Tunnels for Protein Export from the Endoplasmic Reticulum.

The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum.

Quality Control of Procollagen in Cells.

Collagen is the most abundant protein in mammals. A unique feature of collagen is its triple-helical structure formed by the Gly-Xaa-Yaa repeats. Three single chains of procollagen make a trimer, and the triple-helical structure is then folded in the endoplasmic reticulum (ER). This unique structure is essential for collagen's functions in vivo, including imparting bone strength, allowing signal transduction, and forming basement membranes. The triple-helical structure of procollagen is stabilized by posttranslational modifications and intermolecular interactions, but collagen is labile even at normal body temperature. Heat shock protein 47 (Hsp47) is a collagen-specific molecular chaperone residing in the ER that plays a pivotal role in collagen biosynthesis and quality control of procollagen in the ER. Mutations that affect the triple-helical structure or result in loss of Hsp47 activity cause the destabilization of procollagen, which is then degraded by autophagy. In this review, we present the current state of the field regarding quality control of procollagen.

Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Regulation of the CUL3 Ubiquitin Ligase by a Calcium-Dependent Co-adaptor.

The ubiquitin ligase CUL3 is an essential regulator of neural crest specification whose aberrant activation has been linked to autism, schizophrenia, and hypertension. CUL3 exerts its roles by pairing with ∼90 distinct substrate adaptors, yet how the different CUL3-complexes are activated is poorly understood. Here, we show that CUL3 and its adaptor KLHL12 require two calcium-binding proteins, PEF1 and ALG2, for recognition of their substrate SEC31. PEF1 and ALG2 form a target-specific co-adaptor that translates a transient rise in cytosolic calcium levels into more persistent SEC31 ubiquitylation, which in turn triggers formation of large COPII coats and promotes collagen secretion. As calcium also instructs chondrocyte differentiation and collagen synthesis, calcium-dependent control of CUL3KLHL12 integrates collagen secretion into broader programs of craniofacial bone formation. Our work, therefore, identifies both calcium and CUL3 co-adaptors as important regulators of ubiquitylation events that control human development.

Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Bromine is an essential trace element for assembly of collagen IV scaffolds in tissue development and architecture.

Bromine is ubiquitously present in animals as ionic bromide (Br(-)) yet has no known essential function. Herein, we demonstrate that Br(-) is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a posttranslational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br deficiency is lethal in Drosophila, whereas Br replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br(-), collagen IV, and peroxidasin. We establish that Br(-) is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals, and its deficiency may be relevant to BM alterations observed in nutritional and smoking-related disease. PAPERFLICK:

The extracellular matrix dictates regional competence for tumour initiation.

The skin epidermis is constantly renewed throughout life1,2. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation3. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2-a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma-affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.

The pathway of collagen secretion.

COPII vesicles mediate export of secretory cargo from the endoplasmic reticulum (ER). However, a standard COPII vesicle with a diameter of 60-90 nm is too small to export collagens that are composed of rigid triple helices of up to 400 nm in length. How do cells pack and secrete such bulky molecules? This issue is fundamentally important, as collagens constitute approximately 25% of our dry body weight and are essential for almost all cell-cell interactions. Recently, a potential mechanism for the biogenesis of mega-transport carriers was identified, involving packing collagens and increasing the size of COPII coats. Packing is mediated by TANGO1, which binds procollagen VII in the lumen and interacts with the COPII proteins Sec23/Sec24 on the cytoplasmic side of the ER. Cullin3, an E3 ligase, and its specific adaptor protein, KLHL12, ubiquitinate Sec31, which could increase the size of COPII coats. Recruitment of these proteins and their specific interactors into COPII-mediated vesicle biogenesis may be all that is needed for the export of bulky collagens from the ER. Nonetheless, we present an alternative pathway in which TANGO1 and COPII cooperate to export collagens without generating a mega-transport carrier.

The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.

Human neocortical expansion involves glutamatergic neuron diversification.

The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.

Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion.

