A stomatin and a degenerin interact in lipid rafts of the nervous system of Caenorhabditis elegans

In Caenorhabditis elegans, the gene unc-1 controls anesthetic sensitivity and normal locomotion. The protein UNC-1 is a close homolog of the mammalian protein stomatin and is expressed primarily in the nervous system. Genetic studies in C. elegans have shown that the UNC-1 protein interacts with a sodium channel subunit, UNC-8. In humans, absence of stomatin is associated with abnormal sodium and potassium levels in red blood cells. Stomatin also has been postulated to participate in the formation of lipid rafts, which are membrane microdomains associated with protein complexes, cholesterol, and sphingolipids. In this study, we isolated a low-density, detergent-resistant fraction from cell membranes of C. elegans. This fraction contains cholesterol, sphingolipids, and protein consistent with their identification as lipid rafts. We then probed Western blots of protein from the rafts and found that the UNC-1 protein is almost totally restricted to this fraction. The UNC-8 protein is also found in rafts and coimmunoprecipitates UNC-1. A second stomatin-like protein, UNC-24, also affects anesthetic sensitivity, is found in lipid rafts, and regulates UNC-1 distribution. Mutations in the unc-24 gene alter the distribution of UNC-1 in lipid rafts. Each of these mutations alters anesthetic sensitivity in C. elegans. Because lipid rafts contain many of the putative targets of volatile anesthetics, they may represent a novel class of targets for volatile anesthetics.     

A stomatin and a degenerin interact to control anesthetic sensitivity in Caenorhabditis elegans

The mechanism of action of volatile anesthetics is unknown. In Caenorhabditis elegans, mutations in the gene unc-1 alter anesthetic sensitivity. The protein UNC-1 is a close homologue of the mammalian protein stomatin. Mammalian stomatin is thought to interact with an as-yet-unknown ion channel to control sodium flux. Using both reporter constructs and translational fusion constructs for UNC-1 and green fluorescent protein (GFP), we have shown that UNC-1 is expressed primarily within the nervous system. The expression pattern of UNC-1 is similar to that of UNC-8, a sodium channel homologue. We examined the interaction of multiple alleles of unc-1 and unc-8 with each other and with other genes affecting anesthetic sensitivity. The data indicate that the protein products of these genes interact, and that an UNC-1/UNC-8 complex is a possible anesthetic target. We propose that membrane-associated protein complexes may represent a general target for volatile anesthetics.     

The Caenorhabditis elegans Behavioral Gene unc-24 Encodes a Novel Bipartite Protein Similar to Both Erythrocyte Band 7.2 (Stomatin) and Nonspecific Lipid Transfer Protein

Abstract: We report here the positional cloning and molecular characterization of the unc-24 gene of Caenorhabditis elegans . This gene is required for normal locomotion and interacts with genes that affect the worm's response to volatile anesthetics. The predicted gene product contains a domain similar to part of two ion channel regulators (the erythrocyte integral membrane protein stomatin and the C. elegans neuronal protein MEC-2) juxtaposed to a domain similar to nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2). Sequence analysis suggests that the nsLTP-like domain of UNC-24 provides lipid carrier function and is tethered to the plasma membrane by the stomatin-like domain, which may be regulatory. We postulate that UNC-24 may be involved in lipid transfer between closely apposed membranes.     

Sulfated signal from ASJ sensory neurons modulates stomatin-dependent coordination in Caenorhabditis elegans

The neuronal stomatin-like proteins UNC-1 and UNC-24 play important roles in the nervous system of Caenorhabditis elegans. These neuronal stomatin-like proteins are putative chaperone proteins that can modify volatile anesthetic sensitivity and disrupt coordinated locomotion. A suppressor of unc-1 and unc-24, named ssu-1(fc73) (for suppressor of stomatin uncoordination), suppresses three phenotypes of neuronal stomatin-like protein deficiency as follows: volatile anesthetic sensitivity, uncoordinated locomotion, and a constitutive alternative developmental phenotype known as dauer. Here we provide the first phenotypic characterization of ssu-1, predicted to be the only C. elegans cytosolic alcohol sulfotransferase, a family of enzymes that catalyze a sulfate linkage with the alcohol group of small molecules for the purposes of detoxification or modification of signaling. In vitro enzyme analysis of bacterially expressed SSU-1 demonstrates sulfotransferase activity and thus confirms the function predicted by protein sequence similarities. Whereas unc-1 is expressed in the majority of neurons of C. elegans, expression of SSU-1 protein in only the two ASJ amphid interneurons is sufficient to restore the wild type phenotype. This work demonstrates that SSU-1 is a functional sulfotransferase that likely modifies endocrine signaling in C. elegans. The expression of SSU-1 in the ASJ neurons refines the understanding of the function of these cells and supports their classification as endocrine tissue. The relationship of unc-1, unc-24, and ssu-1 is the first association of neuronal stomatin-like proteins sharing regulatory roles with a sulfotransferase enzyme.     

Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues

Human stomatin (band 7.2b) is a 31-kDa erythrocyte membrane protein of unknown function but implicated in the control of ion channel permeability, mechanoreception, and lipid domain organization. Although absent in erythrocytes from patients with hereditary stomatocytosis, stomatin is not linked to this disorder. A second stomatin homologue, termed SLP-1, has been identified in nonerythroid tissues, and other stomatin related proteins are found in Drosophila, Caenorhabditis elegans, and plants. We now report the cloning and characterization of a new and unusual stomatin homologue, human SLP-2 (stomatin-like protein 2). SLP-2 is encoded by an approximately 1.5-kilobase mRNA (GenBank(TM) accession no. AF190167). The gene for human SLP-2, HUSLP2, is present on chromosome 9p13. Its derived amino acid sequence predicts a 38,537-kDa protein that is overall approximately 20% similar to human stomatin. Northern and Western blots for SLP-1 and SLP-2 reveal a wide but incompletely overlapping tissue distribution. Unlike SLP-1, SLP-2 is also present in mature human erythrocytes ( approximately 4,000 +/- 5,600 (+/- 2 S.D.) copies/cell). SLP-2 lacks a characteristic NH(2)-terminal hydrophobic domain found in other stomatin homologues and (unlike stomatin) is fully extractable from erythrocyte membranes by NaOH, pH 11. SLP-2 partitions into both Triton X-100-soluble and -insoluble pools in erythrocyte ghost membranes or when expressed in cultured COS cells and migrates anomalously on SDS-polyacrylamide gel electrophoresis analysis with apparent mobilities of approximately 45,500, 44,600, and 34,300 M(r). The smallest of these protein bands is believed to represent the product of alternative translation initiated at AUGs beginning with nt 217 or 391, although this point has not been rigorously proven. Collectively, these findings identify a novel and unusual member of the stomatin gene superfamily that interacts with the peripheral erythrocyte cytoskeleton and presumably other integral membrane proteins but not directly with the membrane bilayer. We hypothesize that SLP-2 may link stomatin or other integral membrane proteins to the peripheral cytoskeleton and thereby play a role in regulating ion channel conductances or the organization of sphingolipid and cholesterol-rich lipid rafts.     

The Caenorhabditis elegans Avermectin Resistance and Anesthetic Response Gene unc-9 Encodes a Member of a Protein Family Implicated in Electrical Coupling of Excitable Cells

Abstract: Mutations in the unc-9 gene of the nematode Caenorhabditis elegans cause abnormal forward locomotion and an egg-retention phenotype. unc-9 mutations also reduce the worms' sensitivity to avermectin and block a form of hypersensitivity to volatile anesthetics. We report here the cloning and molecular characterization of unc-9 and show that it encodes a member of the OPUS family of proteins that is 56% identical to another OPUS protein, UNC-7. It is significant that unc-9 mutants share all phenotypes with unc-7 mutants. Mutants in another gene, unc-124 , also share all tested phenotypes with unc-9 mutants, including identical locomotory and egg-laying defects, suggesting that multiple genes are required for the same biochemical function. OPUS proteins are implicated in the function of invertebrate gap junctions, and, based on a new alignment including 24 members from C. elegans , we present a refined model for the structure of OPUS proteins suggesting that oligomers could form a hydrophilic pore. We also show that alteration of highly conserved proline residues in UNC-9 leads to a cold sensitivity that likely affects a step in protein expression rather than function. Finally, we speculate on the basis of the avermectin resistance and anesthetic response phenotypes.     

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.     

Caenorhabditis elegans UNC-96 is a new component of M-lines that interacts with UNC-98 and paramyosin and is required in adult muscle for assembly and/or maintenance of thick filaments

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent "needles." By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel approximately 47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.     

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.     

UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development.

Nuclear migrations are essential for metazoan development. Two nuclear migrations that occur during C. elegans development require the function of the unc-84 gene. unc-84 mutants are also defective in the anchoring of nuclei within the hypodermal syncytium and in the migrations of the two distal tip cells of the gonad. Complementation analyses of 17 unc-84 alleles defined two genetically separable functions. Both functions are required for nuclear and distal tip cell migrations, but only one is required for nuclear anchorage. The DNA lesions associated with these 17 mutations indicate that the two genetically defined functions correspond to two distinct regions of the UNC-84 protein. The UNC-84 protein has a predicted transmembrane domain and a C-terminal region with similarity to the S. pombe spindle pole body protein Sad1 and to two predicted mammalian proteins. Analysis of a green fluorescent protein reporter indicated that UNC-84 is widely expressed and localized to the nuclear envelope. We propose that UNC-84 functions to facilitate a nuclear-centrosomal interaction required for nuclear migration and anchorage.     

Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans

The organization of the motor protein myosin into motile cellular structures requires precise temporal and spatial control. Caenorhabditis elegans UNC-45 facilitates this by functioning both as a chaperone and as a Hsp90 cochaperone for myosin during thick filament assembly. Consequently, mutations in C. elegans unc-45 result in paralyzed animals with severe myofibril disorganization in striated body wall muscles. Here, we report a new E3/E4 complex, formed by CHN-1, the C. elegans ortholog of CHIP (carboxyl terminus of Hsc70-interacting protein), and UFD-2, an enzyme known to have ubiquitin conjugating E4 activity in yeast, as necessary and sufficient to multiubiquitylate UNC-45 in vitro. The phenotype of unc-45 temperature-sensitive animals is partially suppressed by chn-1 loss of function, while UNC-45 overexpression in worms deficient for chn-1 results in severely disorganized muscle cells. These results identify CHN-1 and UFD-2 as a functional E3/E4 complex and UNC-45 as its physiologically relevant substrate.     

The Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1 and UNC-16 complex and regulates synaptic vesicle localization

Kinesin-1 is a heterotetramer composed of kinesin heavy chain (KHC) and kinesin light chain (KLC). The Caenorhabditis elegans genome has a single KHC, encoded by the unc-116 gene, and two KLCs, encoded by the klc-1 and klc-2 genes. We show here that UNC-116/KHC and KLC-2 form a complex orthologous to conventional kinesin-1. KLC-2 also binds UNC-16, the C. elegans JIP3/JSAP1 JNK-signaling scaffold protein, and the UNC-14 RUN domain protein. The localization of UNC-16 and UNC-14 depends on kinesin-1 (UNC-116 and KLC-2). Furthermore, mutations in unc-16, klc-2, unc-116, and unc-14 all alter the localization of cargos containing synaptic vesicle markers. Double mutant analysis is consistent with these four genes functioning in the same pathway. Our data support a model whereby UNC-16 and UNC-14 function together as kinesin-1 cargos and regulators for the transport or localization of synaptic vesicle components.     

Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

The Caenorhabditis elegans unc-78 gene encodes a homologue of actin-interacting protein 1 required for organized assembly of muscle actin filaments

Assembly and maintenance of myofibrils require dynamic regulation of the actin cytoskeleton. In Caenorhabditis elegans, UNC-60B, a muscle-specific actin depolymerizing factor (ADF)/cofilin isoform, is required for proper actin filament assembly in body wall muscle (Ono, S., D.L. Baillie, and G.M. Benian. 1999. J. Cell Biol. 145:491--502). Here, I show that UNC-78 is a homologue of actin-interacting protein 1 (AIP1) and functions as a novel regulator of actin organization in myofibrils. In unc-78 mutants, the striated organization of actin filaments is disrupted, and large actin aggregates are formed in the body wall muscle cells, resulting in defects in their motility. Point mutations in unc-78 alleles change conserved residues within different WD repeats of the UNC-78 protein and cause less severe phenotypes than a deletion allele, suggesting that these mutations partially impair the function of UNC-78. UNC-60B is normally localized in the diffuse cytoplasm and to the myofibrils in wild type but mislocalized to the actin aggregates in unc-78 mutants. Similar Unc-78 phenotypes are observed in both embryonic and adult muscles. Thus, AIP1 is an important regulator of actin filament organization and localization of ADF/cofilin during development of myofibrils.     

MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin

BACKGROUND: The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. RESULTS: We show here that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. CONCLUSIONS: mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance.     

Membrane raft actin deficiency and altered Ca-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to < 10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca²⁺-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

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.