Brasher BB et al. (2001) Oncogene "Mutational analysis of the regulatory function of the c-Abl Src homology 3 domain."

Roumiantsev S, Van Etten RA, Brasher BB

[Oncogene, 2001]

The catalytic activity of the c-Abl tyrosine kinase is tightly regulated by its Src homology 3 (SH3) domain through a complex mechanism that may involve intramolecular binding to Pro242 in the linker region between the SH2 and catalytic domains as well as interactions with a trans-inhibitor. We analysed the effect of mutation or replacement of SH3 on c-Abl tyrosine kinase activity and transformation. Random mutagenesis of SH3 identified several novel point mutations that dysregulated c-Abl kinase activity in vivo, but the RT loop was insensitive to mutational activation. Activating SH3 mutations abolished binding of proline-rich SH3 ligands in vitro, while mutations at Ser140 in the connector between the SH3 and SH2 domains activated Abl kinase activity in vivo and in vitro but did not impair SH3 ligand-binding. Abl was regulated efficiently when its SH3 domain was replaced with a heterologous SH3 from c-Src that binds a different spectrum of proline-rich ligands, but not by substitution of a modular WW domain with similar ligand-binding specificity. These results suggest that the SH3 domain regulates Abl principally by binding to the atypical intramolecular ligand Pro242 rather than a canonical PxxP ligand. Coordination between the SH3 and SH2 domains mediated by the connector region may be required for regulation of Abl even in the absence of SH2 ligand binding.

Nechipurenko, Inna et al. (2015) International Worm Meeting "A new role for the conserved centrosomal protein Girdin in the regulation of primary cilia biogenesis in C. elegans and mammalian cells."

Blacque, Oliver, Olivier-Mason, Anique, Sengupta, Piali, Nechipurenko, Inna, Kennedy, Julie

[International Worm Meeting, 2015]

Precise regulation of centrosome position is critical for diverse processes including asymmetric cell division, neurogenesis, directed cell migration, and ciliogenesis. Primary cilia, present on the surface of most cell types in metazoans, are key mediators of diverse signaling pathways essential for embryonic development, sensory signal transduction, and adult tissue homeostasis. Cilia from all studied organisms share a conserved core organization comprised of a microtubule-based axoneme anchored by the basal body (BB) and surrounded by a specialized membrane. Primary cilium assembly is a multi-step process that relies on mother centriole-to-BB transition, BB migration and anchoring to membrane, establishment of the 'ciliary gate', and axoneme elongation from the BB via intraflagellar transport of ciliary components. Considerable progress has been made in identifying molecular components of distinct centrosomal and ciliary sub-compartments; however, the mechanisms regulating centrosome positioning during early steps of ciliogenesis are poorly understood. We find that the highly conserved signaling protein Girdin localizes to the BB in C. elegans sensory neurons and mammalian cells. Loss of grdn-1 function in C. elegans results in defective sensory cilia morphology, ultrastructure, and localization of BB, transition zone, and signaling proteins. Similarly, Girdin knockdown in mammalian RPE-1 cells leads to a complete loss of, or shortened cilia, and dissociation of the BB from the nucleus. These findings suggest a conserved function for Girdin in regulating centrosome positioning, and thereby ciliogenesis, in both C. elegans sensory neurons and mammalian cells. Mammalian Girdin has been previously implicated in regulating the actin cytoskeleton, and in coupling multiple classes of surface receptors to downstream signaling events in different contexts (e.g. cell migration, synaptic plasticity, and tumorigenesis). Therefore, Girdin is perfectly positioned to link extracellular cues to the cytoskeleton to ensure correct centrosome/BB positioning during primary cilia formation - a previously uncharacterized role for this protein in any species. We will present our ongoing efforts to further characterize the mechanism, by which Girdin regulates BB positioning and primary cilia biogenesis in C. elegans sensory neurons and mammalian cells.

Nechipurenko IV et al. (2016) Dev Cell "A Conserved Role for Girdin in Basal Body Positioning and Ciliogenesis."

Blacque OE, Kazatskaya A, Nechipurenko IV, Olivier-Mason A, Heiman MG, Kennedy J, Sengupta P, McLachlan IG

[Dev Cell, 2016]

Primary cilia are ubiquitous sensory organelles that mediate diverse signaling pathways. Cilia position on the cell surface is determined by the location of the basal body (BB) that templates the cilium. The mechanisms that regulate BB positioning in the context of ciliogenesis are largely unknown. Here we show that the conserved signaling and scaffolding protein Girdin localizes to the proximal regions of centrioles and regulates BB positioning and ciliogenesis in Caenorhabditis elegans sensory neurons and human RPE-1 cells. Girdin depletion alters localization of the intercentriolar linker and ciliary rootlet component rootletin, and rootletin knockdown in RPE-1 cells mimics Girdin-dependent phenotypes. C.elegans Girdin also regulates localization of the apical junction component AJM-1, suggestingthat in nematodes Girdin may position BBs via rootletin- and AJM-1-dependent anchoring to the cytoskeleton and plasma membrane, respectively. Together, our results describe a conserved role for Girdin in BB positioning and ciliogenesis.

