Control of Autophagosome Axonal Retrograde Flux by Presynaptic Activity Unveiled Using Botulinum Neurotoxin Type A

Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity

Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts

Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

International audienceMyosin Va (MyoVa) is a prime candidate for controlling actin-based organelle motion in neurons and neuroendocrine cells. Its function in secretory granule (SG) trafficking was investigated in enterochromaffin cells by wide-field and total internal reflection fluorescence microscopy. The distribution of endogenous MyoVa partially overlapped with SGs and microtubules. Impairing MyoVa function by means of a truncated construct (MyoVa tail) or RNA interference prevented the formation of SG-rich regions at the cell periphery and reduced SG density in the subplasmalemmal region. Individual SG trajectories were tracked to analyze SG mobility. A wide distribution of their diffusion coefficient, Dxy, was observed. Almost immobile SGs (Dxy < 5 × 10−4 μm2 · s−1) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a Dxy below this threshold value for at least 2 s before fusion; (2) a negative autocorrelation of the vertical motion was found in subtrajectories with a Dxy below the threshold. Using this criterion of docking, we found that the main effect of MyoVa inhibition was to reduce the number of docked granules, leading to reduced secretory responses. Surprisingly, this reduction was not attributable to a decreased transport of SGs toward release sites. In contrast, MyoVa silencing reduced the occurrence of long-lasting, but not short-lasting, docking periods. We thus propose that, despite its known motor activity, MyoVa directly mediates stable attachment of SGs at the plasma membrane

Surface trafficking of neurotransmitter receptor: Comparison between single-molecule/quantum dot strategies

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C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles.

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers

Differential Nanoscale Topography and Functional Role of GluN2-NMDA Receptor Subtypes at Glutamatergic Synapses

NMDA receptors (NMDARs) play key roles in the use-dependent adaptation of glutamatergic synapses underpinning memory formation. In the forebrain, these plastic processes involve the varied contributions of GluN2A- and GluN2B-containing NMDARs that have different signaling properties. Although the molecular machinery of synaptic NMDAR trafficking has been under scrutiny, the postsynaptic spatial organization of these two receptor subtypes has remained elusive. Here, we used super-resolution imaging of NMDARs in rat hippocampal synapses to unveil the nanoscale topography of native GluN2A- and GluN2B-NMDARs. Both subtypes were found to be organized in separate nanodomains that vary over the course of development. Furthermore, GluN2A- and GluN2B-NMDAR nanoscale organizations relied on distinct regulatory mechanisms. Strikingly, the selective rearrangement of GluN2A- and GluN2B-NMDARs, with no overall change in NMDAR current amplitude, allowed bi-directional tuning of synaptic LTP. Thus, GluN2A- and GluN2B-NMDAR nanoscale organizations are differentially regulated and seem to involve distinct signaling complexes during synaptic adaptation

Fast live simultaneous multiwavelength four-dimensional optical microscopy

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4 Delta and bud6 Delta alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis