Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity.

The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

SDmixer-a versatile software tool for spectral demixing of multicolor single molecule localization data.

Spectral demixing (SD) offers multicolor single molecule localization microscopy (SMLM) with low crosstalk and without the need to correct for registration errors. Here, we present SDmixer, a versatile, open-source software tool that enables any laboratory to perform rapid SD-based multicolor SMLM. A graphic user interface allows non-experts to process 2D or 3D data sets from any SML software and to reconstruct the super-resolved multicolor images with flexible output options.

Spectral demixing avoids registration errors and reduces noise in multicolor localization-based super-resolution microscopy.

Multicolor single molecule localization-based super-resolution microscopy (SMLM) approaches are challenged by channel crosstalk and errors in multi-channel registration. We recently introduced a spectral demixing-based variant of direct stochastic optical reconstruction microscopy (SD-dSTORM) to perform multicolor SMLM with minimal color crosstalk. Here, we demonstrate that the spectral demixing procedure is inherently free of errors in multicolor registration and therefore does not require multicolor channel alignment. Furthermore, spectral demixing significantly reduces single molecule noise and is applicable to astigmatism-based 3D multicolor imaging achieving 25 nm lateral and 66 nm axial resolution on cellular nanostructures.

A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling.

Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.

The specific monomer/dimer equilibrium of the corticotropin-releasing factor receptor type 1 is established in the endoplasmic reticulum.

G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.

Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.

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.

Multi-colour direct STORM with red emitting carbocyanines.

BACKGROUND INFORMATION: Single molecule-based super-resolution methods have become important tools to study nanoscale structures in cell biology. However, the complexity of multi-colour applications has prevented them from being widely used amongst biologists. Direct stochastic optical reconstruction microscopy (dSTORM) offers a simple way to perform single molecule super-resolution imaging without the need for an activator fluorophore and compatible with many conventionally used fluorophores. The search for the ideal dye pairs suitable for dual-colour dSTORM has been compromised by the fact that fluorophores spectrally apt for dual-colour imaging differ with respect to the optimal buffer conditions required for photoswitching and the generation of prolonged non-fluorescent (OFF) states. RESULTS: We present a novel variant of dSTORM that combines advantages of spectral demixing with the buffer compatible blinking properties of red emitting carbocyanine dyes, spectral demixing dSTORM (SD-dSTORM). In contrast to previously published work, SD-dSTORM requires reduced laser power and fewer imaging frames for the faithful reconstruction of super-resolved biological nanostructures. In addition, SD-dSTORM allows the use of commercially available rather than custom-made probes and does not rely on potentially error-prone cross-talk correction, thus allowing reliable co-localisation. CONCLUSIONS: SD-dSTORM presents a significant advance towards user-friendly single molecule localisation-based super-resolution microscopy combining advantages of state-of-the-art methodologies to perform fast, reliable and efficient multi-colour dSTORM.