Nechipurenko IV et al. (2017) Elife "Centriolar remodeling underlies basal body maturation during ciliogenesis in Caenorhabditis elegans."

Berciu C, Sengupta P, Nicastro D, Nechipurenko IV

[Elife, 2017]

The primary cilium is nucleated by the mother centriole-derived basal body (BB) via as yet poorly characterized mechanisms. BBs have been reported to degenerate following ciliogenesis in the C. elegans embryo, although neither BB architecture nor early ciliogenesis steps have been described in this organism. In a previous study (Doroquez et al., 2014), we described the three-dimensional morphologies of sensory neuron cilia in adult C. elegans hermaphrodites at high resolution. Here, we use serial section electron microscopy and tomography of staged C. elegans embryos to demonstrate that BBs remodel to support ciliogenesis in a subset of sensory neurons. We show that centriolar singlet microtubules are converted into BB doublets which subsequently grow asynchronously to template the ciliary axoneme, visualize degeneration of the centriole core, and define the developmental stage at which the transition zone is established. Our work provides a framework for future investigations into the mechanisms underlying BB remodeling.

Kotsis F et al. (2021) Biochem Biophys Res Commun "Ift88, but not Kif3a, is required for establishment of the periciliary membrane compartment."

Kuehn EW, Boehlke C, Nitschke R, Kramer-Zucker A, Epting D, Janusch H, Lorentzen E, Neumann-Haefelin E, Viau A, Walz G, Li Y, Ganner A, Kotsis F

[Biochem Biophys Res Commun, 2021]

The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C.elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.

Song CX et al. (2022) J Cell Sci "WDR47 facilitates ciliogenesis by modulating intraflagellar transport."

Zeng WX, Liu R, Zeng XT, Song CX, Tong XJ, Li Q

[J Cell Sci, 2022]

Cilia are conserved organelles found in many cell types in eukaryotes, and their dysfunction causes defects in environmental sensing and signaling transduction; such defects are termed ciliopathies. Distinct cilia have cell-specific morphologies and exert distinct functions. However, the underlying mechanisms of cell-specific ciliogenesis and regulation are unclear. Here we identified a WD40-repeat (WDR) protein, WDR47/NMTN-1, and show that it is specifically required for ciliogenesis of AWB chemosensory neurons in C. elegans. WDR47/NMTN-1 is expressed in the AWB chemosensory neuron pair, and is enriched at the basal body (BB) of the AWB cilia. Knockout of wdr47/nmtn-1 causes abnormal AWB neuron cilia morphology, structural integrity, and induces aberrant AWB-mediated aversive behaviors. We further demonstrate that wdr47/nmtn-1 deletion affects movement of intraflagellar transport (IFT) particles and their cargo delivery in AWB neurons. Our results indicate that WDR47/NMTN-1 is essential for AWB neuron ciliary morphology and function, which reveal a novel mechanism for cell-specific ciliogenesis. Since WDR47/NMTN-1 is conserved in mammals, our findings may help understand the process of cell-specific ciliogenesis and provide insights for treating ciliopathies.

Thomas JH et al. (1996) East Coast Worm Meeting "LINEAGE ANALYSIS OF SYNTHETIC MULTIVULVA MUTANTS"

Horvitz HR, Thomas JH

[East Coast Worm Meeting, 1996]

Synthetic Multivulva (synMuv) genes comprise two distinct classes: A and B. AB double mutants are Muv, whereas AA and BB double mutants, like A and B single mutants, are wildtype for vulval development at the gross anatomical level. These data suggest that the synMuv genes define two functionally redundant pathways that negatively regulate vulval development (Ferguson and Horvitz, 1989, Genetics 123: 109121). If the synMuv genes are completely redundant for the regulation of vulval development, they should exhibit no abnormalities at the cellular level. To test this hypothesis, we analyzed the lineages of selected class A and class B mutants. The lineages of the six Pn.p cells of the vulval equivalence group in animals carrying a strong class A mutation, lin-15(n767), were identical to those of wild-type animals. These Pn.p cell lineages were analyzed in animals carrying a strong class B mutation, lin-36(n766), and were also found to be identical to those of wild-type animals. All six Pn.p cells of the vulval equivalence group in lin-36(n766); lin-15(n767) double mutants adopted vulval like fates. These results suggest that the class A and class B genes are completely redundant at the lineage level, and that in double mutants, all six Pn.p cells are at least partially induced to form vulval tissue.

