Vertebrate gap junctions, composed of proteins from the connexin gene family, play critical roles in embryonic development, co-ordinated contraction of excitable cells, tissue homoeostasis, normal cell growth and differentiation. Phosphorylation of connexin43, the most abundant and ubiquitously expressed connexin, has been implicated in the regulation of gap junctional communication at several stages of the connexin ‘life cycle’, including hemichannel oligomerization, export of the protein to the plasma membrane, hemichannel activity, gap junction assembly, gap junction channel gating and connexin degradation. Consistent with a short (1–5 h) protein half-life, connexin43 phosphorylation is dynamic and changes in response to activation of many different kinases. The present review assesses our current understanding of the effects of phosphorylation on connexin43 structure and function that in turn regulate gap junction biology, with an emphasis on events occurring in heart and skin.

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell–cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with ³²P_i yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43^−/− mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of ∼50-pS channel events and a concomitant loss of ∼100-pS channel events. This change to ∼50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

Localizing gene expression: Single-cell RNA sequencing can provide information about cellular relationships based on shared transcriptomes, but most methods lose spatial information. Those methods that do retain spatial information can be limited to a specific set of genes and/or a small area. Srivatsan et al. introduce sci-Space, a spatial transcriptomic method that uses a grid of barcoded oligos on a slide that can be transferred to nuclei of an overlaid frozen tissue section to obtain both the spatial origin and the transcriptome of thousands of single cells per slide. The researchers used sci-Space to create a spatial atlas of mouse E14 sagittal sections, revealing spatially expressed genes across cell types. This application illustrates how sci-Space complements existing approaches in spatial genomics. Science , abb9536, this issue p. 111

Luteinizing hormone (LH) acts on ovarian follicles to reinitiate meiosis in prophase-arrested mammalian oocytes, and this has been proposed to occur by interruption of a meioisis-inhibitory signal that is transmitted through gap junctions into the oocyte from the somatic cells that surround it. To investigate this idea, we microinjected fluorescent tracers into live antral follicle-enclosed mouse oocytes, and we demonstrate for the first time that LH causes a decrease in the gap junction permeability between the somatic cells,prior to nuclear envelope breakdown (NEBD). The decreased permeability results from the MAP kinase-dependent phosphorylation of connexin 43 on serines 255,262 and 279/282. We then tested whether the inhibition of gap junction communication was sufficient and necessary for the reinitiation of meiosis. Inhibitors that reduced gap junction permeability caused NEBD, but an inhibitor of MAP kinase activation that blocked gap junction closure in response to LH did not prevent NEBD. Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH.