The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.

Key points• Hypertonicity-induced cation channels (HICCs) are key players in proliferation and apoptosis but their molecular identity has remained elusive.• We report that in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as activators of TRPM2 cation channels, elicited currents that are identical to the osmotic activation of HICCs.• siRNA-mediated silencing of the expression of TRPM2 and CD38 (as the supposed source of ADPr and cADPr) inhibited HICC and nucleotide-induced currents and the osmotic volume response of cells.• Quantification of intracellular cADPr and extracellular application of nucleotides revealed that the outwardly directed gradient rather than the intracellular activity of ADPr and cADPr is the triggering factor for TRPM2.• Cloning of TRPM2 identified the C-splice variant as the molecular correlate of the HICC, which was supported by quantification of Ca 2+ selectivity.• Immunoprecipation and FRET/FLIM assays revealed the interaction of TRPM2 and CD38 and we propose a transport-related nucleotide export via CD38 as a novel mechanism of TRPM2 activation.Abstract Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Furthermore, the activation profile of HICCs is not well defined yet. We report here that, in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as supposed activators of TRPM2, elicited cation currents that were virtually identical to the osmotic activation of HICCs. Silencing of the expression of TRPM2 and of the ecto-enzyme CD38 (as a likely source of ADPr and cADPr) inhibited HICC as well as nucleotide-induced currents and, in parallel, the hypertonic volume response of cells (the regulatory volume increase, RVI) was attenuated. Quantification of intracellular cADPr levels and the systematic application of extra-vs. intracellular nucleotides indicate that the outwardly directed gradient rather than the cellular activity of ADPr and cADPr triggers TRPM2 activation, probably along with a simultaneous biotransformation of nucleotides. Cloning of TRPM2 identified the C-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca 2+ selectivity. Finally, immunoprecipitation and high-resolution FRET/FLIM imaging revealed the interaction of TRPM2 and K. Sato and J. Christmann contributed equally to this work. CD38 in the native as well as in a heterologous (HEK293T) expression system. We propose transport-related nucleotide export via CD38 as a novel mechanism of TRPM2/HICC activation. With the biotransformation of nucleotides running in parallel, continuous zero trans-conditions are achieved which will render the system infinitely sensitive. (

Cytokinesis is the final step of cell division. Increasing evidence suggests failure of cytokinesis might contribute to the development of cancer. Here, we demonstrate that the serologically defined colon cancer antigen-3 (SDCCAG3) forms a complex with PTPN13, a protein tyrosine phosphatase known to be involved in the regulation of cytokinesis, carcinogenesis and tumor aggressiveness. We show that SDCCAG3 is a novel endosomal protein, primarily localized at the early/recycling endosomal compartment. SDCCAG3 undergoes dynamic localization during cell division with strong accumulation at the midbody during cytokinesis. Overexpression as well as downregulation correlates with the generation of multinucleate cells. Furthermore, we show interaction of SDCCAG3 with the Arf GTPase activating protein GIT1 (G protein-coupled receptor kinase interactor-1). Overexpression of an ArfGAP-negative version of GIT1 also results in an increased number of multinucleate cells suggesting regulation of Arf-mediated vesicular trafficking or signaling via SDCCAG3. Finally, we demonstrate that SDCCAG3 expression levels are elevated in colon cancers. In summary, we have established SDCCAG3 as a novel endosomal protein, which is involved in the regulation of cytokinesis.