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.

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.

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.

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.