Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice [version 2; referees: 2 approved]

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and use visual scoring for immunocytochemistry staining to determine the extent of the antibodies’ specificity. Thus, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts

Characterization of the livestock production system and potential for enhancing productivity through improved feeding in Amoni Division, Mweiga District, Central Kenya, May 2010

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M^–1 cm^–1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication

Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform

The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily

Post-synaptic Release of the Neuronal Tissue-Type Plasminogen Activator (tPA)

The neuronal serine protease tissue-type Plasminogen Activator (tPA) is an important player of the neuronal survival and of the synaptic plasticity. Thus, a better understanding the mechanisms regulating the neuronal trafficking of tPA is required to further understand how tPA can influence brain functions. Using confocal imaging including living cells and high-resolution cell imaging combined with an innovating labeling of tPA, we demonstrate that the neuronal tPA is contained in endosomal vesicles positives for Rabs and in exosomal vesicles positives for synaptobrevin-2 (VAMP2) in dendrites and axons. tPA-containing vesicles differ in their dynamics with the dendritic tPA containing-vesicles less mobile than the axonal tPA-containing vesicles, these laters displaying mainly a retrograde trafficking. Interestingly spontaneous exocytosis of tPA containing-vesicles occurs largely in dendrites