Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans

Mutations in the Caenorhabditis elegans unc-84 gene cause defects in nuclear migration and anchoring. We show that endogenous UNC-84 protein colocalizes with Ce-lamin at the nuclear envelope and that the envelope localization of UNC-84 requires Ce-lamin. We also show that during mitosis, UNC-84 remains at the nuclear periphery until late anaphase, similar to known inner nuclear membrane proteins. UNC-84 protein is first detected at the 26-cell stage and thereafter is present in most cells during development and in adults. UNC-84 is properly expressed in unc-83 and anc-1 lines, which have phenotypes similar to unc-84, suggesting that neither the expression nor nuclear envelope localization of UNC-84 depends on UNC-83 or ANC-1 proteins. The envelope localization of Ce-lamin, Ce-emerin, Ce-MAN1, and nucleoporins are unaffected by the loss of UNC-84. UNC-84 is not required for centrosome attachment to the nucleus because centrosomes are localized normally in unc-84 hyp7 cells despite a nuclear migration defect. Models for UNC-84 localization are discussed.     

Multiple signaling mechanisms of the UNC-6/netrin receptors UNC-5 and UNC-40/DCC in vivo.

Cell and growth cone migrations along the dorsoventral axis of Caenorhabditis elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, we have examined genetic interactions between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. We report that cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. We show that hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, we observed that unc-5 hypomorphs exhibit dominant negative effects. We propose that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes.     

SDQR migrations in Caenorhabditis elegans are controlled by multiple guidance cues and changing responses to netrin UNC-6.

The netrin guidance cue, UNC-6, and the netrin receptors, UNC-5 and UNC-40, guide SDQR cell and axon migrations in C. elegans. In wild-type larvae, SDQR migrations are away from ventral UNC-6-expressing cells, suggesting that UNC-6 repels SDQR. In unc-6 null larvae, SDQR migrations are towards the ventral midline, indicating a response to other guidance cues that directs the migrations ventrally. Although ectopic UNC-6 expression dorsal to the SDQR cell body would be predicted to cause ventral SDQR migrations in unc-6 null larvae, in fact, more migrations are directed dorsally, suggesting that SDQR is not always repelled from the dorsal source of UNC-6. UNC-5 is required for dorsal SDQR migrations, but not for the ventral migrations in unc-6 null larvae. UNC-40 appears to moderate both the response to UNC-6 and to the other cues. Our results show that SDQR responds to multiple guidance cues and they suggest that, besides UNC-6, other factors influence whether an UNC-6 responsive cell migrates toward or away from an UNC-6 source in vivo. We propose that multiple signals elicited by the guidance cues are integrated and interpreted by SDQR and that the response to UNC-6 can change depending on the combination of cues encountered during migration. These responses determine the final dorsoventral position of the SDQR cell and axon.     

UNC-52/perlecan affects gonadal leader cell migrations in C. elegans hermaphrodites through alterations in growth factor signaling

The unc-52 gene of Claenorhabditis elegans encodes a homologue of the basement membrane heparan sulfate proteoglycan perlecan. Viable alleles reduce the abundance of UNC-52 in late larval stages and increase the frequency of distal tip cell (DTC) migration defects caused by mutations disrupting the UNC-6/netrin guidance system. These unc-52 alleles do not cause circumferential DTC migration defects in an otherwise wild-type genetic background. The effects of unc-52 mutations on DTC migrations are distinct from effects on myofilament organization and can be partially suppressed by mutations in several genes encoding growth factor-like molecules, including EGL-17/FGF, UNC-129/TGF-beta, DBL-1/TGF-beta, and EGL-20/WNT. We propose that UNC-52 serves dual roles in C. elegans larval development in the maintenance of muscle structure and the regulation of growth factor-like signaling pathways.     

UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.

The unc-5 gene is required for guiding pioneering axons and migrating cells along the body wall in C. elegans. In mutants, dorsal migrations are disrupted, but ventral and longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by transposon insertions. Based on genomic and cDNA sequencing, the gene encodes UNC-5, a transmembrane protein of 919 aa. The predicted extracellular N-terminus comprises two immunoglobulin and two thrombospondin type 1 domains. Except for an SH3-like motif, the large intracellular C-terminus is novel. Mosaic analysis shows that unc-5 acts in migrating cells and pioneering neurons. We propose that UNC-5 is a transmembrane receptor expressed on the surface of motile cells and growth cones to guide dorsal movements.     

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.     

