A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host

Abstract

In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide-sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs. Syntaxin-1A/SNAP-25/VAMP-2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin-1A/SNAP-25/VAMP-2 synthesis provoked increased length of E. coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin coexpression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs.

Keywords: SNARE proteins; caveolin; membrane fusion; membrane reconstitution; synthetic biology.

Figure 1

A system to reconstitute synaptic SNAREs in Caveolin-expressing Escherichia coli. A, structure of recombinant plasmids driving expression, respectively, of Ce caveolin (pASK background), named plasmid C and of four combinations of SNARE (Syntaxin-1A, VAMP-2, SNAP-25) and accessory proteins (Munc-18a, TeNT), named plasmids 1 to 4 (modified pDuet background). The positions and nature of ORFs and DNA modules for antibiotic resistance, replication, and transcription/translation are depicted: RBS, ribosomal-binding site; Amp R and Kana R, ampicillin and kanamycin resistances; AHT pro, anhydro-tetracycline promotor; t7 pro, t promoter; t7 ter, t7 terminator; Ori, origin of replication. Leader sequences and tags are indicated: MBP, maltose-binding protein; Strep, Strep-tag; His, 6 histidine tag; CBP tag; Flag, flag-tag. Immunoblots of cell extracts showing synthesis of CeCav (left panel) and SNAREs proteins (right panel) upon expression from plasmid C or from plasmids 1 to 4 after AHT or IPTG induction, respectively. Reactive proteins were visualized by ECL. B–D, biochemical characterization of SNARE complexes present in Escherichia coli in the absence or presence of cytoplasmic-cisternae. Affinity chromatography on Ni-NTA matrix of soluble His₆-syntaxin–containing complexes present in six A₆₀₀-equivalent of bacterial cultures. Bacteria were transformed as indicated with one of the following plasmids: 1, 2, or 3, not combined (B) or combined (C) with plasmid C. Matrix-bound complexes were recovered in boiling Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting using anti-syntaxin-1A, anti-Munc-18a, anti-VAMP-2, anti-SNAP-25, and anti-MBP antibodies. Affinity chromatography on amylose resin was performed using the same samples (D) to identify proteins associated with CeCav. Note that CeCav could capture Syntaxin-1A and/or SNAP-25 but not complexes containing VAMP-2 or Munc-18a. For ECL detection, in order to avoid signal saturation of probed proteins, only 1% of the starting material (Input) and 30% of the recovered bound material (Ni-NTA– or amylose-bound) were loaded on the same gel and transferred on the same membrane. Molecular weights are indicated (kDa). Right panels in (B–D) show the quantification of the indicated proteins in the matrix-bound material. ECL signals determined for the indicated proteins (X) by densitometry (using ImageJ software) and obtained from 4 to 5 independent experiments are plotted as Bound X/Bound Syntaxin (B and C) or Bound X/Bound CeCav after normalization. Each p value corresponds to the statistical one-way ANOVA (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.). Red dots in boxplots represent the standardized individual values of all experimental replicates and black squares correspond to the mean value of the data. SNARE, soluble N-Ethylmaleimide–sensitive attachment protein receptor.

Figure 2

Increased size of bacteria coexpressing Caveolin and synaptic SNAREs. The morphology of bacteria transformed with the indicated plasmids was analyzed both at times 0 and 3 h of IPTG exposure, in the absence (A–C) and in the presence of caveolin synthesis (D–F), after overnight culture in the presence of AHT. SNARE expression induced bacterial cell elongation only in the presence of CeCav expression. A, D, and G, phase contrast microscopy appearance of bacteria (A, D, and G). Note the abundance of the elongated morphology acquired by cells coexpressing caveolin and SNAREs in the absence of Munc-18a. Boxplot of the quantification of cell length for all combinations of expressed proteins (B, E, and H) and as cumulative frequency distribution means ± SD (C, F, and I). The effect of TeNT-LC expression in the absence of Munc-18a synthesis (G–I) was analyzed in bacteria containing caveolin or not. Note that cell elongation is prevented upon TeNT-LC expression in the presence of caveolin (compare C, F, and I). Bacteria length was measured with ImageJ software using the plug-in ObjectJ. Data were obtained from four (E and H) and three (B) independent experiments, and between 500 and 1000 cells were measured for each condition. Bar represents 5 μm. B and H, one-way ANOVA with Tukey’s multiple comparison test. E, ANOVA orthogonal contrast. (∗p ≤ 0.05; ∗∗∗p ≤ 0.001). Dots in boxplot represent the mean value of each experimental replicate and black squares correspond to the mean of the experimental replicates. AHT, anhydrotetracycline; SNARE, soluble N-Ethylmaleimide–sensitive attachment protein receptor; TeNT-LC, light chain of tetanus neurotoxin.

