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

[This corrects the article DOI: 10.1371/journal.pone.0240571.].

Repulsive guidance molecule acts in axon branching in Caenorhabditis elegans.

Repulsive guidance molecules (RGMs) are evolutionarily conserved proteins implicated in repulsive axon guidance. Here we report the function of the Caenorhabditis elegans ortholog DRAG-1 in axon branching. The axons of hermaphrodite-specific neurons (HSNs) extend dorsal branches at the region abutting the vulval muscles. The drag-1 mutants exhibited defects in HSN axon branching in addition to a small body size phenotype. DRAG-1 expression in the hypodermal cells was required for the branching of the axons. Although DRAG-1 is normally expressed in the ventral hypodermis excepting the vulval region, its ectopic expression in vulval precursor cells was sufficient to induce the branching. The C-terminal glycosylphosphatidylinositol anchor of DRAG-1 was important for its function, suggesting that DRAG-1 should be anchored to the cell surface. Genetic analyses suggested that the membrane receptor UNC-40 acts in the same pathway with DRAG-1 in HSN branching. We propose that DRAG-1 expressed in the ventral hypodermis signals via the UNC-40 receptor expressed in HSNs to elicit branching activity of HSN axons.

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.

Control of the basement membrane and cell migration by ADAMTS proteinases: Lessons from C. elegans genetics.

The members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of secreted proteins, MIG-17 and GON-1, play essential roles in Caenorhabditis elegans gonadogenesis. The genetic and molecular analyses of these proteinases uncovered novel molecular interactions regulating the basement membrane (BM) during the migration of the gonadal leader cells. MIG-17, which is localized to the gonadal BM recruits or activates fibulin-1 and type IV collagen, which then recruits nidogen, thereby inducing the remodeling of the BM that is required for directional control of leader cell migration. GON-1 acts antagonistically with fibulin-1 to regulate the levels of type IV collagen accumulation in the gonadal BM, which facilitates active migration of the leader cells. The cooperative action of MIG-17 and GON-1 represents an excellent model for understanding the mechanisms of organogenesis mediated by ADAMTS proteinases.

The BED finger domain protein MIG-39 halts migration of distal tip cells in Caenorhabditis elegans.

Organs are often formed by the extension and branching of epithelial tubes. An appropriate termination of epithelial tube extension is important for generating organs of the proper size and morphology. However, the mechanism by which epithelial tubes terminate their extension is mostly unknown. Here we show that the BED-finger domain protein MIG-39 acts to stop epithelial tube extension in Caenorhabditis elegans. The gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern during larval development and stop migrating at the young adult stage, generating a gonad with anterior and posterior U-shaped arms. In mig-39 mutants, however, DTCs overshot their normal stopping position. MIG-39 promoted the deceleration of DTCs, leading to the proper timing and positioning of the cessation of DTC migration. Among three Rac GTPase genes, mutations in ced-10 and rac-2 enhanced the overshoot of anterior DTCs, while they suppressed that of posterior DTCs of mig-39 mutants. On the other hand, the mutation in mig-2 suppressed both the anterior and posterior DTC defects of mig-39. Genetic analyses suggested that MIG-39 acts in parallel with Rac GTPases in stopping DTC migration. We propose a model in which the anterior and posterior DTCs respond in an opposite manner to the levels of Rac activities in the cessation of DTC migration.

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.

Nuclear positioning in the gonadal distal tip cells of C. elegans.

Morphogenesis of the hermaphrodite gonad of Caenorhabditis elegans is directed by the U-shaped migration of the gonadal leader cells, which are called distal tip cells (DTCs). The nuclei of migrating DTCs are always positioned at the leading edge of the cells, even as these cells turn dorsally to contact the hypodermis and intestine. When the DTCs turn dorsally, VAB-10B1/spectraplakin acts in nuclear translocation by regulating the polarized growth of microtubules. The function of spectraplakin in nuclear positioning may be evolutionarily conserved. Here we discuss the possible reason for leading-edge positioning of the DTC nucleus.

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.

Recruitment of CG-NAP to the Golgi apparatus through interaction with dynein-dynactin complex.

