Biology subjects

Colon-Ramos, D.

Publications and source records attributed to Colon-Ramos, D..

27 records · Page 2Linked to original sources

bioRxiv2021

The active zone protein Clarinet regulates ATG-9 trafficking at synapses and presynaptic autophagy

In neurons, autophagy is temporally and spatially regulated to occur near presynaptic sites. How trafficking of autophagy proteins is regulated to support synaptic autophagy is not well understood. From forward genetic screens, we identify a role for the long isoform of the active zone protein Clarinet (CLA-1L) in regulating trafficking of autophagy protein ATG-9 at synapses, and presynaptic autophagy. ATG-9 is a transmembrane protein that undergoes activity-dependent exo-endocytosis at synapses, and mutations in CLA-1L result in abnormal accumulation of ATG-9 into clathrin-rich endocytic intermediates. CLA-1L extends from the active zone to the periactive zone, and genetically interacts with periactive zone proteins required for clathrin-dependent endocytosis. We find that CLA-1L is specifically required for sorting of ATG-9 at synapses, likely via endosome-mediated endocytosis, and for activity-dependent presynaptic autophagy. Our findings provide mechanistic insights into how active zone proteins regulate key steps of ATG-9 exo-endocytosis, a process that could couple the activity state of the neuron and autophagy. HighlightsO_LI* The long isoform of the active zone protein Clarinet (CLA-1L) regulates ATG-9 trafficking at synapses C_LIO_LI* CLA-1L extends from the active zone to the periactive zone and cooperates with the periactive zone endocytic proteins EHS-1/EPS15 and ITSN-1/ intersectin 1 in ATG-9 trafficking during exo-endocytosis C_LIO_LI* Mutations in CLA-1L, or in clathrin-associated adaptor molecules, result in abnormal accumulation of ATG-9 into clathrin-rich endocytic intermediates C_LIO_LI* CLA-1L mutants which affect ATG-9 trafficking are also defective in activity-dependent presynaptic autophagy C_LI

neuroscience↗

bioRxiv2020

Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9

Autophagy is a cellular degradation pathway essential for neuronal health and function. Autophagosome biogenesis occurs at synapses, is locally regulated and increases in response to neuronal activity. The mechanisms that couple autophagosome biogenesis to synaptic activity remain unknown. In this study we determine that trafficking of ATG-9, the only transmembrane protein in the core autophagy pathway, links the synaptic vesicle cycle with autophagy. ATG-9 positive vesicles in C. elegans are generated from the trans-Golgi network via AP3-dependent budding, and delivered to presynaptic sites. At presynaptic sites, ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt endocytosis, including one associated with Parkinsons disease, result in abnormal ATG-9 accumulation at clathrin-rich synaptic foci and defects in activity-dependent presynaptic autophagy. Our findings uncover regulated key steps of ATG-9 trafficking at presynaptic sites, and provide evidence that ATG-9 exo-endocytosis couples autophagosome biogenesis at presynaptic sites with the activity-dependent synaptic vesicle cycle. HighlightsO_LIIn C. elegans, ATG-9 is delivered to presynaptic sites in vesicles generated from the trans-Golgi network via AP-3-dependent budding C_LIO_LIATG-9 vesicles undergo activity-dependent exo-endocytosis at presynaptic sites C_LIO_LIMutations in endocytic proteins, including a mutation associated with Parkinsons disease, result in abnormal ATG-9 accumulation at clathrin-rich foci C_LIO_LIAbnormal accumulation of ATG-9 at clathrin-rich foci is associated with defects in activity-dependent presynaptic autophagy C_LI

neuroscience↗

bioRxiv2020

A neurite-zippering mechanism, mediated by layer-specific expression of IgCAMs, regulates synaptic laminar specificity in the C. elegans nerve ring neuropil

During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil, and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally-regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.

neuroscience↗

bioRxiv2020

HySyn: A genetically-encoded synthetic synapse to rewire neural circuits in vivo

Here we introduce HySyn, a system designed to rewire neural connectivity in vivo by reconstituting a functional heterologous synapse. We demonstrate that genetically targeted expression of the two HySyn components, a Hydra-derived neuropeptide and its receptor, creates de novo neuromodulatory transmission in a mammalian neuronal tissue culture model and rewires a behavioral circuit in vivo in the nematode Caenorhabditis elegans. HySyn can interface with existing optogenetic, chemogenetic and pharmacological approaches to functionally probe synaptic transmission, dissect neuropeptide signaling, or modulate specific neural circuits.

neuroscience↗

bioRxiv2019

Accelerating iterative deconvolution and multiview fusion by orders of magnitude

We describe theoretical and practical advances in algorithm and software design, resulting in ten to several thousand-fold faster deconvolution and multiview fusion than previous methods. First, we adapt methods from medical imaging, showing that an unmatched back projector accelerates Richardson-Lucy deconvolution by at least 10-fold, in most cases requiring only a single iteration. Second, we show that improvements in 3D image-based registration with GPU processing result in speedups of 10-100-fold over CPU processing. Third, we show that deep learning can provide further accelerations, particularly for deconvolution with a spatially varying point spread function. We illustrate the power of our methods from the subcellular to millimeter spatial scale, on diverse samples including single cells, nematode and zebrafish embryos, and cleared mouse tissue. Finally, we show that our methods facilitate the use of new microscopes that improve spatial resolution, including dual-view cleared tissue light-sheet microscopy and reflective lattice light-sheet microscopy.

