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Robby M Weimer

Publications and source records attributed to Robby M Weimer.

12 recordsLinked to original sources
PubMed2006

UNC-13 and UNC-10/rim localize synaptic vesicles to specific membrane domains.

Synaptic vesicles undergo a maturation step, termed priming, in which they become competent to fuse with the plasma membrane. To morphologically define the site of vesicle priming and identify fusion-competent synaptic vesicles, we combined a rapid physical-fixation technique with immunogold staining and high-resolution morphometric analysis at Caenorhabditis elegans neuromuscular junctions. In these presynaptic terminals, a subset of synaptic vesicles contact the plasma membrane within approximately 100 nm of a presynaptic dense projection. UNC-13, a protein required for vesicle priming, localizes to this same region of the plasma membrane. In an unc-13 null mutant, few synaptic vesicles contact the plasma membrane, suggesting that membrane-contacting synaptic vesicles represent the morphological correlates of primed vesicles. Interestingly, a subpopulation of membrane-contacting vesicles, located within 30 nm of a dense projection, are unperturbed in unc-13 mutants. We show that UNC-10/Rim, a protein implicated in presynaptic plasticity, localizes to dense projections and that loss of UNC-10/Rim causes an UNC-13-independent reduction in membrane-contacting synaptic vesicles within 30 nm of the dense projections. Our data together identify a discrete domain for vesicle priming within 100 nm of dense projections and further suggest that UNC-10/Rim and UNC-13 separately contribute to the membrane localization of synaptic vesicles within this domain.

Animals↗
PubMed2006

Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans.

Tetraspan vesicle membrane proteins (TVPs) comprise a major portion of synaptic vesicle proteins, yet their contribution to the synaptic vesicle cycle is poorly understood. TVPs are grouped in three mammalian gene families: physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In Caenorhabditis elegans, only a single member of each of these families exists. These three nematode TVPs colocalize to the same vesicular compartment when expressed in mammalian cells, suggesting that they could serve overlapping functions. To examine their function, C. elegans null mutants were isolated for each gene, and a triple mutant was generated. Surprisingly, these animals develop normally and exhibit normal neuronal architecture and synaptic contacts. In addition, functions of the motor and sensory systems are normal as determined by pharmacological, chemotaxis, and thermotaxis assays. Finally, direct electrophysiological analysis of the neuromuscular junction revealed no phenotype in the TVP mutants. We therefore conclude that TVPs are not needed for the basic neuronal machinery and instead may contribute to subtle higher order functions.

Animals↗
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↗
PubMed2006

Rapid redistribution of synaptic PSD-95 in the neocortex in vivo.

Most excitatory synapses terminate on dendritic spines. Spines vary in size, and their volumes are proportional to the area of the postsynaptic density (PSD) and synaptic strength. PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organizes the associated signaling complexes. PSD-95 is thought to determine the size and strength of synapses. Although spines and their synapses can persist for months in vivo, PSD-95 and other PSD proteins have shorter half-lives in vitro, on the order of hours. To probe the mechanisms underlying synapse stability, we measured the dynamics of synaptic PSD-95 clusters in vivo. Using two-photon microscopy, we imaged PSD-95 tagged with GFP in layer 2/3 dendrites in the developing (postnatal day 10-21) barrel cortex. A subset of PSD-95 clusters was stable for days. Using two-photon photoactivation of PSD-95 tagged with photoactivatable GFP (paGFP), we measured the time over which PSD-95 molecules were retained in individual spines. Synaptic PSD-95 turned over rapidly (median retention times tau(r) is approximately 22-63 min from P10-P21) and exchanged with PSD-95 in neighboring spines by diffusion. PSDs therefore share a dynamic pool of PSD-95. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. Furthermore, retention times increased with developmental age (tau(r) is approximately 100 min at postnatal day 70) and decreased dramatically following sensory deprivation. Our data suggest that individual PSDs compete for PSD-95 and that the kinetic interactions between PSD molecules and PSDs are tuned to regulate PSD size.

Animals↗
PubMed2006

Preservation of C. elegans tissue via high-pressure freezing and freeze-substitution for ultrastructural analysis and immunocytochemistry.

