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1 syx D syx M13 F % with cluster G 3 rnd Syx rnd M13 ** rnd M13 H E % with anule bg syx rnd F cell S a c Supplementary Fig 1. Quantification of membrane protein clusters beneath anules. TIRF-M images of Ins1 cell transfect with Syx-EGFP (Syx and anule marker NPY-cherry (; different magnifications of the same cell are shown. White circles indicate anule locations that were includ in the analysis. Scalebar 1µm. as in, but for munc18 (. as in, but for munc13 (M13. D Spatially align average images center on anule positions, for anule channel (; 1843 anule positions in 175 cells, syntaxin (syx; 1843 anules, 175 cells, munc18 (; 221 anules, 19 cells, and munc13 (M13; 752 anules, 59 cells anule positions in 175 cells were averag. Scaling to 8-bit for display was done with identical settings for all images, after first normalizing for the average backound-subtract annulus fluorescence (S. Scalebar.5µm E Fraction of syx clusters that visually colocaliz with anules at any time during 6s from 5 cells. F Fraction of anules ( that visually colocaliz with Syx-EGFP (Syx, munc18 ( or munc13 (M13, at any time during 6 s. olocalization with random locations (rnd was quantifi as control. 8, 278, and 5 anules, from 5 cells each. H Definition of parameters us during analysis. ell is the area containing the footprint of the cell, bg and area with no cell, c and a are center on the position of a anule. The excess anule relat fluorescence is c-a= F, and the surrounding fluorescence not influenc by the anule is a-bg=s. ll values are read as pixel-averag fluorescence. Scalebar 1µm. G Quantification of -: Plot of F vs. S for syntaxin-egfp; see Methods for details. Shown is the nearly linear range <F<. Each symbol represents the average of 5 cells.
2 events/cell 6 exo exocytosis (stimulat at 1s ing (non-stimulat uning (non-stimulat exocytosis (non-stimulat Supplementary Fig 2 (arg Supplementary Fig 2. dditional analysis for experiments in Fig 2. Trajectories of anules (> frames long in the cell shown in ; total observation time was 5s. Scalebar 2µm. Normaliz rates of ing (purple, uning (een and exocytosis in unstimulat conditions (ey or when increasing K+ to 75 mm (arrow, and data in purple from 9 cells each.
3 trl lifeact Lifeact (F16 trl ** Lat -2 ( m.6 n.s. Lat density.4.2. trl Lat lifeact Supplementary Fig 3. Destruction of actin cortex does not affect anule density. TIRF images of cells expressing the EGFP-tagg actin-marker Lifect (lifeact and NPYcherry (, in control conditions and 15 min after treatment with 2 µm latrunculin (Lat. Scalebar 2µm. Quantification of the Lifeact signal (F=cell-bg. The smaller signal after treatment with Lat indicates destruction of cortical actin in 43 cells each. Quantification of anule density in conditions as in in 43 cells each.
4 .1 syntaxin..1.1 efo exo ( re - 15 s. SNP SNP25.15 re syntaxin d o ck re (> 1 2 s efo exo ( re -15s munc18 munc efo exo ( re -15s -.1 **. r e.5 munc13.1 munc ** do ck re d o ck (>12 s exo ( -15s.5 rab3a.1 rab3a. * efo exo ( re -15s. - re D # anules 5 < Supplementary Fig 4. dditional analysis and controls for experiments in Fig 3. - Representative traces for the protein signal ( at individual anules during ing ( and ing ( events. verage signal ( for indicat proteins at anules (, at the site of a ing event b ( b or after ( afte arrival of the anule, during presence of a or ( afte, b and after exocytosis (exo b and exo -15 s, and at failures (fail b. Note absence of syntaxin, SNP25 and munc13 b ing. -68 anules from 9-44 cells for each condition. D Distribution of the lag between the onset of syntaxin loss and uning of the anule. Negative times indicate that syntaxin loss prec uning. 25 events from 15 cells.