bioengineering↗

bioRxiv2019

The Glycolytic Protein Phosphofructokinase Dynamically Relocalizes into Subcellular Compartments with Liquid-like Properties in vivo

While much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. Here we built a hybrid microfluidic-hydrogel device for use in Caenorhabditis elegans to systematically examine and quantify the dynamic subcellular localization of the rate-limiting enzyme of glycolysis, phosphofructokinase-1/PFK-1.1. We determine that endogenous PFK-1.1 localizes to distinct, tissue-specific subcellular compartments in vivo. In neurons, PFK-1.1 is diffusely localized in the cytosol, but capable of dynamically forming phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in the dispersion of PFK-1.1 in the cytosol, indicating that PFK-1.1 reversibly organizes into biomolecular condensates in response to cues within the cellular environment. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Prolonged conditions of energy stress during sustained hypoxia alter the biophysical properties of PFK-1.1 in vivo, affecting its viscosity and mobility within phase-separated condensates. PFK-1.1s ability to form tetramers is critical for its capacity to form condensates in vivo, and heterologous self-association domain such as cryptochrome 2 (CRY2) is sufficient to constitutively induce the formation of PFK-1.1 condensates. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically compartmentalize in vivo to specific subcellular compartments in response to acute energy stress via multivalency as phase-separated condensates.

cell biology↗

bioRxiv2018

Retrograde Transport and ATG-4.2-Mediated Maturation Cooperate to Remove Autophagosomes from the Synapse

Autophagy is spatially compartmentalized in neurons, with autophagosome biogenesis occurring in the axon and degradation in the cell body. The mechanisms that coordinate autophagosome formation, trafficking and degradation across the polarized structure of the neuron are not well understood. Here we use genetic screens and in vivo imaging in single neurons of C. elegans to demonstrate that specific steps of autophagy are differentially required in distinct subcellular compartments of the neuron. We demonstrate that completion of autophagosome biogenesis and closure at the synapse are necessary for dynein-mediated retrograde transport. We uncover a role for UNC-16/JIP3/Sunday Driver in facilitating autophagosome retrograde transport. Through forward genetic screens we then determine that autophagosome maturation and degradation in the cell body depend on removal of LGG-1/Atg8/GABARAP from autophagosomes by the protease ATG-4.2. Our studies reveal that regulation of distinct ATG4 proteases contributes to the coordination of autophagy across subcellular regions of the neuron.\n\nHIGHLIGHTS and eTOC BlurbO_LIAutophagosome closure, but not maturation, occurs locally at presynaptic sites\nC_LIO_LIRetrograde transport of autophagosomes requires the motor adaptor UNC-16/JIP3\nC_LIO_LIThe autophagy protease ATG-4.2, but not the related ATG-4.1, is required for autophagosome maturation and degradation\nC_LIO_LIDefects in retrograde transport and maturation genetically interact and enhance accumulation of autophagosomes in presynaptic regions\nC_LI

cell biology↗

bioRxiv2017

Integration of plasticity mechanisms within a single sensory neuron of C. elegans actuates a memory

Neural plasticity--the ability of a neuron to change its cellular properties in response to past experiences--underpins the nervous systems capacity to form memories and actuate behaviors. How different plasticity mechanisms act together in vivo and at a cellular level to transform sensory information into behavior is not well understood. Here we show that in the nematode C. elegans two plasticity mechanisms--sensory adaptation and presynaptic plasticity--act within a single cell to encode thermosensory information and actuate a temperature-preference memory. Sensory adaptation enables the primary thermosensory neuron, AFD, to adjust the temperature range of its sensitivity to the local environment, thereby optimizing its ability to detect temperature fluctuations associated with migration. Presynaptic plasticity transforms this thermosensory information into a behavioral preference by gating synaptic communication between sensory neuron AFD and its postsynaptic partner, AIY. The gating of synaptic communication is regulated at AFD presynaptic sites by the conserved kinase nPKC{varepsilon}. Bypassing or altering AFD presynaptic plasticity predictably changes the learned behavioral preferences without affecting sensory responses. Our findings indicate that two distinct and modular neuroplasticity mechanisms function together within a single sensory neuron to encode multiple components of information required to enact thermotactic behavior. The integration of these plasticity mechanisms result in a single-cell logic system that can both represent sensory stimuli and guide memory-based behavioral preference.

neuroscience↗

bioRxiv2017

Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release

Active zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens we isolated a novel C. elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, reduced spontaneous neurotransmitter release, increased synaptic depression and reduced synapse number. Ultrastructurally, cla-1 mutants have fewer synaptic vesicles adjacent to the dense projection and an increased number of docked vesicles. Cla-1 encodes 3 isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The short isoform localizes exclusively to the active zone while a longer ~9000 amino acid isoform colocalizes with synaptic vesicles. Specific loss of CLA-1L results in synaptic vesicle clustering defects and increased synaptic depression, but not in reduced synapse number or mini frequency. Together our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, synaptic vesicle clustering and release.

neuroscience↗