N-terminally endogenous mKate2-tagged HIM-4 in Caenorhabditis elegans.

The gene him-4 encodes the C. elegans homolog of hemicentin, an evolutionarily conserved extracellular matrix protein. Despite being an extracellular matrix protein, mutations in him-4 result in pleiotropic defects during development and demonstrate tissue fragility. While previous studies in C. elegans have confirmed the localization of either transgenic HIM-4::GFP or endogenous C-terminally tagged HIM-4::mNeonGreen, only green or green/yellow fluorescent protein-tagged HIM-4 are available to the C. elegans community. Here, I used CRISPR/Cas9 technology to insert the far-red fluorescence protein mKate2 at the him-4 genomic locus, and established worms expressing mKate2::HIM-4. As expected, localization of mkate2::HIM-4 at the rachis was observed at the L4 stage. In contrast to the localization of type IV collagen or nidogen such as major components of the basement membrane, mkate2::HIM-4 was polarized at the anterior part of the pharyngeal basement membrane. A unique polarized localization pattern of pharyngeal basement membrane is maintained throughout the L1-L4 stages.

PIGN prevents protein aggregation in the endoplasmic reticulum independently of its function in the GPI synthesis.

Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases.

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane.

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.

In situ imaging in C. elegans reveals developmental regulation of microtubule dynamics.

Microtubules (MTs) are cytoskeletal polymers that undergo dynamic instability, the stochastic transition between growth and shrinkage phases. MT dynamics are required for diverse cellular processes and, while intrinsic to tubulin, are highly regulated. However, little is known about how MT dynamics facilitate or are regulated by tissue biogenesis and differentiation. We imaged MT dynamics in a smooth muscle-like lineage in intact developing Caenorhabditis elegans. All aspects of MT dynamics change significantly as stem-like precursors exit mitosis and, secondarily, as they differentiate. We found that suppression, but not enhancement, of dynamics perturbs differentiated muscle function in vivo. Distinct ensembles of MT-associated proteins are specifically required for tissue biogenesis versus tissue function. A CLASP family MT stabilizer and the depolymerizing kinesin MCAK are differentially required for MT dynamics in the precursor or differentiated cells, respectively. All of these multidimensional phenotypic comparisons were facilitated by a data display method called the diamond graph.

The novel secreted factor MIG-18 acts with MIG-17/ADAMTS to control cell migration in Caenorhabditis elegans.

The migration of Caenorhabditis elegans gonadal distal tip cells (DTCs) offers an excellent model to study the migration of epithelial tubes in organogenesis. mig-18 mutants cause meandering or wandering migration of DTCs during gonad formation, which is very similar to that observed in animals with mutations in mig-17, which encodes a secreted metalloprotease of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family. MIG-18 is a novel secreted protein that is conserved only among nematode species. The mig-17(null) and mig-18 double mutants exhibited phenotypes similar to those in mig-17(null) single mutants. In addition, the mutations in fbl-1/fibulin-1 and let-2/collagen IV that suppress mig-17 mutations also suppressed the mig-18 mutation, suggesting that mig-18 and mig-17 function in a common genetic pathway. The Venus-MIG-18 fusion protein was secreted from muscle cells and localized to the gonadal basement membrane, a tissue distribution reminiscent of that observed for MIG-17. Overexpression of MIG-18 in mig-17 mutants and vice versa partially rescued the relevant DTC migration defects, suggesting that MIG-18 and MIG-17 act cooperatively rather than sequentially. We propose that MIG-18 may be a cofactor of MIG-17/ADAMTS that functions in the regulation of the gonadal basement membrane to achieve proper direction of DTC migration during gonadogenesis.

Basement membrane sliding and targeted adhesion remodels tissue boundaries during uterine-vulval attachment in Caenorhabditis elegans.

Large gaps in basement membrane occur at sites of cell invasion and tissue remodelling in development and cancer. Though never followed directly in vivo, basement membrane dissolution or reduced synthesis have been postulated to create these gaps. Using landmark photobleaching and optical highlighting of laminin and type IV collagen, we find that a new mechanism, basement membrane sliding, underlies basement membrane gap enlargement during uterine-vulval attachment in Caenorhabditis elegans. Laser ablation and mutant analysis reveal that the invaginating vulval cells promote basement membrane movement. Further, an RNA interference and expression screen identifies the integrin INA-1/PAT-3 and VAB-19, homologue of the tumour suppressor Kank, as regulators of basement membrane opening. Both concentrate within vulval cells at the basement membrane gap boundary and halt expansion of the shifting basement membrane. Basement membrane sliding followed by targeted adhesion represents a new mechanism for creating precise basement membrane breaches that can be used by cells to break down compartment boundaries.

Identification of various types of α2-HS glycoprotein in sera of patients with pancreatic cancer: Possible implication in resistance to protease treatment.

