Biomedical subjects

Michael L Nonet

Publications and source records attributed to Michael L Nonet.

14 recordsLinked to original sources

PubMed2025

Parameters that influence bipartite reporter system expression in Caenorhabditis elegans.

The development of bipartite reporter systems in Caenorhabditis elegans has lagged by more than a decade behind its adoption in Drosophila, the other invertebrate model commonly used to dissect biological mechanisms. Here, we characterize many parameters that influence expression in recently developed C. elegans bipartite systems. We examine how DNA binding site number and spacing influence expression and characterize how these expression parameters vary in distinct tissue types. Furthermore, we examine how both basal promoters and 3' UTR influence the specificity and level of expression. These studies provide both a framework for the rational design of driver and reporter transgenes and molecular and genetic tools for the creation, characterization, and optimization of bipartite system components for expression in other cell types.

Animals↗

PubMed2023

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling.

BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

Animals↗

PubMed2006

SYD-2 Liprin-alpha organizes presynaptic active zone formation through ELKS.

A central event in synapse development is formation of the presynaptic active zone in response to positional cues. Three active zone proteins, RIM, ELKS (also known as ERC or CAST) and Liprin-alpha, bind each other and are implicated in linking active zone formation to synaptic vesicle release. Loss of function in Caenorhabditis elegans syd-2 Liprin-alpha alters the size of presynaptic specializations and disrupts synaptic vesicle accumulation. Here we report that a missense mutation in the coiled-coil domain of SYD-2 causes a gain of function. In HSN synapses, the syd-2(gf) mutation promotes synapse formation in the absence of syd-1, which is essential for HSN synapse formation. syd-2(gf) also partially suppresses the synaptogenesis defects in syg-1 and syg-2 mutants. The activity of syd-2(gf) requires elks-1, an ELKS homolog; but not unc-10, a RIM homolog. The mutant SYD-2 shows increased association with ELKS. These results establish a functional dependency for assembly of the presynaptic active zone in which SYD-2 plays a key role.

Animals↗

PubMed2006

Regulators of kinesin involved in polarized trafficking and axon outgrowth.

Proteins such as UNC-76 that associate with kinesin motors are important in directing neurite extension. A small Caenorhabditis elegans coiled-coil protein, UNC-69, has now been shown to interact with UNC-76 and to be involved in axonal (but not dendritic) transport and outgrowth, as well as synapse formation.

Animals↗

PubMed2006

Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans.

Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function.

Animals↗

PubMed2006

SNARE complex zero layer residues are not critical for N-ethylmaleimide-sensitive factor-mediated disassembly.

Membrane-anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNARE complexes are parallel four-helix bundles stabilized in part by hydrophobic interactions within their core. At the center of SNARE complexes is a distinctive zero layer that consists of one arginine and three glutamines. This zero layer is thought to play a special role in the biology of the SNARE complex. One proposal is that the polar residues of the zero layer enable N-ethylmaleimide-sensitive factor (NSF)-mediated SNARE complex disassembly. Here, we studied the effects of manipulating the zero layer of the well studied synaptic SNARE complex in vitro and in vivo. Using a fluorescence-based assay to follow SNARE complex disassembly in real time, we found that the maximal rate at which NSF disassembles complexes was unaffected by mutations in the zero layer, including single replacement of the syntaxin glutamine with arginine as well as multiple replacement of all four layer residues with non-polar amino acids. To determine whether syntaxin with arginine instead of glutamine in its zero layer can support SNARE function in vivo, we introduced it as a transgene into a Caenorhabditis elegans syntaxin-null strain. Mutant syntaxin rescued viability and locomotory defects similarly to wild-type syntaxin, demonstrating that SNARE complexes with two glutamines and two arginines in the zero layer can support neurotransmission. These findings show that residues of the zero layer do not play an essential role in NSF-mediated disassembly.

Aldicarb↗

PubMed2006

Tomosyn inhibits synaptic vesicle priming in Caenorhabditis elegans.

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.

Amino Acid Sequence↗

PubMed2005

Presynaptic ryanodine receptors are required for normal quantal size at the Caenorhabditis elegans neuromuscular junction.

Analyses of the effect of ryanodine in vertebrate brain slices have led to the conclusion that presynaptic ryanodine receptors (RYRs) may have several functions in synaptic release, including causing large-amplitude miniature postsynaptic currents (mPSCs) by promoting concerted multivesicular release. However, the role of RYRs in synaptic release is controversial. To better understand the role of RYRs in synaptic release, we analyzed the effect of RYR mutation on mPSCs and evoked postsynaptic currents (ePSCs) at the Caenorhabditis elegans neuromuscular junction (NMJ). Amplitudes of mPSCs varied greatly at the C. elegans NMJ. Loss-of-function mutations of the RYR gene unc-68 (uncoordinated 68) essentially abolished large-amplitude mPSCs. The amplitude of ePSCs was also greatly suppressed. These defects were completely rescued by expressing wild-type UNC-68 specifically in neurons but not in muscle cells, suggesting that RYRs acted presynaptically. A combination of removing extracellular Ca2+ and UNC-68 function eliminated mPSCs, suggesting that influx and RYR-mediated release are likely the exclusive sources of Ca2+ for synaptic release. Large-amplitude mPSCs did not appear to be caused by multivesicular release, as has been suggested to occur at vertebrate central synapses, because the rise time of mPSCs was constant regardless of the amplitude but distinctive from that of ePSCs, and because large-amplitude mPSCs persisted under conditions that inhibit synchronized synaptic release, including elimination of extracellular Ca2+, and mutations of syntaxin and SNAP25 (soluble N-ethylmaleimide-sensitive factor attachment protein 25). These observations suggest that RYRs are essential to normal quantal size and are potential regulators of quantal size.

