The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.

Several lines of evidence suggest that cyclic GMP might be involved in long-term potentiation (LTP) in the hippocampus. Arachidonic acid, nitric oxide and carbon monoxide, three molecules that have been proposed to act as retrograde messengers in LTP, all activate soluble guanylyl cyclase. We report here that an inhibitor of guanylyl cyclase blocks the induction of LTP in the CA1 region of hippocampal slices. Conversely, cGMP analogues produce long-lasting enhancement of the excitatory postsynaptic potential if they are applied at the same time as weak tetanic stimulation of the presynaptic fibres. The enhancement is spatially restricted, is not blocked by valeric acid (APV), nifedipine, or picrotoxin, and partially occludes LTP. This synaptic enhancement may be mediated by the cGMP-dependent protein kinase (PKG). Inhibitors of PKG block the induction of LTP, and activators of PKG produce activity-dependent long-lasting enhancement. These results suggest that guanylyl cyclase and PKG contribute to LTP, possibly as activity-dependent presynaptic effectors of retrograde messengers.

The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.

Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site‐specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans‐cyclooctene units that react with tetrazine derivatives in an inverse‐electron‐demand Diels–Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide–cyclooctyne click reaction (right).