Smyth, J. | bioRxiv Science

bioRxiv2023

EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

RationaleCardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular volume. Ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs. ObjectiveDetermine the relationship between ventricular CV and structural changes to micro and nano-scale extracellular spaces. MethodsConduction and connexin43 (Cx43) gap junction protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with albumin, mannitol, dextran 70kDa, or dextran 2MDa. Peak sodium current was quantified from isolated guinea pig ventricular myocytes. Extracellular resistance (Re) was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. ResultsCV was significantly reduced by mannitol, and increased by albumin, dextran 70kDa and 2MDa. The combination of albumin and dextran 70kDa decreased CV relative to albumin alone. Re was reduced by mannitol, not significantly changed by albumin, and increased by both dextran 70kDa and dextran 2MDa. Cx43 gap junction expression and conductance, and peak sodium current were not significantly altered by the osmotic agents. The perinexal width in response to osmotic agents, in order of narrowest to widest, was: albumin with dextran 70kDa, albumin or dextran 2MDa alone, dextran 70kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. ConclusionsCardiac conduction does not correlate with bulk tissue impedance, but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume in ventricular myocardium is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

biophysics↗

bioRxiv2021

The oncogenic transcription factor FUS-CHOP can undergo nuclear liquid-liquid phase separation

Myxoid liposarcoma is caused by a chromosomal translocation resulting in a fusion protein comprised of the N-terminus of FUS (fused in sarcoma) and the full-length transcription factor CHOP (CCAAT/Enhancer Binding Protein Homologous Protein). FUS functions in RNA metabolism and CHOP is a stress-induced transcription factor. The FUS-CHOP fusion protein causes unique gene expression and oncogenic transformation. The FUS segment is required for oncogenic transformation, but the mechanism of FUS-CHOP-induced transcriptional activation is unknown. Recently, some transcription factors and super enhancers were proposed to undergo liquid-liquid phase separation and form membraneless compartments that recruit transcription machinery to gene promoters. Since phase separation of FUS depends on its N-terminus, transcriptional activation by FUS-CHOP could result from the N-terminus driving nuclear phase transitions. Here, we characterized FUS-CHOP in cells and in vitro, and observed novel phase-separating properties relative to unmodified CHOP. Our data indicate FUS-CHOP forms phase-separated condensates at super enhancer transcriptional sites. We provide strong evidence that the FUS-CHOP phase transition is a novel oncogenic mechanism and potential therapeutic target for treatment of myxoid liposarcoma.

cell biology↗