Identification of a role for CLASP2 in insulin action.

Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. We present evidence that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin stimulation. Using mass spectrometry-based protein identification combined with phosphoantibody immunoprecipitation in L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the anti-phosphoserine immunoprecipitates upon insulin stimulation. Western blotting of CLASP2 immunoprecipitates with the phosphoantibody confirmed the finding that CLASP2 undergoes insulin-stimulated phosphorylation, and a number of novel phosphorylation sites were identified. Confocal imaging of L6 myotubes revealed that CLASP2 colocalizes with GLUT4 at the plasma membrane within areas of insulin-mediated cortical actin remodeling. CLASP2 is responsible for directing the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the concept that CLASP2 plays a role in the trafficking of GLUT4 at the cell periphery, CLASP2 knockdown by siRNA in L6 myotubes interfered with insulin-stimulated GLUT4 localization to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibited insulin-stimulated glucose transport. We therefore propose a new model for CLASP2 in insulin action, where CLASP2 directs the delivery of GLUT4 to cell cortex landing zones important for insulin action.

A semi-automated analysis method of small sensory nerve fibers in human skin-biopsies.

Computerized detection method (CDM) software programs have been extensively developed in the field of astronomy to process and analyze images from nearby bright stars to tiny galaxies at the edge of the Universe. These object-recognition algorithms have potentially broader applications, including the detection and quantification of cutaneous small sensory nerve fibers (SSNFs) found in the dermal and epidermal layers, and in the intervening basement membrane of a skin punch biopsy. Here, we report the use of astronomical software adapted as a semi-automated method to perform density measurements of SSNFs in skin-biopsies imaged by Laser Scanning Confocal Microscopy (LSCM). In the first half of the paper, we present a detailed description of how the CDM is applied to analyze the images of skin punch biopsies. We compare the CDM results to the visual classification results in the second half of the paper. Abbreviations used in the paper, description of each astronomical tools, and their basic settings and how-tos are described in the appendices. Comparison between the normalized CDM and the visual classification results on identical images demonstrates that the two density measurements are comparable. The CDM therefore can be used - at a relatively low cost - as a quick (a few hours for entire processing of a single biopsy with 8-10 scans) and reliable (high-repeatability with minimum user-dependence) method to determine the densities of SSNFs.