The structural organization and position of the Golgi apparatus are highly regulated by microtubule cytoskeleton and microtubule motor proteins. The mechanisms linking these proteins to the Golgi apparatus remain elusive. Here, we found that centrosome and Golgi-localized PKN associated protein (CG-NAP) was localized to the Golgi apparatus in a microtubule-dependent manner. Microtubule-binding experiments revealed that CG-NAP possessed two microtubule-binding domains. We also found that CG-NAP was well co-localized with cytoplasmic dynein subunits during recovery from the on-ice treatment of cells that induced dissociation of CG-NAP from the Golgi. Similar co-localization was observed during recovery from the acetate treatment, which has been reported to inhibit the dynein-mediated transport. CG-NAP was co-immunoprecipitated with a dynactin subunit p150(Glued). Expressing the p150(Glued)-binding region of CG-NAP fused with mitochondria-targeting sequence induced recruitment of mitochondria to the pericentriolar area, suggesting that this region interacts with functional cytoplasmic dynein in vivo. Moreover, over-expression of this region caused fragmentation of the Golgi similar to that of dynamitin. These results suggest that CG-NAP is recruited to the minus ends of microtubules by interacting with cytoplasmic dynein, thereby localizes to the Golgi apparatus in a microtubule-dependent manner and possibly involved in the formation of the Golgi near the centrosomes.

Centrosome-targeting region of CG-NAP causes centrosome amplification by recruiting cyclin E-cdk2 complex.

Centrosome duplication occurs once per cell cycle and is thought to be triggered by cyclin E-cdk2. However, it is largely unknown how the duplication is regulated. Here, we found that the expression of the centrosome-targeting region of CG-NAP (centrosome and Golgi-localized PKN-associated protein), which we designate as CG-NAP/D, increased the number of centrosomes in Chinese hamster ovary (CHO)-K1 cells. The amplified centrosomes co-localized with centrosome markers gamma-tubulin, centrin-2 and kendrin as well as endogenous CG-NAP. When CG-NAP/D was dislocated from centrosomes by deleting the centrosome-targeting domain or by fusing with a membrane-targeting sequence, centrosome amplification was suppressed. CG-NAP/D interacted with exogenously expressed cyclin E, which co-localized at centrosomes. The immunoprecipitates of CG-NAP/D exhibited histone H1 kinase activity, suggesting the co-immunoprecipitation of active cyclin-cdk complexes. Furthermore, centrosome fractions prepared from cells expressing CG-NAP/D contained increased amount of cdk2 compared with those from control cells. Centrosome amplification by CG-NAP/D was suppressed by co-expression of a mutant cyclin E unable to interact with cdk2. These results suggest that CG-NAP/D causes centrosome amplification by anchoring excess amount of cyclin E-cdk2 to centrosomes and, possibly, CG-NAP participates in centrosome duplication by recruiting cyclin E-cdk2 to centrosomes in normal cell cycle.

Regulation of a mitogen-activated protein kinase kinase kinase, MLTK by PKN.

PKNalpha is a fatty acid- and Rho-activated serine/threonine protein kinase having a catalytic domain homologous to members of the protein kinase C family. Recently it was reported that PKNalpha is involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway. To date, however, how PKNalpha regulates the p38gamma MAPK signaling pathway is unclear. Here we demonstrate that PKNalpha efficiently phosphorylates MLTKalpha (MLK-like mitogen-activated protein triple kinase), which was recently identified as a MAPK kinase kinase (MAPKKK) for the p38 MAPK cascade. Phosphorylation of MLTKalpha by PKNalpha enhances its kinase activity in vitro. Expression of the kinase-negative mutant of PKNalpha inhibited the mobility shift of MLTKalpha caused by osmotic shock in SDS-PAGE. Furthermore, PKNalpha associates with each member of the p38gamma MAPK signaling pathway (p38gamma, MKK6, and MLTKalpha). These results suggest that PKNalpha functions as not only an upstream activator of MLTKalpha but also a putative scaffold protein for the p38gamma MAPK signaling pathway.