Kiyoji Nishiwaki

A fibulin-1 homolog interacts with an ADAM protease that controls cell migration in C. elegans.

ADAM (a disintegrin and metalloprotease) family proteins play important roles in animal development and pathogenesis. In C. elegans, a secreted ADAM protein, MIG-17, acts from outside the gonad to control the migration of gonadal distal tip cells (DTCs) that promote gonad morphogenesis. Here, we report that dominant mutations in the fbl-1 gene encoding fibulin-1 spliced isoforms, which are calcium binding extracellular matrix proteins, bypass the requirement for MIG-17 activity in directing DTC migration. Specific amino acid substitutions in the third EGF-like motif of one of the two isoforms, FBL-1C, which corresponds to mammalian fibulin-1C, suppress mig-17 mutations. FBL-1C is synthesized in the gut cells and localizes strongly to the gonadal basement membrane in a MIG-17-dependent manner. Localization of mutant FBL-1C is weaker than that of the wild-type protein and is insensitive to MIG-17 activity, suggesting that it gains a novel function that compensates for its reduced molecular density. We propose that proteolysis by MIG-17 recruits FBL-1C to the gonadal basement membrane, where it is required for the guidance of DTCs, and that mutant FBL-1C acts in a manner that mimics the downstream events of MIG-17-mediated proteolysis.

The conserved oligomeric Golgi complex acts in organ morphogenesis via glycosylation of an ADAM protease in C. elegans

In C. elegans, the gonad acquires two U-shaped arms through directed migration of gonadal distal tip cells (DTCs). A member of the ADAM (a disintegrin and metalloprotease) family, MIG-17, is secreted from muscle cells and localizes to the gonadal basement membrane where it functions in DTC migration. Mutations in cogc-3 and cogc-1 cause misdirected DTC migration similar to that seen in mig-17 mutants. Here, we report that COGC-3 and COGC-1 proteins are homologous to mammalian COG-3/Sec34 and COG-1/ldlBp, respectively, two of the eight components of the conserved oligomeric Golgi (COG) complex required for Golgi function. Knockdown of any of the other six components by RNA interference also produces DTC migration defects, suggesting that the eight components function in a common pathway. COGC-3 and COGC-1 are required for the glycosylation and gonadal localization of MIG-17, but not for secretion of MIG-17 from muscle cells. Furthermore, COGC-3 requires MIG-17 activity for its action in DTC migration. Our findings demonstrate that COG complex-dependent glycosylation of an ADAM protease plays a crucial role in determining organ shape.

MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in Caenorhabditis elegans

MRG15, a mammalian protein related to the mortality factor MORF4, is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the Caenorhabditis elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). By contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ versus soma distinction.

C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV

The gonad arms of C. elegans hermaphrodites acquire invariant shapes by guided migrations of distal tip cells (DTCs), which occur in three phases that differ in the direction and basement membrane substrata used for movement. We found that mig-6 encodes long (MIG-6L) and short (MIG-6S) isoforms of the extracellular matrix protein papilin, each required for distinct aspects of DTC migration. Both MIG-6 isoforms have a predicted N-terminal papilin cassette, lagrin repeats and C-terminal Kunitz-type serine proteinase inhibitory domains. We show that mutations affecting MIG-6L specifically and cell-autonomously decrease the rate of post-embryonic DTC migration, mimicking a post-embryonic collagen IV deficit. We also show that MIG-6S has two separable functions - one in embryogenesis and one in the second phase of DTC migration. Genetic data suggest that MIG-6S functions in the same pathway as the MIG-17/ADAMTS metalloproteinase for guiding phase 2 DTC migrations, and MIG-17 is abnormally localized in mig-6 class-s mutants. Genetic data also suggest that MIG-6S and non-fibrillar network collagen IV play antagonistic roles to ensure normal phase 2 DTC guidance.

A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc‐5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc‐5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc‐5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc‐5 interacts genetically with mig‐2, which encodes Rho GTPase. These results suggest that EXC‐5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.

SRC-1, a non-receptor type of protein tyrosine kinase, controls the direction of cell and growth cone migration in C. elegans

Src family tyrosine kinase (SFK) has been implicated in the regulation of cell adhesion and migration during animal development. We show that SRC-1, an ortholog of SFK, plays an essential role in directing cell migration in Caenorhabditis elegans. The mutation in the src-1 gene results in defective distal tip cell (DTC)-directed gonad morphogenesis in an activity-dependent and DTC cell-autonomous manners. In the src-1 mutants, DTCs fail to turn and continue their centrifugal migration along the ventral muscles. The effect of the src-1 mutation is suppressed by mutations in genes that function in the CED/Rac pathway, suggesting that SRC-1 in DTCs is an upstream regulator of a Rac pathway that controls cytoskeletal remodeling. In the src-1 mutant, the expression of unc-5/netrin receptor is normally regulated, and neither the precocious expression of UNC-5 nor the mutation in the unc-5 gene significantly affects the DTC migration defect. These data suggest that SRC-1 acts in the netrin signaling in DTCs. The src-1 mutant also exhibits cell-autonomous defects in the migration and growth cone path-finding of Q neuroblast descendants AVM and PVM. However, these roles of SRC-1 do not appear to involve the CED/Rac pathway. These findings show that SRC-1 functions in responding to various extracellular guidance cues that direct the cell migration via disparate signaling pathways in different cell types.