Nuclear envelope localization of human UNC84A does not require nuclear lamins

The SUN proteins are a conserved family of proteins in eukaryotes. Human UNC84A (Sun1) is a homolog of Caenorhabditis elegans UNC-84, a protein involved in nuclear anchorage and migration. We have analyzed targeting of UNC84A to the nuclear envelope (NE) and show that the N-terminal 300 amino acids are crucial for efficient NE localization of UNC84A whereas the conserved C-terminal SUN domain is not required. Furthermore, we demonstrate by combining RNA interference with immunofluorescence and fluorescence recovery after photobleaching analysis that localization and anchoring of UNC84A is not dependent on the lamin proteins, in contrast to what had been observed for C. elegans UNC-84.     

UNC-83 IS a KASH protein required for nuclear migration and is recruited to the outer nuclear membrane by a physical interaction with the SUN protein UNC-84

UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even if they localized normally. At least two separable portions of the C-terminal half of UNC-84 were found to interact with the UNC-83 KASH domain in a membrane-bound, split-ubiquitin yeast two-hybrid system. However, the SUN domain was essential for UNC-84 function and UNC-83 localization in vivo. These data support the model that KASH and SUN proteins bridge the nuclear envelope, connecting the nuclear lamina to cytoskeletal components. This mechanism seems conserved across eukaryotes and is the first proposed mechanism to target proteins specifically to the outer nuclear membrane.     

SRC-1 mediates UNC-5 signaling in Caenorhabditis elegans

The secreted molecule unc-6/netrin is important for guiding axon projections and cell migrations. unc-5 and unc-40/DCC are identified as receptors for unc-6/netrin. The downstream factors of unc-6 receptors are beginning to be elucidated, and some key factors have been identified in various organisms. Here, we showed that SRC-1 interacts with the cytosolic domain of UNC-5 through its SH2 domain. This interaction also requires the intact kinase activity of SRC-1. Downregulation of src-1 by RNA interference decreases the biological processes initiated by the UNC-5 protein and decreases UNC-5 tyrosine phosphorylation. We also generated a chimeric protein consisting of the extracellular domain and transmembrane domain of UNC-5 and an intracellular domain of SRC-1. This fusion protein is able to partially rescue mutant phenotypes caused by unc-5 but not unc-6, unc-40, and unc-34. Our results support a model in which SRC-1 is required for UNC-5-induced axon repulsion and gonad migration signaling pathways and in which localizing SRC-1 activity to UNC-5 is crucial for proper signal transduction in response to unc-6/netrin.     

UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans.

The unc-6 gene is required for the guidance of pioneer axons and migrating cells along the body wall in C. elegans. In mutants, dorsal and ventral migrations are disrupted, but longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by two independent transposon insertions. Based on genomic and cDNA sequencing, the gene encodes a novel laminin-related protein, UNC-6 (591 amino acids). The N-terminus is homologous to the N-termini (i.e., domains V1, V-1, V-2, and V-3) of laminin subunits, while the C-terminus is a unique domain. We propose that UNC-6 is a component of an extracellular matrix cue that guides dorsoventral migrations on the epidermis.     

Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei

Kinesin-1 and dynein are recruited to the nuclear envelope by the Caenorhabditis elegans klarsicht/ANC-1/Syne homology (KASH) protein UNC-83 to move nuclei. The mechanisms of how these motors are coordinated to mediate nuclear migration are unknown. Time-lapse differential interference contrast and fluorescence imaging of embryonic hypodermal nuclear migration events were used to characterize the kinetics of nuclear migration and determine microtubule dynamics and polarity. Wild-type nuclei display bidirectional movements during migration and are also able to roll past cytoplasmic granules. unc-83, unc-84, and kinesin-1 mutants have severe nuclear migration defects. Without dynein, nuclear migration initiates normally but lacks bidirectional movement and shows defects in nuclear rolling, implicating dynein in resolution of cytoplasmic roadblocks. Microtubules are highly dynamic during nuclear migration. EB1::green fluorescence protein imaging demonstrates that microtubules are polarized in the direction of nuclear migration. This organization of microtubules fits with our model that kinesin-1 moves nuclei forward and dynein functions to move nuclei backward for short stretches to bypass cellular roadblocks.     

Multiple mechanisms actively target the SUN protein UNC-84 to the inner nuclear membrane

Approximately 100 proteins are targeted to the inner nuclear membrane (INM), where they regulate chromatin and nuclear dynamics. The mechanisms underlying trafficking to the INM are poorly understood. The Caenorhabditis elegans SUN protein UNC-84 is an excellent model to investigate such mechanisms. UNC-84 recruits KASH proteins to the outer nuclear membrane to bridge the nuclear envelope (NE), mediating nuclear positioning. UNC-84 has four targeting sequences: two classical nuclear localization signals, an INM sorting motif, and a signal conserved in mammalian Sun1, the SUN--nuclear envelope localization signal. Mutations in some signals disrupt the timing of UNC-84 nuclear envelope localization, showing that diffusion is not sufficient to move all UNC-84 to the NE. Thus targeting UNC-84 requires an initial step that actively transports UNC-84 from the peripheral endoplasmic reticulum to the NE. Only when all four signals are simultaneously disrupted does UNC-84 completely fail to localize and to function in nuclear migration, meaning that at least three signals function, in part, redundantly to ensure proper targeting of UNC-84. Multiple mechanisms might also be used to target other proteins to the INM, thereby ensuring their proper and timely localization for essential cellular and developmental functions.     

