Publications by authors named "Kevin M Collins"

19 Publications

  • Page 1 of 1

Directed movement changes coexistence outcomes in heterogeneous environments.

Ecol Lett 2021 Nov 24. Epub 2021 Nov 24.

Department of Environmental Science and Policy, University of California, Davis, Davis, California, USA.

Understanding mechanisms of coexistence is a central topic in ecology. Mathematical analysis of models of competition between two identical species moving at different rates of symmetric diffusion in heterogeneous environments show that the slower mover excludes the faster one. The models have not been tested empirically and lack inclusions of a component of directed movement toward favourable areas. To address these gaps, we extended previous theory by explicitly including exploitable resource dynamics and directed movement. We tested the mathematical results experimentally using laboratory populations of the nematode worm, Caenorhabditis elegans. Our results not only support the previous theory that the species diffusing at a slower rate prevails in heterogeneous environments but also reveal that moderate levels of a directed movement component on top of the diffusive movement allow species to coexist. Our results broaden the theory of species coexistence in heterogeneous space and provide empirical confirmation of the mathematical predictions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ele.13925DOI Listing
November 2021

Presynaptic Gαo (GOA-1) signals to depress command neuron excitability and allow stretch-dependent modulation of egg laying in Caenorhabditis elegans.

Genetics 2021 May 25. Epub 2021 May 25.

Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL USA 33136.

Egg laying in the nematode worm Caenorhabditis elegans is a two-state behavior modulated by internal and external sensory input. We have previously shown that homeostatic feedback of embryo accumulation in the uterus regulates bursting activity of the serotonergic HSN command neurons that sustains the egg-laying active state. How sensory feedback of egg release signals to terminate the egg-laying active state is less understood. We find that Gαo, a conserved Pertussis Toxin-sensitive G protein, signals within HSN to inhibit egg-laying circuit activity and prevent entry into the active state. Gαo signaling hyperpolarizes HSN, reducing HSN Ca2+ activity and input onto the postsynaptic vulval muscles. Loss of inhibitory Gαo signaling uncouples presynaptic HSN activity from a postsynaptic, stretch-dependent homeostat, causing precocious entry into the egg-laying active state when only a few eggs are present in the uterus. Feedback of vulval opening and egg release activates the uv1 neuroendocrine cells which release NLP-7 neuropeptides which signal to inhibit egg laying through Gαo-independent mechanisms in the HSNs and Gαo-dependent mechanisms in cells other than the HSNs. Thus, neuropeptide and inhibitory Gαo signaling maintains a bi-stable state of electrical excitability that dynamically controls circuit activity in response to both external and internal sensory input to drive a two-state behavior output.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/genetics/iyab080DOI Listing
May 2021

The Sex-Specific VC Neurons Are Mechanically Activated Motor Neurons That Facilitate Serotonin-Induced Egg Laying in .

J Neurosci 2021 04 9;41(16):3635-3650. Epub 2021 Mar 9.

Department of Biology, University of Miami, Coral Gables, Florida 33143

Successful execution of behavior requires coordinated activity and communication between multiple cell types. Studies using the relatively simple neural circuits of invertebrates have helped to uncover how conserved molecular and cellular signaling events shape animal behavior. To understand the mechanisms underlying neural circuit activity and behavior, we have been studying a simple circuit that drives egg-laying behavior in the nematode worm Here we show that the sex-specific, ventral C (VC) motor neurons are important for vulval muscle contractility and egg laying in response to serotonin. Ca imaging experiments show the VCs are active during times of vulval muscle contraction and vulval opening, and optogenetic stimulation of the VCs promotes vulval muscle Ca activity. Blocking VC neurotransmission inhibits egg laying in response to serotonin and increases the failure rate of egg-laying attempts, indicating that VC signaling facilitates full vulval muscle contraction and opening of the vulva for efficient egg laying. We also find the VCs are mechanically activated in response to vulval opening. Optogenetic stimulation of the vulval muscles is sufficient to drive VC Ca activity and requires muscle contractility, showing the presynaptic VCs and the postsynaptic vulval muscles can mutually excite each other. Together, our results demonstrate that the VC neurons facilitate efficient execution of egg-laying behavior by coordinating postsynaptic muscle contractility in response to serotonin and mechanosensory feedback. Many animal motor behaviors are modulated by the neurotransmitters, serotonin and ACh. Such motor circuits also respond to mechanosensory feedback, but how neurotransmitters and mechanoreceptors work together to coordinate behavior is not well understood. We address these questions using the egg-laying circuit in where we can manipulate presynaptic neuron and postsynaptic muscle activity in behaving animals while recording circuit responses through Ca imaging. We find that the cholinergic VC motoneurons are important for proper vulval muscle contractility and egg laying in response to serotonin. Muscle contraction also activates the VCs, forming a positive feedback loop that promotes full contraction for egg release. In all, mechanosensory feedback provides a parallel form of modulation that shapes circuit responses to neurotransmitters.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.2150-20.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055074PMC
April 2021

Identification of Toxicity Effects of CuO Materials on as a Function of Environmental Ionic Composition.

Environ Sci Nano 2020 Feb 8;7(2):645-655. Epub 2020 Jan 8.

Department of Biology, University of Miami, 1301 Memorial Drive, Coral Gables, Florida, 33146, USA.

Previous work has shown that spherical CuO nanomaterials show negative effects on cell and animal physiology. The biological effects of CuO materials, which posess unique chemical features compared to CuO nanomaterials and can be synthesized in a similarly large variety of shapes and sizes, are comparatively less studied. Here, we synthesized truncated octahedral CuO particles and characterized their structure, stability, and physiological effects in the nematode worm animal model, . CuO particles were found to be generally stable in aqueous media, although the particles did show signs of oxidation and leaching of Cu within hours in worm growth media. The particles were found to be especially sensitive to inorganic phosphate (PO ) found in standard NGM nematode growth medium. CuO particles were observed being taken up into the nematode pharynx and detected in the lumen of the gut. Toxicity experiments revealed that treatment with CuO particles caused a significant reduction in animal size and lifespan. These toxic effects resembled treatment with Cu, but measurements of Cu leaching, worm size, and long-term behavior experiments show the particles are more toxic than expected from Cu ion leaching alone. These results suggest worm ingestion of intact CuO particles enhances their toxicity and behavior effects while particle exposure to environmental phosphate precipitates leached Cu into biounavailable phosphate salts. Interestingly, the worms showed an acute avoidance of bacterial food with CuO particles, suggesting that animals can detect chemical features of the particles and/or their breakdown products and actively avoid areas with them. These results will help to understand how specific, chemically-defined particles proposed for use in polluted soil and wastewater remediation affect animal toxicity and behaviors in their natural environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051015PMC
February 2020

Serotonin and neuropeptides are both released by the HSN command neuron to initiate Caenorhabditis elegans egg laying.

PLoS Genet 2019 01 24;15(1):e1007896. Epub 2019 Jan 24.

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut, United States of America.

Neurons typically release both a small-molecule neurotransmitter and one or more neuropeptides, but how these two types of signal from the same neuron might act together remains largely obscure. For example, serotonergic neurons in mammalian brain express the neuropeptide Substance P, but it is unclear how this co-released neuropeptide might modulate serotonin signaling. We studied this issue in C. elegans, in which all serotonergic neurons express the neuropeptide NLP-3. The serotonergic Hermaphrodite Specific Neurons (HSNs) are command motor neurons within the egg-laying circuit which have been shown to release serotonin to initiate egg-laying behavior. We found that egg-laying defects in animals lacking serotonin were far milder than in animals lacking HSNs, suggesting that HSNs must release other signal(s) in addition to serotonin to stimulate egg laying. While null mutants for nlp-3 had only mild egg-laying defects, animals lacking both serotonin and NLP-3 had severe defects, similar to those of animals lacking HSNs. Optogenetic activation of HSNs induced egg laying in wild-type animals, and in mutant animals lacking either serotonin or NLP-3, but failed to induce egg laying in animals lacking both. We recorded calcium activity in the egg-laying muscles of animals lacking either serotonin, NLP-3, or both. The single mutants, and to a greater extent the double mutant, showed muscle activity that was uncoordinated and unable to expel eggs. Specifically, the vm2 muscles cells, which are direct postsynaptic targets of the HSN, failed to contract simultaneously with other egg-laying muscle cells. Our results show that the HSN neurons use serotonin and the neuropeptide NLP-3 as partially redundant co-transmitters that together stimulate and coordinate activity of the target cells onto which they are released.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1007896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363226PMC
January 2019

Homeostatic Feedback Modulates the Development of Two-State Patterned Activity in a Model Serotonin Motor Circuit in .