Immune exclusion predicts poor patient outcomes in multiple malignancies, including triple-negative breast cancer (TNBC)1. The extracellular matrix (ECM) contributes to immune exclusion2. However, strategies to reduce ECM abundance are largely ineffective or generate undesired outcomes3,4. Here we show that discoidin domain receptor 1 (DDR1), a collagen receptor with tyrosine kinase activity5, instigates immune exclusion by promoting collagen fibre alignment. Ablation of Ddr1 in tumours promotes the intratumoral penetration of T cells and obliterates tumour growth in mouse models of TNBC. Supporting this finding, in human TNBC the expression of DDR1 negatively correlates with the intratumoral abundance of anti-tumour T cells. The DDR1 extracellular domain (DDR1-ECD), but not its intracellular kinase domain, is required for immune exclusion. Membrane-untethered DDR1-ECD is sufficient to rescue the growth of Ddr1-knockout tumours in immunocompetent hosts. Mechanistically, the binding of DDR1-ECD to collagen enforces aligned collagen fibres and obstructs immune infiltration. ECD-neutralizing antibodies disrupt collagen fibre alignment, mitigate immune exclusion and inhibit tumour growth in immunocompetent hosts. Together, our findings identify a mechanism for immune exclusion and suggest an immunotherapeutic target for increasing immune accessibility through reconfiguration of the tumour ECM.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

A myeloid-stromal niche and gp130 rescue in NOD2-driven Crohn's disease.

Crohn's disease is a chronic inflammatory intestinal disease that is frequently accompanied by aberrant healing and stricturing complications. Crosstalk between activated myeloid and stromal cells is critical in the pathogenicity of Crohn's disease1,2, and increases in intravasating monocytes are correlated with a lack of response to anti-TNF treatment3. The risk alleles with the highest effect on Crohn's disease are loss-of-function mutations in NOD24,5, which increase the risk of stricturing6. However, the mechanisms that underlie pathogenicity driven by NOD2 mutations and the pathways that might rescue a lack of response to anti-TNF treatment remain largely uncharacterized. Here we use direct ex vivo analyses of patients who carry risk alleles of NOD2 to show that loss of NOD2 leads to dysregulated homeostasis of activated fibroblasts and macrophages. CD14+ peripheral blood mononuclear cells from carriers of NOD2 risk alleles produce cells that express high levels of collagen, and elevation of conserved signatures is observed in nod2-deficient zebrafish models of intestinal injury. The enrichment of STAT3 regulation and gp130 ligands in activated fibroblasts and macrophages suggested that gp130 blockade might rescue the activated program in NOD2-deficient cells. We show that post-treatment induction of the STAT3 pathway is correlated with a lack of response to anti-TNF treatment in patients, and demonstrate in vivo in zebrafish the amelioration of the activated myeloid-stromal niche using the specific gp130 inhibitor bazedoxifene. Our results provide insights into NOD2-driven fibrosis in Crohn's disease, and suggest that gp130 blockade may benefit some patients with Crohn's disease-potentially as a complement to anti-TNF therapy.

Dual topologies of myotomal collagen XV and Tenascin C act in concert to guide and shape developing motor axons.

During development, motor axons are guided toward muscle target by various extrinsic cues including extracellular matrix (ECM) proteins whose identities and cellular source remain poorly characterized. Here, using single-cell RNAseq of sorted GFP+ cells from smyhc1:gfp-injected zebrafish embryos, we unravel the slow muscle progenitors (SMP) pseudotemporal trajectory at the single-cell level and show that differentiating SMPs are a major source of ECM proteins. The SMP core-matrisome was characterized and computationally predicted to form a basement membrane-like structure tailored for motor axon guidance, including basement membrane-associated ECM proteins, as collagen XV-B, one of the earliest core-matrisome gene transcribed in differentiating SMPs and the glycoprotein Tenascin C. To investigate how contact-mediated guidance cues are organized along the motor path to exert their function in vivo, we used microscopy-based methods to analyze and quantify motor axon navigation in tnc and col15a1b knock-out fish. We show that motor axon shape and growth rely on the timely expression of the attractive cue Collagen XV-B that locally provides axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Importantly, bioprinted micropatterns that mimic this in vivo ECM topology were sufficient to drive directional motor axon growth. Our study offers evidence that not only the composition of ECM cues but their topology critically influences motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury as regenerating nerves follow their original path.

A model for collagen secretion by intercompartmental continuities.

Newly synthesized secretory proteins are exported from the endoplasmic reticulum (ER) at specialized subcompartments called exit sites (ERES). Cargoes like procollagen are too large for export by the standard COPII-coated vesicle of 60 nm average diameter. We have previously suggested that procollagen is transported from the ER to the next secretory organelle, the ER-Golgi intermediate compartment (ERGIC), in TANGO1-dependent interorganelle tunnels. In the theoretical model presented here, we suggest that intrinsically disordered domains of TANGO1 in the ER lumen induce an entropic contraction, which exerts a force that draws procollagen toward the ERES. Within this framework, molecular gradients of pH and/or HSP47 between the ER and ERGIC create a force in the order of tens of femto-Newtons. This force is substantial enough to propel procollagen from the ER at a speed of approximately 1 nm · s-1. This calculated speed and the quantities of collagen secreted are similar to its observed physiological secretion rate in fibroblasts, consistent with the proposal that ER export is the rate-limiting step for procollagen secretion. Hence, the mechanism we propose is theoretically adequate to explain how cells can utilize molecular gradients and export procollagens at a rate commensurate with physiological needs.

