SYP72 interacts with the mechanosensitive channel MSL8 to protect pollen from hypoosmotic shock during hydration

In flowering plants, hydration of desiccated pollen grains on stigma is a prerequisite for pollen germination, during which pollen increase markedly in volume through water uptake, requiring them to survive hypoosmotic shock to maintain cellular integrity. However, the mechanisms behind the adaptation of pollen to this hypoosmotic challenge are largely unknown. Here, we identify the Qc-SNARE protein SYP72, which is specifically expressed in male gametophytes, as a critical regulator of pollen survival upon hypoosmotic shock during hydration. SYP72 interacts with the MSCS-LIKE 8 (MSL8) and is required for its localization to the plasma membrane. Intraspecies and interspecies genetic complementation experiments reveal that SYP72 paralogs and orthologs from green algae to angiosperms display conserved molecular functions and rescue the defects of Arabidopsis syp72 mutant pollen facing hypoosmotic shock following hydration. Our findings demonstrate a critical role for SYP72 in pollen resistance to hypoosmotic shock through the MSL8 cascade during pollen hydration.

Syntaxin 12 and COMMD3 are new factors that function with VPS33B in the biogenesis of platelet α-granules

Platelet a-granules regulate hemostasis and myriad other physiological processes but their biogenesis is unclear. Mutations in only three proteins are known to cause a-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized a-granule proteins through megakaryocyte endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Stx12, a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12 deficient megakaryocytes display reduced a-granule numbers and overall levels of a-granule proteins, thus revealing Stx12 as new component of the a-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in megakaryocytes contains COMMD3, one of ten COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced a-granule numbers and overall levels of a-granule proteins, establishing the COMMD3/CCC complex as a new factor in a-granule biogenesis. Furthermore, P-Selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-Selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in a-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of a-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in megakaryocytes to make a-granules.

Prefused lysosomes cluster on autophagosomes regulated by VAMP8

Lysosome–autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes—vesicle-associated membrane protein 8 (VAMP8)—plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

The EXO70B2 exocyst subunit contributes to papillae and encasement formation in anti-fungal defence in Arabidopsis

In the reaction to non-adapted Blumeria graminis f. sp. hordei (Bg), Arabidopsis thaliana leaf epidermal cells deposit cell wall reinforcements called papillae or seal fungal haustoria in encasements, both of which involve intensive exocytosis. A plant syntaxin SYP121/PEN1 has been found to be of key importance for the timely formation of papillae, and the vesicle tethering complex exocyst subunit EXO70B2 has been found to contribute to their morphology. Here, we identify a specific role for the EXO70B2-containing exocyst complex in the papillae membrane domains important for the callose deposition and GFP-SYP121 delivery to the focal attack sites, as well as its contribution to encasement formation. The mRuby2-EXO70B2 co-localises with the exocyst core subunit SEC6 and GFP-SYP121 in the membrane domain of papillae, and both proteins have the capacity to directly interact. The exo70B2/syp121 double mutant has a reduced number of papillae and haustorial encasements in response to Bg, indicating an additive role of the exocyst in SYP121 coordinated non-host resistance. In summary, we report cooperation between the plant exocyst and a SNARE protein in penetration resistance against non-adapted fungal pathogens.

Syntaxin-1 is necessary for UNC5/Netrin-1-dependent macropinocytosis and chemorepulsion

Brain connectivity requires correct axonal guidance to drive axons to their appropriate targets. This process is orchestrated by guidance cues that exert attraction or repulsion to developing axons. However, the intricacies of the cellular machinery responsible for the correct response of growth cones are just being unveiled. Netrin-1 is a bifunctional molecule involved in axon pathfinding and cell migration that induces repulsion during postnatal cerebellar development. This process is mediated by Uncoordinated locomotion 5 (UNC5) receptors located on external granule layer (EGL) tracts. Here, we demonstrate that this response is characterized by enhanced membrane internalization through macropinocytosis, but not clathrin-mediated endocytosis. We show that UNC5 receptors form a protein complex with the t-SNARE syntaxin-1 (Stx1). By combining botulinum neurotoxins, a shRNA knock-down strategy and Stx1 knock-out mice, we demonstrate that this SNARE protein is required for Netrin-1-induced macropinocytosis and chemorepulsion, suggesting that Stx1 is crucial in regulating Netrin-1-mediated axonal guidance.

Activity of the SNARE Protein SNAP29 at the Endoplasmic Reticulum and Golgi Apparatus

Snap29 is a conserved regulator of membrane fusion essential to complete autophagy and to support other cellular processes, including cell division. In humans, inactivating SNAP29 mutations causes CEDNIK syndrome, a rare multi-systemic disorder characterized by congenital neuro-cutaneous alterations. The fibroblasts of CEDNIK patients show alterations of the Golgi apparatus (GA). However, whether and how Snap29 acts at the GA is unclear. Here we investigate SNAP29 function at the GA and endoplasmic reticulum (ER). As part of the elongated structures in proximity to these membrane compartments, a pool of SNAP29 forms a complex with Syntaxin18, or with Syntaxin5, which we find is required to engage SEC22B-loaded vesicles. Consistent with this, in HeLa cells, in neuroepithelial stem cells, and in vivo, decreased SNAP29 activity alters GA architecture and reduces ER to GA trafficking. Our data reveal a new regulatory function of Snap29 in promoting secretory trafficking.

The Lack of SNARE Protein Homolog Syn8 Influences Biofilm Formation of Candida glabrata

Biofilm formation of Candida species is considered to be a pathogenic factor of host infection. Since biofilm formation of Candida glabrata has not been as well studied as that of Candida albicans, we performed genetic screening of C. glabrata, and three candidate genes associated with biofilm formation were identified. Candida glabrata SYN8 (CAGL0H06325g) was selected as the most induced gene in biofilm cells for further research. Our results indicated that the syn8Δ mutant was defective not only in biofilm metabolic activity but also in biofilm morphological structure and biomass. Deletion of SYN8 seemed to have no effect on extracellular matrix production, but it led to a notable decrease in adhesion ability during biofilm formation, which may be linked to the repression of two adhesin genes, EPA10 and EPA22. Furthermore, hypersensitivity to hygromycin B and various ions in addition to the abnormal vacuolar morphology in the syn8Δ mutant suggested that active vacuolar function is required for biofilm formation of C. glabrata. These findings enhance our understanding of biofilm formation in this fungus and provide information for the development of future clinical treatments.