Fast structural responses of gap junction membrane domains to AB5 toxins

Abstract

Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast (∼500 ms) formation of connexin-depleted regions (CDRs) inside GJ plaques between cells exposed to AB5 toxins. CDR formation appears as a fast redistribution of connexin channels within GJ plaques with minor changes in outline or geometry. CDR formation does not depend on membrane trafficking or submembrane cytoskeleton and has no effect on GJ conductance. However, CDR responses depend on membrane lipids, can be modified by cholesterol-clustering agents and extracellular K + ion concentration, and influence cAMP signaling. The CDR response of GJ plaques to bacterial toxins is a phenomenon observed for all tested connexin isoforms. Through signaling, the CDR response may enable cells to sense exposure to AB5 toxins. CDR formation may reflect lipid-phase separation events in the biological membrane of the GJ plaque, leading to increased connexin packing and lipid reorganization. Our data demonstrate very fast dynamics (in the millisecond-to-second range) within GJ plaques, which previously were considered to be relatively stable, long-lived structures.membrane traffic | connexin actin | cell-cell junctions | cholesterol H ighly permeable biological membrane domains composed of assemblies of connexin channels form gap junction (GJ) plaques at cell-cell interfaces (1-3). The cell-cell connectivity provided by GJs helps individual cells integrate functionally into tissues via electrical coupling (4, 5) and diffusion of signaling molecules and metabolites (6, 7). GJs critically determine permeability to Ca 2+ , cAMP, cGMP, IP3, glutathione, and other metabolites smaller than 1.5 kDa (6,8,9). Formation of GJs requires vesicular transport and in some cases Golgi-dependent posttranslational modifications of connexins (10). GJ turnover involves endocytosis and proteasomal/lysosomal degradation (11)(12)(13)(14). During infection and under stress conditions the level of signaling molecules is elevated dramatically, and how signaling via GJs is modulated between affected cells remains unclear.High-speed live-cell microscopy revealed early responses of GJ plaques to purified AB5 toxins normally released by the human pathogens Vibrio cholerae, Shigella dysenteriae, and enterohemorrhagic Escherichia coli (EHEC). In this study we used both Bessel beam plane illumination microscopy (15) and spinning disk confocal microscopy. Because GJ responses are rapid, and because GJ plaques often are curved, the high-speed and near-isotropic 3D resolution (∼300 nm) of Bessel beam plane illumination microscopy was beneficial in revealing the responses in 4D spatiotemporal detail. (The experimental setup is shown in Fig. 1 A and B; also see SI Appendix, Fig. S1. For further details, see ref. 15.) To study GJ responses, we applied purified fluorescently ...

3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy

“…Nevertheless, it can still address important biological questions 18,19 when the side lobe background is not a concern. The TP mode (TP Bessel beam plane illumination) offers high-speed and near-isotropic 3D resolution, but with limited multicolor capability 20 . It is the mode of choice for deep imaging, owing to the better penetration of its IR excitation in scattering and/or aberrating tissues.…”

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

“…Based on these findings, it was proposed that Cx43 hemi-channels are inserted into the general plasma membrane and quickly diffuse to the edge of GJ plaque, before slowly diffusing into the plaque center. Subsequently, it was observed that Cx43 can be inserted directly at the plaque and that membrane fluidity exists within the GJ space (Falk et al, 2009; Shaw et al, 2007; Majoul et al, 2013). Plaques can also be internalized from regions away from the plaque center, and that full GJ plaque internalization can occur in a single step (Falk et al, 2009; Piehl et al, 2007).…”

Visualizing Whole-Brain Activity and Development at the Single-Cell Level Using Light-Sheet Microscopy

“…Advances in imaging speed, up to rates of hundreds of millions of volume elements per second, have enabled the design of light-sheet microscopes suitable for whole-brain functional imaging experiments at volumetric imaging rates that match the time scales of genetically encoded calcium indicators (Ahrens et al, 2013;Panier et al, 2013). Breakthroughs in spatial resolution-enabling up to 10-fold improvement in axial resolution and, therefore, isotropic spatial resolution on the order of 300 nm-have been demonstrated for single cells (Majoul et al, 2013;Planchon et al, 2011) and even small multicellular organisms (Gao et al, 2012;Wu et al, 2013). Advances in deep-tissue imaging and physical coverage of large biological specimens have been achieved through the introduction of multiphoton excitation in light-sheet microscopy (Truong et al, 2011) and through the implementation of light-sheet microscopes capable of imaging multiple complementary views of the sample simultaneously (Krzic et al, 2012;Schmid et al, 2013;Tomer et al, 2012).…”

“…Using live imaging approaches, light-sheet microscopes have been used to image dynamic processes over a wide range of spatial and temporal scales, from fast structural changes in the architecture of gap junctions (Majoul et al, 2013) and filopodial dynamics during axonal morphogenesis (Tomer et al, 2012) to whole-embryo imaging of C. elegans and D. melanogaster nervous system development with subcellular resolution (Tomer et al, 2012;Wu et al, 2011Wu et al, , 2013. In parallel to these efforts in the domain of developmental live imaging, several other groups developed strategies for structural wholebrain imaging, with a particular focus on rapid imaging of fixed and cleared mammalian brains (Dodt et al, 2007;Susaki et al, 2014;Tomer et al, 2014).…”

“…Using Bessel beam plane illumination microscopy with nearisotropic resolution in the 300 nm range, Majoul et al (2013) imaged the remodeling of proteins and lipids within gap junction plaques in response to bacterial AB5 toxin exposure. They achieved volumetric imaging speeds of one volume per 20 s and observed rapid rearrangements in connexin channels that lead to increased connexin packing and lipid reorganization.…”