Biology subjects

Schwartz, M. L.

Publications and source records attributed to Schwartz, M. L..

3 recordsLinked to original sources

bioRxiv2025

A Toolkit for In Vivo Mapping and Modulating Neurotransmission at Single-Cell Resolution

Understanding the organization and regulation of neurotransmission at the level of individual neurons and synapses requires tools that can track and manipulate transmitter-specific vesicles in vivo. Here, we present SynaptoTagMe, a suite of genetic tools in Caenorhabditis elegans to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the C. elegans nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use in vivo. The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication in vivo.

neuroscience↗

bioRxiv2021

High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple GFP insertions, 60 - 100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits. Author SummaryGermlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.

genetics↗

bioRxiv2017

Sec17 (α-SNAP) And An SM-Tethering Complex Control The Outcome Of SNARE Zippering In Vitro And In Vivo

Zippering of SNARE complexes spanning docked membranes is essential for most intracellular fusion events. Here we explore how SNARE regulators operate on discrete zippering states. The formation of a metastable trans-complex, catalyzed by HOPS and its SM subunit Vps33, is followed by subsequent zippering transitions that increase the probability of fusion. Operating independently of Sec18 catalysis, Sec17 either inhibits or stimulates SNARE-mediated fusion. If HOPS or Vps33 are absent, Sec17 inhibits fusion at an early stage. Thus, HOPS and Vps33 accelerate SNARE zippering, particularly in the presence of otherwise inhibitory Sec17. Once SNAREs are partially-zipped, Sec17 promotes fusion in either the presence or absence of HOPS -- but with faster kinetics when HOPS is absent. Our data further indicate that Sec17 promotes fusion both through its direct penetration of the membrane and by enhancing C-terminal SNARE zippering. In a working model, the interplay among Sec17, Sec18, SMs, and SNARE zippering can explain why SM proteins are indispensable for SNARE-mediated fusion in vivo.\n\nImpact statementSec17 is shown to have divergent effects on pre-fusion SNARE complex activity, depending on the state of SNARE zippering. HOPS, an SM-tether complex, controls the outcome of Sec17-SNARE engagement. The results suggest a coherent working model for SM activity in vivo.

biochemistry↗