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CAV1 (caveolin 1) expression and secretion is associated with prostate cancer (PCa) disease progression, but the mechanisms underpinning CAV1 release remain poorly understood. Numerous studies have shown CAV1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) CAV1 topology. Here we show that CAV1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive CAV1 secretion. Characterization of the isolated vesicles by electron microscopy, single-molecule fluorescence microscopy and proteomics reveals they represent a novel class of exosomes ~40 nm in diameter containing ~50-60 copies of CAV1 and, strikingly, are released via a non-canonical secretory macroautophagy/autophagy pathway. This study provides novel insights into a mechanism whereby CAV1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression. Abbreviations: 3-MA: 3-methyladenine; APEX: a modified soybean ascorbate peroxidase; ATG5: autophagy related 5; ATG9A: autophagy related 9A; ATG12: autophagy related 12; BHK: baby hamster kidney; C-exosomes: caveolin-exosomes; CAMKK2/CAMKKβ: calckum/calmodulin dependent protein kinase kinase 2; CAV1: caveolin 1; DAB: 3,3′-diaminobenzidine; DAPK: death associated protein kinase; EEA1: early endosome antigen 1; EM: electron microscopy; FCS: fluorescence correlation spectroscopy; GBP: GFP/YFP-binding peptide; GFP: green fluorescent protein; GOLGA2: golgin A2; ILVs: intralumenal vesicles; LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; MTORC1: mechanistic target of rapamycin kinase complex 1; MVBs: multivesicular bodies; PBS: phosphate-buffered saline; PCa: prostate cancer; PI3K: phosphoinositide 3-kinase; PM: plasma membrane; SFM: serum-free medium; TSG101: tumor susceptibility 101; WCL: whole cell lysates; WT: wild type; YFP: yellow fluorescent protein; βoG: β-octylglucoside

AGU Fall Meeting 2019 in San Francisco , 9-13 December 2019, EIS/InSIght team, InSight is the first planetary mission with a seismometer package, SEIS, since the Apollo Lunar Surface Experiments Package. SEIS is complimented by APSS, which has as a goal to document the atmospheric source of seismic noise and signals. Since June 2019, SEIS has been delivering 6 axis 20 sps continuous seismic data, a rate one order of magnitude larger originally planned. More than 50 events have been detected by the end of July 2019 but only three have amplitudes significantly above the SEIS instrument requirement. Two have clear and coherent arrivals of P and S waves, enabling location, diffusion/attenuation characterization and receiver function analysis. The event¿s magnitudes are likely ¿ 3 and no clear surface waves nor deep interior phases have been identified. This suggests deep events with scattering along their final propagation paths and with large propagation differences as compared to Earth and Moon quakes. Most of the event¿s detections are made possible due to the very low noise achieved by the instrument installation strategy and the very low VBB self-noise. Most of the SEIS signals have amplitudes of spectral densities in the 0.03-5Hz frequency bandwidth ranging from 10-10 m/s2/Hz1/2 to 5 10-9 m/s2/Hz1/2. The smallest noise levels occurs during the early night, with angstrom displacements or nano-radian tilts. This monitors the elastic and seismic interaction of a planetary surface with its atmosphere, illustrated not only by a wide range of SEIS signals correlated with pressure vortexes, dust devils or wind activity but also by modulation of resonances above 1 Hz, amplified by ultra-low velocity surface layers. After about one half of a Martian year, clear seasonal changes appear also in the noise, which will be discussed. One year after landing, the seismic noise is therefore better and better understood, and noise correction techniques begun to be implemented, either thanks to the APSS wind and pressure sensors, or by SEIS only data processing techniques. These data processing techniques open not only the possibility of better signal to noise ratio of the events, but are also used for various noise auto-correlation techniques as well as searches of long period signals. Noise and seismic signals on Mars are therefore completely different from what seismology encountered previously on Earth and Moon.

SDS-PAGE of reduced G. tridactyla venom. Figure S2. Genomic organization of the glycerotoxin gene analyzed in G. tridactyla. Figure S3. Immunolocalization of GLTx in a cross section through a putative venom gland embedded in paraffin. Figure S4. Anti-serotonin (5-HT) staining and phalloidinâ rhodamine counterstaining on everted G. tridactyla pharynges cut into two halves. Figure S5. Quantitative real-time PCR (qPCR) expression levels (shown as Fold Change, RQ) between biological groups (putative venom glands [A], pharyngeal lobes [B], and posterior body wall [C]) per analyzed specimen (nâ =â 10, biological samples). Figure S6. Tissues analyzed in comparative GLTx expression studies (qPCR experiments and transcriptome analyses) on G. tridactyla. (PDF 2931 kb)

Protocol S1â S3. Protocol S1. Protocol: In situ hybridization on pharynx tissue of G. tridactyla (Glyceridae, Annelida). Protocol S2. Protocol: Anti-GLTx staining on pharynx tissue of G. tridactyla (Glyceridae, Annelida). Protocol S3. Protocol: Fluorescence in situ hybridization (FISH) coupled with antibody staining against GLTx on pharynx tissue of G. tridactyla. (PDF 590 kb)

SDS-PAGE of reduced G. tridactyla venom. Figure S2. Genomic organization of the glycerotoxin gene analyzed in G. tridactyla. Figure S3. Immunolocalization of GLTx in a cross section through a putative venom gland embedded in paraffin. Figure S4. Anti-serotonin (5-HT) staining and phalloidinâ rhodamine counterstaining on everted G. tridactyla pharynges cut into two halves. Figure S5. Quantitative real-time PCR (qPCR) expression levels (shown as Fold Change, RQ) between biological groups (putative venom glands [A], pharyngeal lobes [B], and posterior body wall [C]) per analyzed specimen (nâ =â 10, biological samples). Figure S6. Tissues analyzed in comparative GLTx expression studies (qPCR experiments and transcriptome analyses) on G. tridactyla. (PDF 2931 kb)

Kasula et al. use single-molecule imaging to reveal the diffusional signature for the SNARE proteins Munc18-1 and syntaxin-1A during secretory vesicle priming. The authors show that a conformational change in the Munc18-1 domain 3a hinge-loop regulates engagement of syntaxin-1A in the SNARE complex., Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.

In neurosecretory cells, secretory vesicles (SVs) undergo Ca2(+)-dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking. Refereed/Peer-reviewed