High-pressure freezing (HPF) is capable of converting liquid water, to a depth of approx 0.6 mm, into amorphous ice nearly instantaneously. At midbody, an adult Caenorhabditis elegans hermaphrodite approaches its widest girth of approx 0.1 mm. In theory, an entire living adult animal can be physically immobilized instantly in amorphous ice by HPF, thus, providing a unique opportunity to examine cellular architecture with exquisite spatial preservation. The following chapter will discuss, in detail, procedures for freezing C. elegans under high pressure, for embedding frozen samples in resin after a freeze-substitution step, and for the postembedding immunogold labeling of proteins contained within thin sections of embedded animals. These protocols enable high-resolution analysis of both morphological features and molecular domains within most tissues of C. elegans.

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PubMed2005

Diverse modes of axon elaboration in the developing neocortex.

The development of axonal arbors is a critical step in the establishment of precise neural circuits, but relatively little is known about the mechanisms of axonal elaboration in the neocortex. We used in vivo two-photon time-lapse microscopy to image axons in the neocortex of green fluorescent protein-transgenic mice over the first 3 wk of postnatal development. This period spans the elaboration of thalamocortical (TC) and Cajal-Retzius (CR) axons and cortical synaptogenesis. Layer 1 collaterals of TC and CR axons were imaged repeatedly over time scales ranging from minutes up to days, and their growth and pruning were analyzed. The structure and dynamics of TC and CR axons differed profoundly. Branches of TC axons terminated in small, bulbous growth cones, while CR axon branch tips had large growth cones with numerous long filopodia. TC axons grew rapidly in straight paths, with frequent interstitial branch additions, while CR axons grew more slowly along tortuous paths. For both types of axon, new branches appeared at interstitial sites along the axon shaft and did not involve growth cone splitting. Pruning occurred via retraction of small axon branches (tens of microns, at both CR and TC axons) or degeneration of large portions of the arbor (hundreds of microns, for TC axons only). The balance between growth and retraction favored overall growth, but only by a slight margin. Given the identical layer 1 territory upon which CR and TC axons grow, the differences in their structure and dynamics likely reflect distinct intrinsic growth programs for axons of long projection neurons versus local interneurons.

Animals↗
PubMed2005

Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans.

Active zones are presynaptic regions where synaptic vesicles fuse with plasma membrane to release neurotransmitters. Active zones are highly organized structurally and are functionally conserved among different species. Synapse defective-2 (SYD-2) family proteins regulate active zone morphology in Caenorhabditis elegans and Drosophila. Here, we demonstrate by immunoelectron microscopy that at C. elegans synapses, SYD-2 localizes strictly at active zones and can be used as an active zone marker when fused to green fluorescent protein (GFP). By driving expression of SYD-2::GFP fusion protein in GABAergic neurons, we are able to visualize discrete fluorescent puncta corresponding to active zones in living C. elegans. During development, the number of GABAergic synapses made by specific motoneurons increases only slightly from larvae to adult stages. In contrast, the number of SYD-2::GFP puncta doubles, suggesting that individual synapses accommodate the increasing size of their synaptic targets mainly by incorporating more active zone materials. Furthermore, we used this marker to perform a genetic screen to identify genes involved in the development of active zones. We recovered 16 mutants with altered SYD-2::GFP expression, including alleles of five genes that have been implicated previously in synapse formation or nervous-system development. Mapping of 11 additional mutants suggests that they may represent novel genes involved in active zone formation.

Animals↗
PubMed2004

Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing.

The location of a protein labeled by immunogold techniques can be resolved under an electron beam to within nanometers of its epitope, a resolution that makes immunoelectron microscopy a valuable tool for studies of cell biology. However, tissues in the nematode Caenorhabditis elegans are difficult to preserve for immunoelectron microscopic studies. The animal's cuticle slows the diffusion of solutions into the animal and thus makes it difficult to preserve both immunoreactivity and cell morphology. Here we describe a protocol that circumvents these problems. Specifically, we instantly immobilized tissue in vitreous ice by freezing living adult animals under high pressure. Frozen specimens were then chemically fixed, dehydrated, and embedded at low temperatures. As a result, chemical diffusion across the cuticle could occur over an extended period without morphological deterioration. We show that this method is capable of preserving both cell morphology, including fine structures, and immunoreactivity. Therefore, it provides a means to characterize the localization of endogenous proteins and exogenous proteins, such as the green fluorescent protein (GFP), with respect to subcellular compartments in C. elegans tissues by using postembedding immunogold labeling.

Actins↗
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↗
PubMed2003

The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces.

Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.

Actin Cytoskeleton↗