Behavioral genetics of caenorhabditis elegans unc-103-encoded erg-like K(+) channel.

The Caenorhabditis elegans unc-103 gene encodes a potassium channel whose sequence is most similar to the ether-a-go-go related gene (erg) type of K+ channels. We find that the n500 and e1597 gain-of-function (gf) mutations in unc-103 cause reduced excitation in most muscles, while loss-of-function (lf) mutations cause mild muscle hyper-excitability. Both gf alleles change the same residue near the cytoplasmic end of S6, consistent with this region regulating channel activation. We also report additional dominant-negative and lf alleles of unc-103 that can antagonize or reduce the function of both gf and wild-type alleles. The unc-103 locus contains 6 promoter regions that express unc-103 in different combinations of body-wall and sex-specific muscles, motor-, inter- and sensory-neurons. Each promoter drives transcripts containing a unique first exon, conferring sequence variability to the N-terminus of the UNC-103 protein, while three splice variants introduce variability into the UNC-103 C-terminus. unc-103(0) hermaphrodites prematurely lay embryos that would normally be retained in the uterus and lay eggs under conditions that inhibit egg-laying behavior. In the egg-laying circuit, unc-103 is expressed in vulval muscles and the HSN neurons from different promoters. Supplying the proper UNC-103 isoform to the vulval muscles is sufficient to restore regulation to egg-laying behavior.

VAB-10 spectraplakin acts in cell and nuclear migration in Caenorhabditis elegans

Cytoskeletal regulation is important in cell migration. The Caenorhabditis elegans gonadal distal tip cells (DTCs) offer a simple model with which to investigate the mechanism of cell migration in organogenesis. Here, we report that one of the spectraplakin isoforms, VAB-10B1, plays an essential role in cell and nuclear migration of DTCs by regulating the actin and microtubule (MT) cytoskeleton. In the vab-10(tk27) mutant, which lacks VAB-10B1, alignment of filamentous (F)-actin and MTs was weakly and severely disorganized, respectively, which resulted in a failure to translocate the DTC nucleus and a premature termination of DTC migration. An MT growing-tip marker, EBP-2-GFP, revealed that polarized outgrowth of MTs towards the nuclei of migrating DTCs was strikingly impaired in tk27 animals. A vab-10 mini-gene encoding only the actin- and MT-binding domains significantly rescued the gonadal defects, suggesting that VAB-10B1 has a role in linking actin and MT filaments. These results suggest that VAB-10B1/spectraplakin regulates the polarized alignment of MTs, possibly by linking F-actin and MTs, which enables normal nuclear translocation and cell migration of DTCs.

MIG-17/ADAMTS controls cell migration by recruiting nidogen to the basement membrane in C. elegans

Mutations in the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family of secreted proteases cause diseases linked to ECM abnormalities. However, the mechanisms by which these enzymes modulate the ECM during development are mostly unexplored. The Caenorhabditis elegans MIG-17/ADAMTS protein is secreted from body wall muscle cells and localizes to the basement membrane (BM) of the developing gonad where it controls directional migration of gonadal leader cells. Here we show that specific amino acid changes in the ECM proteins fibulin-1C (FBL-1C) and type IV collagen (LET-2) result in bypass of the requirement for MIG-17 activity in gonadal leader cell migration in a nidogen (NID-1)-dependent and -independent manner, respectively. The MIG-17, FBL-1C and LET-2 activities are required for proper accumulation of NID-1 at the gonadal BM. However, mutant FBL-1C or LET-2 in the absence of MIG-17 promotes NID-1 localization. Furthermore, overexpression of NID-1 in mig-17 mutants substantially rescues leader cell migration defects. These results suggest that functional interactions among BM molecules are important for MIG-17 control of gonadal leader cell migration. We propose that FBL-1C and LET-2 act downstream of MIG-17-dependent proteolysis to recruit NID-1 and that LET-2 also activates a NID-1-independent pathway, thereby inducing the remodeling of the BM required for directional control of leader cell migration.

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.