J Neurosci 2018 07 11;38(28):6283-6298. Epub 2018 Jun 11.

Neuroscience Program, University of Miami Miller School of Medicine, Miami, Florida 33136 and

Neuron activity accompanies synapse formation and maintenance, but how early circuit activity contributes to behavior development is not well understood. Here, we use the egg-laying motor circuit as a model to understand how coordinated cell and circuit activity develops and drives a robust two-state behavior in adults. Using calcium imaging in behaving animals, we find the serotonergic hermaphrodite-specific neurons (HSNs) and vulval muscles show rhythmic calcium transients in L4 larvae before eggs are produced. HSN activity in L4 is tonic and lacks the alternating burst-firing/quiescent pattern seen in egg-laying adults. Vulval muscle activity in L4 is initially uncoordinated but becomes synchronous as the anterior and posterior muscle arms meet at HSN synaptic release sites. However, coordinated muscle activity does not require presynaptic HSN input. Using reversible silencing experiments, we show that neuronal and vulval muscle activity in L4 is not required for the onset of adult behavior. Instead, the accumulation of eggs in the adult uterus renders the muscles sensitive to HSN input. Sterilization or acute electrical silencing of the vulval muscles inhibits presynaptic HSN activity and reversal of muscle silencing triggers a homeostatic increase in HSN activity and egg release that maintains ∼12-15 eggs in the uterus. Feedback of egg accumulation depends upon the vulval muscle postsynaptic terminus, suggesting that a retrograde signal sustains HSN synaptic activity and egg release. Our results show that egg-laying behavior in is driven by a homeostat that scales serotonin motor neuron activity in response to postsynaptic muscle feedback. The functional importance of early, spontaneous neuron activity in synapse and circuit development is not well understood. Here, we show in the nematode that the serotonergic hermaphrodite-specific neurons (HSNs) and postsynaptic vulval muscles show activity during circuit development, well before the onset of adult behavior. Surprisingly, early activity is not required for circuit development or the onset of adult behavior and the circuit remains unable to drive egg laying until fertilized embryos are deposited into the uterus. Egg accumulation potentiates vulval muscle excitability, but ultimately acts to promote burst firing in the presynaptic HSNs which results in egg laying. Our results suggest that mechanosensory feedback acts at three distinct steps to initiate, sustain, and terminate egg-laying circuit activity and behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.3658-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041790PMC
July 2018

Photochemical Barcodes.

J Am Chem Soc 2018 04 21;140(13):4485-4488. Epub 2018 Mar 21.

Laboratory for Molecular Photonics, Departments of Biology and Chemistry , University of Miami , 1301 Memorial Drive , Coral Gables , Florida 33146-0431 , United States.

A photochemical strategy to encode fluorescence signals in vivo with spatial control was designed around the unique properties of a photoactivatable borondipyrromethene (BODIPY). The photoinduced disconnection of two oxazines, flanking a single BODIPY, in two consecutive steps produces a mixture of three emissive molecules with resolved fluorescence inside polymer beads. The relative amounts and emission intensities of the three fluorophores can be regulated precisely in each bead by adjusting the dose of activating photons to mark individual particles with distinct codes of fluorescence signals. The visible wavelengths and mild illumination sufficient to induce these transformations permit the photochemical barcoding of beads also in living nematodes. Different regions of the same animal can be labeled with distinct barcodes to allow the monitoring of their dynamics for long times with no toxic effects. Thus, our photochemical strategy for the generation of fluorescence barcodes can produce multiple and distinguishable labels in the same biological sample to enable the spatiotemporal tracking of, otherwise indistinguishable, targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.8b00887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056178PMC
April 2018

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans.

J Vis Exp 2018 02 7(132). Epub 2018 Feb 7.

Neuroscience Program, University of Miami School of Medicine; Department of Biology, University of Miami;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified. Here we describe methods for ratiometric Ca imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca reporter GCaMP5 to record changes in intracellular Ca in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 µm and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/56911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912386PMC
February 2018

Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores.

Bioconjug Chem 2017 05 1;28(5):1519-1528. Epub 2017 May 1.

Laboratory for Molecular Photonics, Departments of Biology and Chemistry, University of Miami , 1301 Memorial Drive, Coral Gables, Florida 33146-0431, United States.

