Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana

Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure-activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants

Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens

A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi

Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake