FEZ2 has acquired additional protein interaction partners relative to FEZ1: functional and evolutionary implications

Background

The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function.

Methodology/Principal Findings

Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom.

Conclusions/Significance

We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins.     

Fasciculation and elongation protein zeta-1 (FEZ1) participates in the polarization of hippocampal neuron by controlling the mitochondrial motility

The fasciculation and elongation protein zeta-1 (FEZ1), a mammalian orthologue of Caenorhabditis elegans UNC-76 protein, is a 45-kDa protein with four coiled-coiled domains and efficiently promotes the neurite elongation in the rat phaeochromocytoma PC12 cells. UNC-76 proteins of C. elegans and Drosophila have been genetically demonstrated to be involved in the axonal guidance. We here show that FEZ1 RNA interference (RNAi) represses the formation of axon in rat embryo hippocampal neurons. An anterograde mitochondrial movement is also retarded in neurites of the RNAi-treated hippocampal neurons. Moreover, the size of mitochondria is considerably elongated by the RNAi treatment. The transport of mitochondria from soma to axon or dendrites is essential for the neuronal differentiation. Therefore, our results strongly suggest that FEZ1 participates in the establishment of neuronal polarity by controlling the mitochondrial motility along axon.     

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.     

Identification of a tissue-non-specific homologue of axonal fasciculation and elongation protein zeta-1

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans UNC-76 protein involved in the axonal outgrowth and fasciculation and promotes neurite extension of PC12 cells through interaction with protein kinase C zeta (PKCzeta). The gene coding for FEZ2, a homologue of FEZ1, has also been reported in rat and human. In this study, we compared mRNA expression of FEZ1 and FEZ2 in adult rat tissues and mouse embryos by Northern blot and in situ hybridization analyses. In contrast to FEZ1 whose mRNA is expressed almost exclusively in rat brain and temporarily around the neurogenesis stage of mouse embryos, the message for FEZ2 is detected weakly in most tissues and abundantly throughout the mouse embryonic stages. Similar to FEZ1, FEZ2 interacted with PKCzeta and induced neurite extension of PC12 cells when coexpressed with a constitutively active mutant of PKCzeta. These results suggest that FEZ2 plays an important role in the morphological changes of various cells by associating with PKCzeta in a tissue-non-specific manner.     

UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ～50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the μ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as μ2 adaptin.     

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.     

Cloning and characterization of SCHIP-1, a novel protein interacting specifically with spliced isoforms and naturally occurring mutant NF2 proteins

The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved in NF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members, implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as a tumor suppressor is unknown. In attempt to shed light on schwannomin function, we have identified a novel coiled-coil protein, SCHIP-1, that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein is homologous to human FEZ proteins and the related Caenorhabditis elegans gene product UNC-76. Immunofluorescent staining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmic membrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can be observed only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and 3, but not with the schwannomin isoform exhibiting growth-suppressive activity. Our observations suggest that SCHIP-1 interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslational modifications, alternative splicing, or mutations.     

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.     

unc-119 homolog required for normal development of the zebrafish nervous system

The UNC-119 proteins, found in all metazoans examined, are highly conserved at both the sequence and functional levels. In the invertebrates Caenorhabditis elegans and Drosophila melanogaster, unc-119 genes are expressed pan-neurally. Loss of function of the unc-119 gene in C. elegans results in a disorganized neural architecture and paralysis. The function of UNC-119 proteins has been conserved throughout evolution, as transgenic expression of the human UNC119 gene in C. elegans unc-119 mutants restores a wild-type phenotype. However, the nature of the conserved molecular function of UNC-119 proteins is poorly understood. Although unc-119 genes are expressed throughout the nervous system of the worm and fly, the analysis of these genes in vertebrates has focused on their function in the photoreceptor cells of the retina. Here we report the characterization of an unc-119 homolog in the zebrafish. The Unc119 protein is expressed in various neural tissues in the developing zebrafish embryo and larva. Morpholino oligonucleotide (MO)-mediated knockdown of Unc119 protein results in a "curly tail down" phenotype. Examination of neural patterning demonstrates that these "curly tail down" zebrafish experience a constellation of neuronal defects similar to those seen in C. elegans unc-119 mutants: missing or misplaced cell bodies, process defasciculation, axon pathfinding errors, and aberrant axonal branching. These findings suggest that UNC-119 proteins may play an important role in the development and/or function of the vertebrate nervous system.     

MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion

The netrin UNC-6 repels motor axons by activating the UNC-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in unc-5 and unc-6, but not in unc-40, dominantly enhance the mutant phenotypes of max-1, whereas overexpression of unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the UNC-5 receptor signaling pathway.     

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.     

unc-53 controls longitudinal migration in C. elegans

Cell migration and outgrowth are thought to be based on analogous mechanisms that require repeated cycles of process extension, reading and integration of multiple directional signals, followed by stabilisation in a preferred direction, and renewed extension. We have characterised a C. elegans gene, unc-53, that appears to act cell autonomously in the migration and outgrowth of muscles, axons and excretory canals. Abrogation of unc-53 function disrupts anteroposterior outgrowth in those cells that normally express the gene. Conversely, overexpression of unc-53 in bodywall muscles leads to exaggerated outgrowth. UNC-53 is a novel protein conserved in vertebrates that contains putative SH3- and actin-binding sites. unc-53 interacts genetically with sem-5 and we demonstrated a direct interaction in vitro between UNC-53 and the SH2-SH3 adaptor protein SEM-5/GRB2. Thus, unc-53 is involved in longitudinal navigation and might act by linking extracellular guidance cues to the intracellular cytoskeleton.     

UNC-87 isoforms,Caenorhabditis elegans calponin-related proteins,interact with both actin and myosin and regulate actomyosin contractility

Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ∼190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.     

The two actin-interacting protein 1 genes have overlapping and essential function for embryonic development in Caenorhabditis elegans

Disassembly of actin filaments by actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1) is a conserved mechanism to promote reorganization of the actin cytoskeleton. We previously reported that unc-78, an AIP1 gene in the nematode Caenorhabditis elegans, is required for organized assembly of sarcomeric actin filaments in the body wall muscle. unc-78 functions in larval and adult muscle, and an unc-78-null mutant is homozygous viable and shows only weak phenotypes in embryos. Here we report that a second AIP1 gene, aipl-1 (AIP1-like gene-1), has overlapping function with unc-78, and that depletion of the two AIP1 isoforms causes embryonic lethality. A single aipl-1-null mutation did not cause a detectable phenotype. However, depletion of both unc-78 and aipl-1 arrested development at late embryonic stages due to severe disorganization of sarcomeric actin filaments in body wall muscle. In vitro, both AIPL-1 and UNC-78 preferentially cooperated with UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament disassembly but not with UNC-60A, a nonmuscle ADF/cofilin. AIPL-1 is expressed in embryonic muscle, and forced expression of AIPL-1 in adult muscle compensated for the function of UNC-78. Thus our results suggest that enhancement of actin filament disassembly by ADF/cofilin and AIP1 proteins is critical for embryogenesis.     

The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane

Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (alpha-integrin), and pat-3 (beta-integrin) genes encode ECM or transmembrane proteins found at the cell-matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell-matrix adhesion complexes. The 720-amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins.We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/beta-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/beta-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/beta-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/beta-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.     

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.