Biology subjects

Lukowicz-Bedford, R. M.

Publications and source records attributed to Lukowicz-Bedford, R. M..

4 recordsLinked to original sources

bioRxiv2024

Electrical synapse molecular diversity revealed by proximity-based proteomic discovery

Neuronal circuits are composed of synapses that are either chemical, where signals are transmitted via neurotransmitter release and reception, or electrical, where signals pass directly through interneuronal gap junction channels. While the molecular complexity that controls chemical synapse structure and function is well appreciated, the proteins of electrical synapses beyond the gap-junction-forming Connexins are not well defined. Yet, electrical synapses are expected to be molecularly complex beyond the gap junctions. Connexins are integral membrane proteins requiring vesicular transport and membrane insertion/retrieval to achieve function, homeostasis, and plasticity. Additionally, electron microscopy of neuronal gap junctions reveals neighboring electron dense regions termed the electrical synapse density (ESD). To reveal the molecular complexity of the electrical synapse proteome, we used proximity-dependent biotinylation (TurboID) linked to neural Connexins in zebrafish. Proteomic analysis of developing and mature nervous systems identifies hundreds of Connexin-associated proteins, with overlapping and distinct representation during development and adulthood. The identified protein classes span cell adhesion molecules, cytoplasmic scaffolds, vesicular trafficking, and proteins usually associated with the post synaptic density (PSD) of chemical synapses. Using circuits with stereotyped electrical and chemical synapses, we define molecular sub-synaptic compartments of ESD localizing proteins, we find molecular heterogeneity amongst electrical synapse populations, and we examine the synaptic intermingling of electrical and chemical synapse proteins. Taken together, these results reveal a new complexity of electrical synapse molecular diversity and highlight a novel overlap between chemical and electrical synapse proteomes. Moreover, human homologs of the electrical synapse proteins are associated with autism, epilepsy, and other neurological disorders, providing a novel framework towards understanding neuro-atypical states.

neuroscience↗

bioRxiv2023

Gap junction mediated bioelectric coordination is required for slow muscle development, organization, and function

Bioelectrical signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the Connexins. The connexin gene family is large and complex, presenting a challenge in identifying the specific Connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for appropriate slow muscle development and function. Through a combination of mutant analysis and in vivo imaging we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging we find that spinal cord generated neural activity is transmitted to developing slow muscle cells and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization, and that disruption leads to defects in behavior. Our work reveals the molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role played by GJ communication in coordinating bioelectric signaling during development.

developmental biology↗

bioRxiv2022

ZO1 scaffold isoforms differentially contribute to electrical synapse formation

Electrical synapses are neuronal gap junction (GJ) channels associated with a macromolecular complex called the electrical synapse density (ESD), which regulates development and dynamically modifies electrical transmission. However, the proteomic makeup and molecular mechanisms utilized by the ESD that direct electrical synapse formation are not well understood. Using the Mauthner cell of zebrafish as a model, we previously found that the intracellular scaffolding protein ZO1b is a member of the ESD, localizing postsynaptically, where it is required for GJ channel localization, electrical communication, neural network function, and behavior. Here, we show that the complexity of the ESD is further diversified by the genomic structure of the ZO1b gene locus. The ZO1b gene is alternatively initiated at three transcriptional start sites resulting in isoforms with unique N-termini that we call ZO1b-Alpha, -Beta, and - Gamma. We demonstrate that ZO1b-Beta and ZO1b-Gamma are broadly expressed throughout the nervous system and localize to electrical synapses. By contrast, ZO1b-Alpha is expressed mainly non-neuronally and is not found at synapses. We generate mutants in all individual isoforms, as well as double mutant combinations in cis on individual chromosomes, and find that ZO1b-Beta is necessary and sufficient for robust GJ channel localization. ZO1b-Gamma, despite its localization to the synapse, plays an auxiliary role in channel localization. This study expands the notion of molecular complexity at the ESD, revealing that an individual genomic locus can contribute distinct isoforms to the macromolecular complex at electrical synapses. Further, independent scaffold isoforms have differential contributions to developmental assembly of the interneuronal GJ channels. We propose that ESD molecular complexity arises both from the diversity of unique genes and from distinct isoforms encoded by single genes. Overall, ESD proteomic diversity is expected to have critical impacts on the development, structure, function, and plasticity of electrical transmission.

neuroscience↗

bioRxiv2021

Connexinplexity: The spatial and temporal expression of connexin genes during vertebrate organogenesis

Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is well appreciated in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we probed the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step towards understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.

developmental biology↗