UNC-5 function requires phosphorylation of cytoplasmic tyrosine 482, but its UNC-40-independent functions also require a region between the ZU-5 and death domains

Members of the UNC-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different UNC-5 domains, we sequenced mutations in seven severe and three weak alleles of unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of UNC-5, wild-type and mutant unc-5::HA transgenes were tested for their ability to rescue the unc-5(e53) null mutant. Our data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, UNC-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, UNC-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to UNC-5 function in vivo. Our data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of UNC-5.     

Role of netrin UNC-6 in patterning the longitudinal nerves of Caenorhabditis elegans

The nervous system of Caenorhabditis elegans comprises circumferential and longitudinal axon tracts. Netrin UNC-6 is required for the guidance of circumferential axon migrations and is expressed by ventral neuroglia and neurons in temporally and spatially regulated patterns. Migrating axons mediate the UNC-6 signal through the UNC-5 and UNC-40 receptors. It is thought that UNC-6 is secreted and becomes associated with basement membranes and cell surfaces to form gradients that direct circumferentially migrating axons toward or away from the ventral UNC-6 sources. Little is known about the effects of UNC-6 on longitudinally migrating axons. In unc-6, unc-5, and unc-40 null mutants, some longitudinal nerves are dorsally or ventrally misdirected. Furthermore, the organization of axons are disrupted within nerves. We show that cells ectopically expressing UNC-6 can redirect the migrations of some neighboring longitudinal axons, suggesting that the gradients postulated to direct circumferential migration also help specify the dorsoventral positions of these longitudinal nerves. We also manipulated the temporal and spatial expression pattern of UNC-6 by two different means. First, we removed the PVT midline neuron which expresses UNC-6 for a short time during axon outgrowths. Second, we expressed UNC-6 uniformly in the nervous system throughout development. The results suggest that changing UNC-6 expression patterns modify the distribution of the cue by providing new localized sources. This new guidance information is critical for organizing the axons of longitudinal nerves.     

A role for septins in cellular and axonal migration in C. elegans

Caenorhabditis elegans has two genes, unc-59 and unc-61, encoding septin-family GTPases. Mutations in the septin genes cause defects in locomotory behavior that have been previously attributed to cytokinesis failures in postembryonic neuroblasts. We find that mutations in either septin gene frequently cause uncoordination in newly hatched larvae in the absence of cytokinesis failures. The septins exhibit developmentally regulated expression, including expression in various neurons at times when processes are extending and synapses are forming. Motor neurons in the mutant larvae display defects in multiple aspects of axonal migration and guidance that are likely to be responsible for the locomotory behavior defects. The septins are also expressed in migrating distal tip cells, which are leaders for gonad arm extension. Septin mutants affect morphology of the distal tip cells, as well as their migration and guidance during gonadogenesis. These results suggest that septins may be generally required for developmental migrations and pathfinding.     

The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and,in parallel,Rac and UNC-115/AbLIM

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, we generated a gain-of-function UNC-40 molecule, MYR::UNC-40. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. Our results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton.     

The conserved kinase UNC-51 acts with VAB-8 and UNC-14 to regulate axon outgrowth in C. elegans

Directional cues guide growth cones. While molecules like UNC-6/netrin direct migrations along the dorsoventral axis of many organisms, it is unclear how anteroposterior guidance is achieved. We describe a physical interaction between VAB-8, a protein both necessary and sufficient for posteriorly directed migrations in C. elegans, and UNC-51, a conserved serine/threonine kinase that functions generally in axon outgrowth. We show that both proteins function in the CAN neurons to direct their axons posteriorly. Expression in the CANs of peptides predicted to interfere with interactions between UNC-51 and both VAB-8 and UNC-14, a second protein that interacts physically with UNC-51, disrupts CAN axon outgrowth. We provide genetic evidence that VAB-8 functions in an UNC-51 pathway for posteriorly directed CAN axon guidance and show that VAB-8 and UNC-14 can be targets of UNC-51 kinase activity. Taken together, our results suggest that VAB-8 and UNC-14 are substrates that mediate the function of UNC-51 in axon outgrowth.