Seven macromolecular constructs incorporating multiple borondipyrromethene (BODIPY) fluorophores along a common poly(methacrylate) backbone with decyl and oligo(ethylene glycol) side chains were synthesized. The hydrophilic oligo(ethylene glycol) components impose solubility in aqueous environment on the overall assembly. The hydrophobic decyl chains effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical properties. As a result, the brightness of these multicomponent assemblies is approximately three times greater than that of a model BODIPY monomer. Such a high brightness level is maintained even after injection of the macromolecular probes in living nematodes, allowing their visualization with a significant improvement in signal-to-noise ratio, relative to the model monomer, and no cytotoxic or behavioral effects. The covalent scaffold of these macromolecular constructs also permits their subsequent conjugation to secondary antibodies. The covalent attachment of polymer and biomolecule does not hinder the targeting ability of the latter and the resulting bioconjugates can be exploited to stain the tubulin structure of model cells to enable their visualization with optimal signal-to-noise ratios. These results demonstrate that this particular structural design for the incorporation of multiple chromophores within the same covalent construct is a viable one to preserve the photophysical properties of the emissive species and enable the assembly of bioimaging probes with enhanced brightness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.bioconjchem.7b00166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906127PMC
May 2017

Local neuropeptide signaling modulates serotonergic transmission to shape the temporal organization of C. elegans egg-laying behavior.

PLoS Genet 2017 04 6;13(4):e1006697. Epub 2017 Apr 6.

Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA United States of America.

Animal behaviors are often composed of distinct alternating behavioral states. Neuromodulatory signals are thought to be critical for establishing stable behavioral states and for orchestrating transitions between them. However, we have only a limited understanding of how neuromodulatory systems act in vivo to alter circuit performance and shape behavior. To address these questions, we have investigated neuromodulatory signaling in the context of Caenorhabditis elegans egg-laying. Egg-laying activity cycles between discrete states-short bursts of egg deposition (active phases) that alternate with prolonged quiescent periods (inactive phases). Here using genetic, pharmacological and optogenetic approaches for cell-specific activation and inhibition, we show that a group of neurosecretory cells (uv1) located in close spatial proximity to the egg-laying neuromusculature direct the temporal organization of egg-laying by prolonging the duration of inactive phases. We demonstrate that the modulatory effects of the uv1 cells are mediated by peptides encoded by the nlp-7 and flp-11 genes that act locally to inhibit circuit activity, primarily by inhibiting vesicular release of serotonin from HSN motor neurons. This peptidergic inhibition is achieved, at least in part, by reducing synaptic vesicle abundance in the HSN motor neurons. By linking the in vivo actions of specific neuropeptide signaling systems with the generation of stable behavioral outcomes, our study reveals how cycles of neuromodulation emanating from non-neuronal cells can fundamentally shape the organization of a behavioral program.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1006697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398689PMC
April 2017

Activity of the egg-laying behavior circuit is controlled by competing activation and feedback inhibition.

Elife 2016 11 16;5. Epub 2016 Nov 16.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States.

Like many behaviors, egg laying alternates between inactive and active states. To understand how the underlying neural circuit turns the behavior on and off, we optically recorded circuit activity in behaving animals while manipulating circuit function using mutations, optogenetics, and drugs. In the active state, the circuit shows rhythmic activity phased with the body bends of locomotion. The serotonergic HSN command neurons initiate the active state, but accumulation of unlaid eggs also promotes the active state independent of the HSNs. The cholinergic VC motor neurons slow locomotion during egg-laying muscle contraction and egg release. The uv1 neuroendocrine cells mechanically sense passage of eggs through the vulva and release tyramine to inhibit egg laying, in part via the LGC-55 tyramine-gated Cl channel on the HSNs. Our results identify discrete signals that entrain or detach the circuit from the locomotion central pattern generator to produce active and inactive states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.21126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142809PMC
November 2016

LIN-12/Notch signaling instructs postsynaptic muscle arm development by regulating UNC-40/DCC and MADD-2 in Caenorhabditis elegans.

Elife 2013 Mar 19;2:e00378. Epub 2013 Mar 19.

Department of Biology , Howard Hughes Medical Institute, Stanford University , Stanford , United States.