α2-Heremans-Schmid glycoprotein (human fetuin) is one of numerous serum proteins produced in the liver. Recently, the biological functions of fetuin, such as calcification and insulin resistance, have been clarified. However, these effects appear to be indirect, occurring through binding to other molecules. When equal amounts of fetuin in sera were treated with chymotrypsin, resistance to the protease treatment was observed in patients with pancreatic cancer, but not in normal volunteers. To investigate the molecular mechanism behind this resistance, gel-filtration chromatography was performed. The results revealed that high molecular types of fetuin showed a resistance to protease treatment. When fetuin was purified from sera of patients with pancreatic cancer and normal volunteers, certain types of proteins, including haptoglobin (which binds to fetuin derived from pancreatic cancer patients), were identified using mass spectrometry. Furthermore, the oligosaccharide structures of fetuin analyzed with lectin microarray differed between pancreatic cancer patients and normal volunteers. This macro/micro heterogeneity of fetuin might contribute to pancreatic cancer resistance to chymotrypsin treatment.

Integrin acts upstream of netrin signaling to regulate formation of the anchor cell's invasive membrane in C. elegans.

Integrin expression and activity have been strongly correlated with developmental and pathological processes involving cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of anchor cell (AC) invasion in C. elegans, we have recently shown that netrin signaling orients a specialized invasive cell membrane domain toward the basement membrane. Here, we demonstrate that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part by targeting the netrin receptor UNC-40 (DCC) to the AC's plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2, and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain.

Stage-specific activation of MIG-17/ADAMTS controls cell migration in Caenorhabditis elegans.

The activation of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family proteases depends on removal of the prodomain. Although several studies suggest that ADAMTS activities play roles in development, homeostasis and disease, it remains unclear when and where the enzymes are activated in vivo. MIG-17, a Caenorhabditis elegans glycoprotein belonging to the ADAMTS family, is secreted from the body wall muscle cells and localizes to the gonadal basement membrane to control the migration of gonadal distal tip cells. Here, we developed a monoclonal antibody that recognizes the N-terminal neo-epitope of the activated MIG-17. In western blotting, the antibody specifically detected the activated form, the signal for which dramatically increased during the third and fourth larval stages, when MIG-17 is required to direct distal tip cell migration. In in situ staining, the monoclonal antibody recognized the activated form in the basement membrane, whereas it failed to detect a processing-resistant mutant form localized to the basement membrane. MIG-17 was activated in the basement membranes of the muscle, intestine and gonad in the third larval stage, and downregulated in nongonadal basement membranes in young adults and in gonadal basement membranes in older adults. Thus, the activation of MIG-17 is regulated in a spatiotemporal manner during C. elegans development. This is the first report demonstrating the regulated activation of an ADAMTS protein in vivo. Our results suggest that monoclonal antibodies against neo-epitopes have potential as powerful tools for detecting activation of ADAMTSs during development and in disease pathogenesis.

Prodomain-dependent tissue targeting of an ADAMTS protease controls cell migration in Caenorhabditis elegans.

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted proteins play important roles in animal development and pathogenesis. However, the lack of in vivo models has hampered elucidation of the mechanisms by which these enzymes are recruited to specific target tissues and the timing of their activation during development. Using transgenic worms and primary cell cultures, here we show that MIG-17, an ADAMTS family protein required for gonadal leader cell migration in Caenorhabditis elegans, is recruited to the gonadal basement membrane in a prodomain-dependent manner. The activation of MIG-17 to control leader cell migration requires prodomain removal, which is suggested to occur autocatalytically in vitro. Although the prodomains of ADAMTS proteases have been implicated in maintaining enzymatic latency, polypeptide folding and secretion, our findings demonstrate that the prodomain has an unexpected function in tissue-specific targeting of MIG-17; this prodomain targeting function may be shared by other ADAMTSs including those in vertebrates.

A secreted type of beta1,6 N-acetylglucosaminyltransferase V (GnT-V), a novel angiogenesis inducer, is regulated by gamma-secretase.

Glycosyltransferases are present in the Golgi apparatus in a membrane-bound form and are released from cells after cleavage by certain proteases. Beta1,6-N-acetylglucosaminyltransferase V (GnT-V), which is cleaved and secreted from the cells, is involved in the biosynthesis of beta1-6GlcNAc branching on N-glycans and has been implicated in tumor progression and metastasis. We recently reported that a secreted type of GnT-V (soluble GnT-V) itself could promote angiogenesis, which is completely different from its original function as a glycosyltransferase, and this might play a role in tumor invasion. In this study, to explore the molecular basis for this functional glycosyltransferase secretion, its cleavage site was examined and the protease(s) involved in that cleavage were identified. The NH2-terminal protein sequence of purified soluble GnT-V (approximately 100 kDa) from GnT-V-overexpressed cells revealed that its terminus started at His31, located at the boundary position between the transmembrane and stem regions. This secretion was not inhibited by a single amino acid mutation at the cleavage site (Leu29, Leu30 to Asp, His31 to Ala), but specifically inhibited by addition of DFK-167, a gamma-secretase inhibitor, suggesting that gamma-secretase is a plausible protease for secretion processing. In addition, transfection of the gene of familial Alzheimer's disease (FAD)[corrected]-linked presenilin-1, a component of gamma-secretase, increased the secretion rate of endogenous GnT-V; the secretion of soluble GnT-V (approximately 100 kDa) was completely inhibited in presenilin-1/2 double-deficient cells, which have no gamma-secretase activity. Collectively, these results demonstrate that Golgi-resident GnT-V is cleaved at the transmembrane region by gamma-secretase, and this might control tumor angiogenesis through a novel pathway.

Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.

The core fucosylation (alpha1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated alpha1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8(-/-) mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8(-/-) mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-beta1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8(-/-) mice have a marked dysregulation of TGF-beta1 receptor activation and signaling, as assessed by TGF-beta1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-beta1 receptor defects found in Fut8(-/-) cells can be rescued by reintroducing Fut8 into Fut8(-/-) cells. Furthermore, exogenous TGF-beta1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8(-/-) lung. We propose that the lack of core fucosylation of TGF-beta1 receptors is crucial for a developmental and progressive/destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.

Attachment of human colon cancer cells to vascular endothelium is enhanced by N-acetylglucosaminyltransferase V.

Expression of N-acetylglucosaminyltransferase V (GnT-V) in colon cancer has been shown to be related to hematogenous metastasis and poor prognosis. To investigate the mechanism by which cancer cells expressing GnT-V metastasize to distant organs, we established GnT-V-overexpressing DLD-1 and WiDr cells (human colon cancer cell lines) by transfecting them with a GnT-V expression vector. Attachment to endothelial cells expressing E-selectin was studied, and expression of the E-selectin ligand, sialyl Lewis x, in colon cancer cells was investigated. Both of the cell lines showed reduced adhesion to fibronectin as compared with mock transfectants. In contrast, attachment to human umbilical vein endothelial cells expressing E-selectin was significantly enhanced by GnT-V expression (p < 0.01). Sialyl Lewis x is a ligand for E-selectin and a marker for poor prognosis of colon cancer. Its synthesis in cells has been shown to involve GnT-V. We demonstrated that expression of sialyl Lewis x in colon cancer cells was induced by GnT-V expression. These results suggest that GnT-V induces sialyl Lewis x expression and leads colon cancer cells to metastasize by enhancing their ability to attach to vascular endothelium in distant organs, such as liver or lung. Inhibition of GnT-V activity may prevent metastasis in colon cancer patients with high sialyl Lewis x expression.

Addition of beta1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin.

beta1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis-related glycoproteins that contain beta1-6 GlcNAc branching have appeared, the precise function of beta1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of beta1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (beta1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes beta1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains beta1-6 GlcNAc branching. The beta1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of beta1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the beta1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between beta1-6 GlcNAc branching and a biological function at the protein level.

Involvement of oligosaccharide changes in alpha5beta1 integrin in a cisplatin-resistant human squamous cell carcinoma cell line.

Multiple mechanisms are involved in the resistance of cancer cells to cisplatin, including the expression of multidrug resistance-associated protein (MRP) and enhanced DNA repair. Here, we report findings to show that oligosaccharide changes in alpha5beta1 integrin are associated with cisplatin resistance in a head and neck squamous cell carcinoma cell line, HSC-2. Cisplatin-resistant HSC-2 (HSC-2/CR) cells were established by stepwise treatment with various concentrations of cisplatin. The oligosaccharides containing beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching, detected by leukoagglutinating phytohemagglutinin (L(4)-PHA) lectin blot, were found to be dramatically decreased in alpha5beta1 integrin immunoprecipitated from HSC-2/CR cells. To better understand the mechanisms underlying cisplatin resistance and oligosaccharide alteration, we analyzed the downstream signaling of alpha5beta1 integrin, one of the target glycoproteins of beta1-6GlcNAc transferase [UDP-GlcNAc:alpha-D-mannoside beta1, 6-N-acetylglucosaminyltransferase (GnT-V)]. Cell adhesion to fibronectin and phosphorylation of focal adhesion kinase (FAK), which are associated with alpha5beta1 integrin and involved in a cell survival signaling, were found to be increased in the cisplatin-resistant cells. Enhancement of the inhibition of cell adhesion and FAK phosphorylation also support the above data in GnT-V transfectants of HSC-2 cells. Interestingly, the differences in sensitivity to cisplatin and FAK phosphorylation between cisplatin-sensitive and -resistant cells were completely abolished by treatment with a neutral antibody of alpha5beta1 integrin. These results suggest that modification of oligosaccharides of alpha5beta1 integrin represents one of the possible mechanisms of drug resistance in head and neck cancer cells.