Journal: The Journal of Cell Biology

Article Title: MT1-MMP recruits the ER-Golgi SNARE Bet1 for efficient MT1-MMP transport to the plasma membrane

doi: 10.1083/jcb.201808149

Figure Lengend Snippet: Bet1 forms novel SNARE complexes with endosomal SNAREs in MDA-MB-231 cells. (A) 3xFLAG-Bet1 interacts with STX4, Vti1b, and VAMP4 in addition to STX5. MDA-MB-231 cells stably expressing 3xFLAG-Bet1 were lysed, and the lysate was subjected to immunoprecipitation using FLAG-beads, and then the precipitate was subjected to immunoblotting for endogenous SNARE proteins. In the following blots, the brightness and contrast were adjusted so that their input bands became comparable to those of other SNAREs: STX3, STX7, STX16, STX17, STX18, VAMP3, VAMP7, and Vti1b. (B) An endogenous complex comprised of STX4, Bet1, Vti1b, and VAMP4 exists in MDA-MB-231 cells, but not in HeLa cells. Lysates of MDA-MB-231 and HeLa cells were subjected to immunoprecipitation of STX4, and then the co-precipitation of Bet1, Vti1b, and VAMP4 was analyzed. In the following blots, the brightness and contrast were adjusted so that their input bands became comparable to those of other SNAREs: Bet1 and VAMP4 in MDA-MB-231 cells and Bet1, VAMP4, and VAMP8 in HeLa cells. (C and D) Bet1-interacting SNAREs are colocalized with Bet1-GFP in endomembrane compartments. MDA-MB-231 cells stably expressing Bet1-GFP were transfected with FLAG-SNAREs and then subjected to immunofluorescence and confocal microscopy (C). Their colocalization was assessed by using the Manders’ overlap coefficient (D). (E–G) Depletion of STX4 and Vti1b reduces the complex of Bet1 with Vti1b and STX4, respectively. MDA-MB-231 cells stably expressing 3xFLAG-Bet1 were transfected with STX4 and Vti1b siRNAs, and then immunoprecipitation and immunoblotting (E) were performed as in A. Co-precipitated endogenous STX4 (F) and Vti1b (G) were quantified. Scale bar: 10 μm in a regular image; 2 μm in an inset. **, P < 0.01; vs. STX4 in D; vs. FLAG (Bet1), mock in F and G.

Article Snippet: The commercial rabbit polyclonal antibodies used were anti-FLAG (Sigma-Aldrich), anti–MT1-MMP (EMD Millipore), anti-STX16 (Synaptic Systems), anti-GFP (Thermo Fisher Scientific), and anti-GPP130 (Covance).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Transfection, Immunofluorescence, Confocal Microscopy

Journal: PLoS ONE

Article Title: Syntaxin 16 Regulates Lumen Formation during Epithelial Morphogenesis

doi: 10.1371/journal.pone.0061857

Figure Lengend Snippet: ( A ) Diagram of the exon and intron organization of zebrafish stx16 . Thick red lines indicate the binding regions of two antisense morpholinos (MOs), one of which targets the start codon (AUG) to block translation of stx16 , and the other the mRNA splice acceptor site of exon 4 (splicing) to block splicing of the stx16 mRNA. The two arrows indicate the locations of primers used in RT-PCR analysis in control and stx16 morphants. ( B ) Western blot analysis showing the expression of Stx16 in control and stx16 -MO AUG -injected embryos at 48 hpf; expression of alpha-actin served as a loading control. ( C ) RT-PCR showing 319 bp and 867 bp amplicons, which result, respectively, from amplification from exon 4 and exon 4 plus unspliced intron 3 in the control uninjected and stx16 -MO sp -injected embryos. ( D ) Expression of stx16 at 48 hrs, as detected by ISH. Left-hand panel: lateral view with anterior to the left. Right-hand panel: cross section of the region shown to left. Arrow: pronephric duct. ( E ) Normarski images (lateral view) of control and stx16 MO sp -injected embryos at 48 hpf. Bar graph at right shows the percentage of embryos showing abnormalities (edema and curvature). Arrow: pericardial edema; arrowhead: body curvature. ( F ) pax2.1 expression at 48 hpf, as detected by ISH in the indicated embryos. Left-hand panels: Lateral view of the trunk region of indicated embryos, with anterior to the left. Right-hand panels: Transverse sections of embryos of the left-hand panel. Arrows: pronephric duct. ( G ) Top panel (i, ii): Representative epifluorescence images of 48-hpf Tg(cldnb:lynEGFP) uninjected control and stx16 -MO sp -injected embryos. Lateral view, anterior to the right, pronephric ducts. Middle panel (iii, iv): Confocal Z-stack images of pronephric ducts of Tg(cldnb:lynEGFP) taken at the regions shown in top panel. Bottom panel (v, vi): transverse sections of Tg(cldnb:lynEGFP) embryos showing the pronephric ducts (arrows). Nucleoli are stained with DAPI. Graph showing the percentages of embryos exhibiting pronephric-duct dilation. ( H , I ) Confocal (Z-stack) images of transverse sections of Tg(cldnb:lynEGFP) embryos at 48 hpf, showing the expression of PKCζ and pan-cadherin on one side of pronephric duct. Arrowheads: lumens.

Article Snippet: Morpholino antisense oligonucleotides (MO) targeting zebrafish syntaxin 16 ( stx16 ) were designed and synthesized by Gene Tools, LLC (Philomath, OR).

Techniques: Binding Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Injection, Amplification, Staining