The diverse cell types and the precise synaptic connectivity between them are the cardinal features of the nervous system. Little is known about how cell fate diversification is linked to synaptic target choices. Here we investigate how presynaptic neurons select one type of muscles, vm2, as a synaptic target and form synapses on its dendritic spine-like muscle arms. We found that the Notch-Delta pathway was required to distinguish target from non-target muscles. APX-1/Delta acts in surrounding cells including the non-target vm1 to activate LIN-12/Notch in the target vm2. LIN-12 functions cell-autonomously to up-regulate the expression of UNC-40/DCC and MADD-2 in vm2, which in turn function together to promote muscle arm formation and guidance. Ectopic expression of UNC-40/DCC in non-target vm1 muscle is sufficient to induce muscle arm extension from these cells. Therefore, the LIN-12/Notch signaling specifies target selection by selectively up-regulating guidance molecules and forming muscle arms in target cells. DOI:http://dx.doi.org/10.7554/eLife.00378.001.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.00378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601818PMC
March 2013

Postsynaptic ERG potassium channels limit muscle excitability to allow distinct egg-laying behavior states in Caenorhabditis elegans.

J Neurosci 2013 Jan;33(2):761-75

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8024, USA.

Caenorhabditis elegans regulates egg laying by alternating between an inactive phase and a serotonin-triggered active phase. We found that the conserved ERG [ether-a-go-go (EAG) related gene] potassium channel UNC-103 enables this two-state behavior by limiting excitability of the egg-laying muscles. Using both high-speed video recording and calcium imaging of egg-laying muscles in behaving animals, we found that the muscles appear to be excited at a particular phase of each locomotor body bend. During the inactive phase, this rhythmic excitation infrequently evokes calcium transients or contraction of the egg-laying muscles. During the serotonin-triggered active phase, however, these muscles are more excitable and each body bend is accompanied by a calcium transient that drives twitching or full contraction of the egg-laying muscles. We found that ERG-null mutants lay eggs too frequently, and that ERG function is necessary and sufficient in the egg-laying muscles to limit egg laying. ERG K(+) channels localize to postsynaptic sites in the egg-laying muscle, and mutants lacking ERG have more frequent calcium transients and contractions of the egg-laying muscles even during the inactive phase. Thus ERG channels set postsynaptic excitability at a threshold so that further adjustments of excitability by serotonin generate two distinct behavioral states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.3896-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542984PMC
January 2013

Trans-SNARE complex assembly and yeast vacuole membrane fusion.

Proc Natl Acad Sci U S A 2007 May 14;104(21):8755-60. Epub 2007 May 14.

Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844, USA.

cis-SNARE complexes (anchored in one membrane) are disassembled by Sec17p (alpha-SNAP) and Sec18p (NSF), permitting the unpaired SNAREs to assemble in trans. We now report a direct assay of trans-SNARE complex formation during yeast vacuole docking. SNARE complex assembly and fusion is promoted by high concentrations of the SNARE Vam7p or Nyv1p or by addition of HOPS (homotypic fusion and vacuole protein sorting), a Ypt7p (Rab)-effector complex with a Sec1/Munc18-family subunit. Inhibitors that target Ypt7p, HOPS, or key regulatory lipids prevent trans-SNARE complex assembly and ensuing fusion. Strikingly, the lipid ligand MED (myristoylated alanine-rich C kinase substrate effector domain) or elevated concentrations of Sec17p, which can displace HOPS from SNARE complexes, permit full trans-SNARE pairing but block fusion. These findings suggest that efficient fusion requires trans-SNARE complex associations with factors such as HOPS and subsequent regulated lipid rearrangements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0702290104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885575PMC
May 2007

Stringent 3Q.1R composition of the SNARE 0-layer can be bypassed for fusion by compensatory SNARE mutation or by lipid bilayer modification.

J Biol Chem 2007 May 30;282(20):14861-7. Epub 2007 Mar 30.

Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

SNARE proteins form bundles of four alpha-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q.1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7(Q283R), yet Vam7(Q283R) is preferred by vacuoles with Nyv1(R191Q). Rotation of the position of the arginine in the 0-layer increases the K(m) for Vam7p but does not affect the maximal rate of fusion. Vam7(Q283R) forms stable 2Q.2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M700971200DOI Listing
May 2007

Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p.

EMBO J 2006 Apr 6;25(8):1579-89. Epub 2006 Apr 6.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.