A macrophage-collagen fragment axis mediates subcutaneous adipose tissue remodeling in mice.

Efficient removal of fibrillar collagen is essential for adaptive subcutaneous adipose tissue (SAT) expansion that protects against ectopic lipid deposition during weight gain. Here, we used mice to further define the mechanism for this collagenolytic process. We show that loss of collagen type-1 (CT1) and increased CT1-fragment levels in expanding SAT are associated with proliferation of resident M2-like macrophages that display increased CD206-mediated engagement in collagen endocytosis compared to chow-fed controls. Blockage of CD206 during acute high-fat diet-induced weight gain leads to SAT CT1-fragment accumulation associated with elevated inflammation and fibrosis markers. Moreover, these SAT macrophages' engagement in collagen endocytosis is diminished in obesity associated with elevated levels collagen fragments that are too short to assemble into triple helices. We show that such short fragments provoke M2-macrophage proliferation and fibroinflammatory changes in fibroblasts. In conclusion, our data delineate the importance of a macrophage-collagen fragment axis in physiological SAT expansion. Therapeutic targeting of this process may be a means to prevent pathological adipose tissue remodeling, which in turn may reduce the risk for obesity-related metabolic disorders.

Native collagen VI delays early muscle stem cell differentiation.

Adult muscle stem cells (MuSCs) are critical for muscle homeostasis and regeneration, and their behavior relies on a finely regulated niche made of specific extracellular matrix (ECM) components and soluble factors. Among ECM proteins, collagen VI (Col6) influences the mechanical properties of the niche and, in turn, MuSC self-renewal capabilities. Here, we investigated whether Col6 can exert a direct function as a biochemical signal for regulating the stemness and differentiation of murine MuSCs and myoblasts. Native Col6, but not its pepsin-resistant fragment, counteracts the early differentiation of myogenic cells by reducing the expression of differentiation marker genes and preserving stemness features, with inhibition of the canonical Wnt pathway. Our data indicate that extracellular Col6 acts as a soluble ligand in delaying early myogenic differentiation by regulating intracellular signals involved in adult myogenesis.

Collagen and actin network mediate antiviral immunity against Orsay virus in C. elegans intestinal cells.

C. elegans is a free-living nematode that is widely used as a small animal model for studying fundamental biological processes and disease mechanisms. Since the discovery of the Orsay virus in 2011, C. elegans also holds the promise of dissecting virus-host interaction networks and innate antiviral immunity pathways in an intact animal. Orsay virus primarily targets the worm intestine, causing enlarged intestinal lumen as well as visible changes to infected cells such as liquefaction of cytoplasm and convoluted apical border. Previous studies of Orsay virus identified that C. elegans is able to mount antiviral responses by DRH-1/RIG-I mediated RNA interference and Intracellular Pathogen Response, a uridylyltransferase that destabilizes viral RNAs by 3' end uridylation, and ubiquitin protein modifications and turnover. To comprehensively search for novel antiviral pathways in C. elegans, we performed genome-wide RNAi screens by bacterial feeding using existing bacterial RNAi libraries covering 94% of the entire genome. Out of the 106 potential antiviral gene hits identified, we investigated those in three new pathways: collagens, actin remodelers, and epigenetic regulators. By characterizing Orsay virus infection in RNAi and mutant worms, our results indicate that collagens likely form a physical barrier in intestine cells to inhibit viral infection by preventing Orsay virus entry. Furthermore, evidence suggests that actin remodeling proteins (unc-34, wve-1 and wsp-1) and chromatin remodelers (nurf-1 and isw-1) exert their antiviral activities by regulating the intestinal actin (act-5), a critical component of the terminal web which likely function as another physical barrier to prevent Orsay infection.

Deletion of mouse lysyl oxidase in megakaryocytes affects bone properties in a sex-dependent manner.

ABSTRACT: Lysyl oxidase (LOX) is a facilitator of extracellular matrix cross-linking. Using newly developed megakaryocyte-specific LOX knockout mice, we show that LOX expressed in these scarce bone marrow cells affects bone volume and collagen architecture in a sex-dependent manner.