Acetylcholine↗

PubMed2005

Redundant localization mechanisms of RIM and ELKS in Caenorhabditis elegans.

Active zone proteins play a fundamental role in regulating neurotransmitter release and defining release sites. The functional roles of active zone components are beginning to be elucidated; however, the mechanisms of active zone protein localization are unknown. Studies have shown that glutamine, leucine, lysine, and serine-rich protein (ELKS), a recently defined member of the active zone complex, acts to localize the active zone protein Rab3a-interacting molecule (RIM) and regulates synaptic transmission in cultured neurons. Here, we test the function of ELKS in vivo. Like mammalian ELKS, Caenorhabditis elegans ELKS is an active zone protein that directly interacts with the postsynaptic density-25/Discs large/zona occludens (PDZ) domain of RIM. However, RIM protein localizes in the absence of ELKS and vice versa. In addition, elks mutants exhibit neither the behavioral nor the physiological defects associated with unc-10 RIM mutants, indicating that ELKS is not a critical component of the C. elegans release machinery. Interestingly, expression of the soluble PDZ domain of RIM disrupts ELKS active zone targeting, suggesting a tight association between the two proteins in vivo. RIM truncations containing only the PDZ and C2A domains target to release sites in an ELKS-dependent manner. Together, these data identify ELKS as a new member of the C. elegans active zone complex, define the role of ELKS in synaptic transmission, and characterize the relationship between ELKS and RIM in vivo. Furthermore, they demonstrate that multiple different protein-protein interactions redundantly anchor both ELKS and RIM to active zones and implicate novel proteins in the formation of the active zone.

Animals↗

PubMed2004

Mutations in Caenorhabditis elegans cytoplasmic dynein components reveal specificity of neuronal retrograde cargo.

We describe Caenorhabditis elegans dynein complex mutants, which misaccumulate synaptic proteins at the ends of neuronal processes. Ultrastructural analysis revealed irregularly sized vesicles that likely represent accumulation of cargo. We propose that synaptobrevin, synaptotagmin, and UNC-104 are specific cargoes of the dynein complex. Many cargoes link to dynein via interactions between dynactin and vesicle-associated spectrin. However, loss of spectrin results in only mild and occasional defects in synaptobrevin localization. Thus, the dynein-dynactin complex shows neuronal cargo selectivity without spectrin being a critical component of cargo binding. We observed parallels to progressive motor neuron disease symptoms in these animals. With age, neuronal misaccumulations increase in size and frequency; locomotion becomes progressively slower; and life span is shortened. These mutants provide a model to assess whether defects in transport of specific cargo mediate neuronal dysfunction.

Aging↗

PubMed2004

Resistance to volatile anesthetics by mutations enhancing excitatory neurotransmitter release in Caenorhabditis elegans.

The molecular mechanisms whereby volatile general anesthetics (VAs) disrupt behavior remain undefined. In Caenorhabditis elegans mutations in the gene unc-64, which encodes the presynaptic protein syntaxin 1A, produce large allele-specific differences in VA sensitivity. UNC-64 syntaxin normally functions to mediate fusion of neurotransmitter vesicles with the presynaptic membrane. The precise role of syntaxin in the VA mechanism is as yet unclear, but a variety of results suggests that a protein interacting with syntaxin to regulate neurotransmitter release is essential for VA action in C. elegans. To identify additional proteins that function with syntaxin to control neurotransmitter release and VA action, we screened for suppressors of the phenotypes produced by unc-64 reduction of function. Loss-of-function mutations in slo-1, which encodes a Ca(2+)-activated K+ channel, and in unc-43, which encodes CaM-kinase II, and a gain-of-function mutation in egl-30, which encodes Gqalpha, were isolated as syntaxin suppressors. The slo-1 and egl-30 mutations conferred resistance to VAs, but unc-43 mutations did not. The effects of slo-1 and egl-30 on VA sensitivity can be explained by their actions upstream or parallel to syntaxin to increase the level of excitatory neurotransmitter release. These results strengthen the link between transmitter release and VA action.

Anesthetics, Inhalation↗

PubMed2003

Defects in synaptic vesicle docking in unc-18 mutants.

Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing unc-18 mutants of the nematode Caenorhabditis elegans. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked vesicles at the active zone in unc-18 mutants, suggesting that UNC-18 functions, directly or indirectly, as a facilitator of vesicle docking.

Animals↗

PubMed2002

Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.

RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.

Animals↗

PubMed2002

AEXpulsing a retrograde signal.

A variety of secreted components have been identified as retrograde signals mediating diverse aspects of synaptic development, maintenance, and plasticity; however, little is known about the mechanisms mediating the release of secreted retrograde signals. Doi and Iwasaki (this issue of Neuron) implicate AEX-1, a protein distantly related to the UNC-13/Munc13 family, as an attractive candidate regulator of the retrograde release machinery in muscle.

Animals↗