP burst from mitochondria at the invasive front. RNAi screening and visualization of a glucose biosensor identified two glucose transporters, FGT-1 and FGT-2, which bathe invasive front mitochondria with glucose and facilitate the ATP burst to form protrusions. FGT-1 localizes at high levels along the invasive membrane, while FGT-2 is adaptive, enriching most strongly during BM breaching and when FGT-1 is absent. Cytosolic glycolytic enzymes that process glucose for mitochondrial ATP production cluster with invasive front mitochondria and promote higher mitochondrial membrane potential and ATP levels. Finally, we show that UNC-6 (netrin), which polarizes invasive protrusions, also orients FGT-1. These studies reveal a robust and integrated energy acquisition, processing, and delivery network that powers BM breaching.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the C. elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor 2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

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.