Figure 3

Invariance of DNA cellular content in elongated bacteria. Map of bicistronic plasmid CC (pASK background) driving expression of CeCav and mCherry. Bacteria transformed with the indicated plasmids combinations were analyzed at time 3 h of IPTG exposure in the presence of caveolin synthesis after overnight culture in the presence of AHT as in Figure 2. A, representative fluorescence images of bacteria after fixation, illustrating either cell morphology as seen in epifluorescence (upper row, mCherry distribution, compare with Fig. 2G) or DNA content after DAPI staining and recording of raw z-stack images in confocal microscopy (lower row, DAPI,). B, boxplots of the quantification of cell length, for the corresponding combination of expressed protein (upper panel) using mCherry images. Note a quasi-identity with Figure 2H. Boxplot of total cellular DAPI fluorescence (lower panel) in arbitrary units. Black squares in boxplot represent the mean value. Data were obtained from three independent experiments, and between 100 and 150 cells were measured for each condition (∗p ≤ 0.05; ∗∗∗p ≤ 0.001). Bar represents 5 μm. AHT, anhydrotetracycline.

Figure 4

Decreased cisternae upon synaptic SNARE induction. Cells were analyzed after overnight production of caveolin (AHT induction) and a 3-h SNARE synthesis (IPTG induction) using the indicated plasmids. A, representative EM images of high-pressure fast-frozen cells. Arrowhead points cytoplasmic cisternea. Inset (black border square) represent the dotted square area containing cisternae-rich regions in the cell. Bar represents 500 nm, zoom magnification bar represents 155 nm. B, chemically fixed, cryosectioned cells were examined after protein expression from indicated plasmids. Immunogold labeling of whole cells with anti-MBP antibody was performed. Note the preferential localization of the gold particles close to the plasma membrane when caveolin and SNAREs are expressed in the absence of TeNT. Careful observation of the cell periphery after the synthesis of CeCav by EM did not reveal contacts of the cisternae and the PM with visible pores allowing continuity between periplasm and cisternae lumen. Bar represents 500 nm. C, STED Imaging of CeCav (anti-MBP antibody) expressed in the same conditions than (B). D, distribution of CeCav in the peripheral and intracellular pools as defined in Experimental procedures and normalized to the total fluorescent intensity of proteins. E and F, distribution of gold particles in Escherichia coli cells immunolabeled with anti-MBP antibody and super-resolution microscopy of CeCav and Syntaxin-1A. The distance between particles and the plasma membrane was measured in sections as illustrated in (B), for the indicated plasmid combinations. Boxplot of the quantification of the distance between particles and the plasma membrane (E) and as frequency distribution means ± SD (F). Data were obtained from three independent experiments Note the decrease of the gold particle to plasma membrane distance only when caveolin and SNAREs are expressed in the absence of TeNT (combination C + 2). G, STED imaging of Star Green– and Star Orange–labeled CeCav and Syntaxin-1A, respectively, in cells transformed with plasmids C + 2 shown in (C), central panel. Note the partial colocalization of the two proteins in clusters mainly present at the cell periphery. Bar represents 1 μm. (One-way ANOVA test, ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). AHT, anhydrotetracycline; MBP, maltose-binding protein; SNARE, soluble N-Ethylmaleimide–sensitive attachment protein receptor.

Figure 5

A model of SNARE-mediated intracellular membrane fusion leading to elongation in Caveolin-expressing Escherichia coli. Bacteria are cotransformed with pASK-CeCav and one of the pDuet-derived plasmids shown in Figure 1A. The production of intracytoplasmic membrane invaginations (cisternae) originating from the plasma membrane (dark line) is first obtained after the selective expression CeCav (AHT induction). In a second phase, v- and t-SNAREs are induced by IPTG and they combine into SNARE complexes. Any functional trans-SNARE complex would mediate fusion of cisternae with cisternae and of cisternae with the plasma membrane. This intracellular membrane mobilization would result in plasma membrane expansion and elongated bacteria. AHT, anhydrotetracycline; SNARE, soluble N-Ethylmaleimide–sensitive attachment protein receptor.