5 syx syx+habc F 15 5 EGFP NHabc-EGFP (% residence ctrl EGFP Habc-EGFP Habc(IRES D E F anule anule F/S NHabc-EGFP EGFP EGFP NHabc-EGFP G WT syx Overlay H Habc syx Overlay I.2 F/S.1. WT syx Habc syx Supplementary Fig 5. Habc domain of syntaxin is important for colocalization with clusters. overexpression of Habc does not affect syntaxin expression, prevents ing and associates with anules Syntaxin expression and targeting to the plasma membrane are unaffect by the Habc fragment. Images showing Syntaxin-herry, in cells coexpressing either EGFP (left or Habc-EGFP (right. Scalebar=1µm. Quantification of ; fluorescence intensity (backound-subtract cell average as measure of syntaxin-herry expression, for cells co-expressing either EGFP or NHabc-EGFP from 91 and 48 cells respectively. Histoam of anule residence times as in Fig 3G, in cells co-expressing Habc-EGFP (black squares, 177 anules, 12 cells, EGFP (open circles, 15 anules, 6 cells, Habc from and IRES construct (triangles, 151 anules, 13 cells, and pires control (diamonds, 89 anules, 12 cells. D verage anule and EGFP images spatially align to anule positions. The dark spot in the anule position is due to exclusion of EGFP by the anule volume (cf ref. Scalebar=.5µm. E verage anule and NHabc-EGFP images spatially align to anule positions. Note accumulation of NHabc- EGFP at the anule site, which overcomes the exclusion effect observ with EGFP in. ll images are individually autoscal. Scalebar=.5µm. F Quantification of anules from 45 cells (NHabc-EGFP, and 687 anules, 29 cells (EGFP. G-H ell expressing syntaxin-egfp (; WT syx or syntaxin- Habc-EGFP (; Habc syx together with munc18- cherry (. Scalebar=2µm. I Quantification of munc18 association at syntaxin clusters; 46 and 38 cells. The
6 syx overlay SN25 overlay overlay M13 overlay rab3 overlay Supplementary Fig 6. Example images of cells expressing the fusion proteins us Representative example TIRF images of cells expressing syntaxin-egfp (syx, EGFP-SNP25 (SN25, Munc18- EGFP, Munc13-EGFP (M13, or EGFP-rab3 (rab3. ll cells also express the anule marker NPY-cherry. Scalebar=2µm.
7 m oti on ( n m / s d (nm z TP NPY F.1s tp-egfp exocytosis 15 NPY-cherry 5 D TP NPY E 4 F tp-egfp G 5 uning F NPY-cherry x-y mobility z-distance Supplementary Fig 7. Differentiation between uning and exocytosis events. During exocytosis the anule fluorescence disappears within one or two frames (-ms, while uning of anules is much slower and these can be observ moving away from the ing site for several seconds. In addition, we do not observe spontaneous exocytosis under the conditions of the ing/uning experiments. Granule behavior was imag in cells coexpressing the two anule markers, een ph-sensitive tissue plasminogen activator (tp-egfp and r ph-insensitive NPYcherry. Example of an exocytosis event in a cell expressing tp-egfp and NPY cherry. Frames are.1 s apart. The increase in the tp signal is due to unquenching of EGFP fluorescence when the acidic ph in the anule lumen is neutraliz during exocytosis Note that tp is retain at the anule site after exocytosis, unlike NPY. Scalebar=.5µm. - Quantification of exocytosis ( events from 6 cells showing F for tp-egfp and NPY-cherry. D Example of an uning event. Note slow lateral movement of the anule and lack of fluorescence transient in the tp-egfp channel. Images are.1 s apart. Scalebar=.5µm. E-F Quantification of uning (24 events from 6 cells. Note slow decay of both signals compar with -. G Example illustrating the selection criteria for ing candidates. xial mobility (frame-to-frame distance; ay and distance from the plasma membrane are shown for a single anule. Time zero is taken as moment of ing.
8 Supplementary References 1. Ril, J., et al. Lifeact: a versatile marker to visualize F-actin. Nat Methods 5, (8. 2. Taraska, J.W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S. & lmers, W. Secretory anules are recaptur largely intact after stimulat exocytosis in cultur endocrine cells. Proc Natl cad Sci U S, 7-75 (3.
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