Vertebrate hemicentin-1 interacts physically and genetically with nidogen-2

Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mammalian hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 for basement membrane integrity. HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately ten-fold lower affinity and in a competitive manner. Immunofluorescence and immunogold labellings reveal that HMCN1/Hmcn1 is localized closely attached to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as loss of Laminin-alpha5 (Lama5) or Hmcn1. Combined partial loss-of-function studies further indicate that nid2a genetically interacts with both hmcn1 and lama5. Together, this suggests that despite their mutually exclusive physical binding, hemicentins, nidogens and laminins tightly cooperate and support each other during formation, maintenance and function of basement membranes to confer tissue linkage.

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.

Chemotherapy-induced collagen IV drives cancer cell invasion through activation of Src/FAK signaling

Triple-negative breast cancer (TNBC) is the most aggressive and deadly subtype of breast cancer, accounting for 30,000 cases annually in the US. While there are several clinical trials ongoing to identify new agents to treat TNBC, the majority of TNBC patients are treated with anthracycline- or taxane-based chemotherapies in the neoadjuvant setting, followed by surgical resection and adjuvant chemotherapy. While many patients respond well to this approach, as many as 25% will suffer local or metastatic recurrence within five years. Understanding the mechanisms that drive recurrence after chemotherapy treatment is critical to improving survival for patients with TNBC. It is well-established that the extracellular matrix, which provides structure and support to tissues, is a major driver of tumor growth, local invasion and dissemination of cancer cells to distant metastatic sites. In the present study, we show that decellularized extracellular matrix (dECM) obtained from chemotherapy-treated mice increases invasion of treatment-naïve breast cancer cells compared to vehicle-treated dECM. Using tandem-mass-tag proteomics, we further demonstrate that anthracycline- and taxane-based chemotherapies induce drug-specific changes in tumor ECM composition. We identify the basement membrane protein collagen IV as significantly upregulated in the ECM of chemotherapy-treated mice and patients treated with neoadjuvant chemotherapy. We show that collagen IV drives invasion via Src/FAK signaling and that inhibiting collagen IV-driven signaling decreases invasion in chemotherapy-treated dECM. These studies provide a novel mechanism by which chemotherapy may induce metastasis via effects on ECM composition.One Sentence SummaryChemotherapy alters the extracellular matrix of breast tumors leading to increased invasion of residual cancer cells.

Kinesin-3 and kinesin-1 motors direct basement membrane protein secretion to a basal sub-region of the basolateral plasma membrane in epithelial cells

SUMMARYBasement membranes (BMs) are sheet-like extracellular matrices that line the basal surfaces of all epithelia. Since BM proteins form networks, they likely need to be secreted near the basal surface. However, the location of their secretion site and how it is selected are unknown. Working in the Drosophila follicular epithelium, we identified two kinesins essential for normal BM formation. Our data suggest the two kinesins work together to transport Rab10+ BM protein-filled secretory vesicles towards the basal surface along the polarized microtubule array common to epithelia. This kinesin transport biases BM protein secretion basally. When kinesins are depleted, BM proteins are mis-secreted to more apical regions of the lateral membrane, creating ectopic BM protein networks between cells that disrupt cell movements and tissue architecture. These results introduce a new transport step in the BM protein secretion pathway and highlight the importance of controlling the sub-cellular exocytic site of network-forming proteins.HighlightsA kinesin-3 and a kinesin-1 are required for normal basement membrane (BM) assemblyKinesins move Rab10+ BM secretory vesicles basally on polarized microtubule arraysTransport biases BM exocytosis to basal subregions of the basolateral membraneLoss of kinesins creates ectopic BM networks that disrupt tissue architecture

Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans

During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1, let-2/collagen IV a2 and fbl-1/fibulin-1, were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9, let-2 and fbl-1 mutations suppressed both mig-17 and gon-1, whereas others acted only on mig-17 or gon-1. These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.

Human cytomegalovirus interactions with the basement membrane protein nidogen 1

In 2000, we reported HCMV induced specific damage on chromosome 1. The virus' capacity to induce DNA breaks indicated potent interaction between viral protein/s and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12-24 hpi, and continuing throughout infection, steady state (ss) NID1 protein levels were decreased in whole cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor, MG132, to culture media stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found ss NID1 was also reduced in infected ECs and hypothesized virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1 knockout EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal CNS/PNS and is important for neuronal migration, neural network excitability and plasticity and regulates Schwann cell proliferation, migration and myelin production. We found NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus. SIGNIFICANCE: We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein, nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cells' (ECs) secretion of basement membrane proteins is critical for vascular wall integrity and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.

RASA1-driven cellular export of collagen IV is required for the development of lymphovenous and venous valves in mice

ABSTRACTRASA1, a negative regulator of Ras-MAPK signaling, is essential for the development and maintenance of lymphatic vessel valves. However, whether RASA1 is required for the development and maintenance of lymphovenous valves (LVV) and venous valves (VV) is unknown. In this study, we show that induced disruption of Rasa1 in mouse embryos did not affect initial specification of LVV or central VV, but did affect their continued development. Similarly, a switch to expression of a catalytically inactive form of RASA1 resulted in impaired LVV and VV development. Blocked development of LVV was associated with accumulation of the basement membrane protein, collagen IV, in LVV-forming endothelial cells (EC), and could be partially or completely rescued by MAPK inhibitors and drugs that promote collagen IV folding. Disruption of Rasa1 in adult mice resulted in venous hypertension and impaired VV function that was associated with loss of EC from VV leaflets. In conclusion, RASA1 functions as a negative regulator of Ras signaling in EC that is necessary for EC export of collagen IV, thus permitting the development of LVV and the development and maintenance of VV.

Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans

During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS (adisintegrin and metalloprotease with thrombospondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1, let-2/collagen IV a2 and fbl-1/fibulin-1, were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9, let-2 and fbl-1 mutations suppressed both mig-17 and gon-1, whereas others acted only on mig-17 or gon-1. These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.