Thomas JH et al. (1995) International C. elegans Meeting "THE SYNTHETIC MULTIVULVA GENES MAY ENCODE COMPONENTS OF A CELL SIGNALING SYSTEM"

Thomas JH, Horvitz HR

[International C. elegans Meeting, 1995]

Synthetic Multivulva (synMuv) genes comprise two distinct classes: A and B. AB double mutants are Muv, whereas AA and BB double mutants, like A and B single mutants, are wild-type for vulval development. These data suggest that the synMuv genes define two functionally redundant pathways that negatively regulate vulval development. To isolate new synMuv genes, we conducted several screens in which animals carrying a mutation in a gene of one class were mutagenized, and their F2 progeny screened for Muv animals. We isolated 19 new class A mutations after screening 23,000 haploid genomes. We defined two new class A complementation groups: lin(n2402) IIL and lin(n2728) IIC. We isolated 30 new class B mutations after screening 16,000 haploid genomes. We defined three new class B complementation groups: lin(n2231) IVR, lin(n2994) IIC, and lin(n2987). We have also characterized three additional class B complementation groups, lin(n770) IIIL, lin(n771) IIIR, and lin(n833) IR, originally identified by Ferguson and Horvitz (Genetics 123: 109-121, 1989). We cloned lin-36, a class B gene, and showed that it encodes a novel protein. We have determined the DNA sequences of six of seven lin-36 mutant alleles; none is an obvious molecular null mutation. We are now analyzing alleles isolated in a noncomplementation screen and are beginning expression studies. Our mosaic analysis suggests that lin-36 acts cell autonomously. By contrast, lin-15, a synMuv locus with both A and B activities, has a cell-non- autonomous focus (Herman and Hedgecock, Nature 348: 169-171, 1990). Together, these observations suggest that the synMuvs encode components of a cell signaling system.

Summers DW et al. (2016) Proc Natl Acad Sci U S A "SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury-induced SARM1 activation."

Summers DW, Gibson DA, DiAntonio A, Milbrandt J

[Proc Natl Acad Sci U S A, 2016]

Axon injury in response to trauma or disease stimulates a self-destruction program that promotes the localized clearance of damaged axon segments. Sterile alpha and Toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is an evolutionarily conserved executioner of this degeneration cascade, also known as Wallerian degeneration; however, the mechanism of SARM1-dependent neuronal destruction is still obscure. SARM1 possesses a TIR domain that is necessary for SARM1 activity. In other proteins, dimerized TIR domains serve as scaffolds for innate immune signaling. In contrast, dimerization of the SARM1 TIR domain promotes consumption of the essential metabolite NAD(+) and induces neuronal destruction. This activity is unique to the SARM1 TIR domain, yet the structural elements that enable this activity are unknown. In this study, we identify fundamental properties of the SARM1 TIR domain that promote NAD(+) loss and axon degeneration. Dimerization of the TIR domain from the Caenorhabditis elegans SARM1 ortholog TIR-1 leads to NAD(+) loss and neuronal death, indicating these activities are an evolutionarily conserved feature of SARM1 function. Detailed analysis of sequence homology identifies canonical TIR motifs as well as a SARM1-specific (SS) loop that are required for NAD(+) loss and axon degeneration. Furthermore, we identify a residue in the SARM1 BB loop that is dispensable for TIR activity yet required for injury-induced activation of full-length SARM1, suggesting that SARM1 function requires multidomain interactions. Indeed, we identify a physical interaction between the autoinhibitory N terminus and the TIR domain of SARM1, revealing a previously unrecognized direct connection between these domains that we propose mediates autoinhibition and activation upon injury.

Ceol CJ et al. (1996) East Coast Worm Meeting "IDENTIFYING INTERACTIONS OF THE SYNTHETIC MULTIVULVA GENES"

Ceol CJ, Horvitz HR

[East Coast Worm Meeting, 1996]

Vulval development in the hermaphrodite occurs in response to an inductive signal provided by the anchor cell. A receptor tyrosine kinase / Ras signaling pathway operating in the vulval precursor cells is required for transmission of this signal. The Synthetic Multivulva (synMuv) genes negatively regulate vulval induction and have been shown to act, in the case of lin-36, cell autonomously in the vulval precursor cells and, in the cases of lin-15 and lin-37, cell non-autonomously in hyp7 (1) (J. Thomas and X. Lu, personal communication). Previous genetic studies have assigned each of the synMuv genes to one of two classes, A or B. AB double mutants exhibit a multivulva phenotype, whereas AA double, BB double, A single and B single mutants are wild-type. Classes A and B are thought to represent functionally redundant pathways (2). We are interested in characterizing interactions used to transmit class A and class B negative regulatory signals to the Ras pathway. Taking a molecular approach, we intend to perform yeast two-hybrid analyses to identify candidate interacting proteins. Products of the five cloned synMuv genes, lin-15A, lin-15B, lin-9, lin-36 and lin-37 (3) (J. Thomas and X. Lu, personal communication), will be used as "baits," first, to search for interactions with cloned vulval induction pathway components and with cloned synMuvs, and, second, to screen C. elegans cDNA libraries for interacting proteins. In addition, we are considering genetic screens to identify activated synMuv alleles. Such gain-of-function alleles may be useful in defining the order of gene action in the synMuv pathways. 1. Herman and Hedgecock, Nature 348: 169-171, 1990. 2. Ferguson and Horvitz, Genetics 123: 109-121, 1989. 3. Clark, Lu and Horvitz, Genetics 137: 987-997, 1994.