Coupling of Rab GTPase activation and SNARE complex assembly during membrane fusion is poorly understood. The homotypic fusion and vacuole protein sorting (HOPS) complex links these two processes: it is an effector for the vacuolar Rab GTPase Ypt7p and is required for vacuolar SNARE complex assembly. We now report that pure, active HOPS complex binds phosphoinositides and the PX domain of the vacuolar SNARE protein Vam7p. These binding interactions support HOPS complex association with the vacuole and explain its enrichment at the same microdomains on docked vacuoles as phosphoinositides, Ypt7p, Vam7p, and the other SNARE proteins. Concentration of the HOPS complex at these microdomains may be a key factor for coupling Rab GTPase activation to SNARE complex assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.emboj.7601051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1440844PMC
April 2006

Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion.

EMBO J 2005 May 5;24(10):1775-86. Epub 2005 May 5.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

SNARE functions during membrane docking and fusion are regulated by Sec1/Munc18 (SM) chaperones and Rab/Ypt GTPase effectors. These functions for yeast vacuole fusion are combined in the six-subunit HOPS complex. HOPS facilitates Ypt7p nucleotide exchange, is a Ypt7p effector, and contains an SM protein. We have dissected the associations and requirements for HOPS, Ypt7p, and Sec17/18p during SNARE complex assembly. Vacuole SNARE complexes bind either Sec17p or the HOPS complex, but not both. Sec17p and its co-chaperone Sec18p disassemble SNARE complexes. Ypt7p regulates the reassembly of unpaired SNAREs with each other and with HOPS, forming HOPS.SNARE complexes prior to fusion. After HOPS.SNARE assembly, lipid rearrangements are still required for vacuole content mixing. Thus, Sec17p and HOPS have mutually exclusive interactions with vacuole SNAREs to mediate disruption of SNARE complexes or their assembly for docking and fusion. Sec17p may displace HOPS from SNAREs to permit subsequent rounds of fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.emboj.7600658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142591PMC
May 2005

A soluble SNARE drives rapid docking, bypassing ATP and Sec17/18p for vacuole fusion.

EMBO J 2004 Jul 8;23(14):2765-76. Epub 2004 Jul 8.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.

Membrane fusion requires priming, the disassembly of cis-SNARE complexes by the ATP-driven chaperones Sec18/17p. Yeast vacuole priming releases Vam7p, a soluble SNARE. Vam7p reassociation during docking allows trans-SNARE pairing and fusion. We now report that recombinant Vam7p (rVam7p) enters into complex with other SNAREs in vitro and bypasses the need for Sec17p, Sec18p, and ATP. Thus, the sole essential function of vacuole priming in vitro is the release of Vam7p from cis-SNARE complexes. In 'bypass fusion', without ATP but with added rVam7p, there are sufficient unpaired vacuolar SNAREs Vam3p, Vti1p, and Nyv1p to interact with Vam7p and support fusion. However, active SNARE proteins are not sufficient for bypass fusion. rVam7p does not bypass requirements for Rho GTPases,Vps33p, Vps39p, Vps41p, calmodulin, specific lipids, or Vph1p, a subunit of the V-ATPase. With excess rVam7p, reduced levels of PI(3)P or functional Ypt7p suffice for bypass fusion. High concentrations of rVam7p allow the R-SNARE Ykt6p to substitute for Nyv1p for fusion; this functional redundancy among vacuole SNAREs may explain why nyv1delta strains lack the vacuole fragmentation seen with mutants in other fusion catalysts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.emboj.7600286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC514947PMC
July 2004

Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion.

J Cell Biol 2003 Feb;160(3):365-74

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

Vacuole tethering, docking, and fusion proteins assemble into a "vertex ring" around the apposed membranes of tethered vacuoles before catalyzing fusion. Inhibitors of the fusion reaction selectively interrupt protein assembly into the vertex ring, establishing a causal assembly hierarchy: (a) The Rab GTPase Ypt7p mediates vacuole tethering and forms the initial vertex ring, independent of t-SNAREs or actin; (b) F-actin disassembly and GTP-bound Ypt7p direct the localization of other fusion factors; (c) The t-SNAREs Vam3p and Vam7p regulate each other's vertex enrichment, but do not affect Ypt7p localization. The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked. Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS). In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy. Our findings provide a unique view of the functional relationships between GTPases, SNAREs, and actin in membrane fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200209095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172665PMC
February 2003
-->