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  • Letter
  • Published:

NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis

Nature Plants volume 9, pages 889–897 (2023)Cite this article

Subjects

Abstract

Eukaryotic membrane fusion requires trans-SNARE complexes bridging the gap between adjacent membranes1. Fusion between a transport vesicle and its target membrane transforms the trans- into a cis-SNARE complex. The latter interacts with the hexameric AAA+-ATPase N-ethylmaleimide-sensitive factor (NSF) and its co-factor alpha-soluble NSF attachment protein (αSNAP), forming a 20S complex2,3. ATPase activity disassembles the SNAP receptor (SNARE) complex into Qa-SNARE, which folds back onto itself, and its partners4,5. The fusion of identical membranes has a different sequence of events6. The fusion partners each have cis-SNARE complexes to be broken up by NSF and αSNAP. The Qa-SNARE monomers are then stabilized by interaction with Sec1/Munc18-type regulators (SM proteins) to form trans-SNARE complexes, as shown for the yeast vacuole7. Membrane fusion in Arabidopsis cytokinesis is formally akin to vacuolar fusion8. Membrane vesicles fuse with one another to form the partitioning membrane known as the cell plate. Cis-SNARE complexes of cytokinesis-specific Qa-SNARE KNOLLE and its SNARE partners are assembled at the endoplasmic reticulum and delivered by traffic via the Golgi/trans-Golgi network to the cell division plane9. The SM protein KEULE is required for the formation of trans-SNARE complexes between adjacent membrane vesicles10. Here we identify NSF and its adaptor αSNAP2 as necessary for the disassembly of KNOLLE cis-SNARE complexes, which is a prerequisite for KNOLLE–KEULE interaction in cytokinesis. In addition, we show that NSF is required for other trafficking pathways and interacts with the respective Q-SNAREs. The SNARE complex disassembly machinery is conserved in plants and plays a unique essential role in cytokinesis.

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Fig. 1: Interference of NSF-EQ–G with secretory traffic and increased interaction of NSF-EQ–G and R–αS2-LA with Q-SNAREs.
Fig. 2: Cytokinesis defects caused by NSF-EQ–G expression.
Fig. 3: Increased interaction of NSF-EQ–G and R–αS2-LA with cytokinesis-specific Qa-SNARE KNOLLE and its SNARE partners, the impact of R–αS2-LA on KEULE, and the role of NSF and αSNAP2 in cell-plate formation.
Fig. 4: A mechanistic model of membrane fusion in Arabidopsis cell-plate formation.

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Data availability

All materials are available from the corresponding author upon request. All data generated or analysed during this study are included in this Letter. Source data are provided with this paper.

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Acknowledgements

We thank K. Romanova for the initial mutant screening, T. Nürnberger and C. Hua for introducing us to size-exclusion chromatography and providing the equipment, R. Stahl and L. Henneberg for electron microscopy technical assistance, the Tübingen Structural Microscopy facility for providing access to their high-pressure freezing machine (EXC 2124-390838134), S. Bednarek (University of Wisconsin–Madison) for anti-SYP31 antiserum, K. Schumacher (COS, Heidelberg University) for anti-SYP81 antiserum, T. Uemura (Ochanomizu University) and A. Nakano (RIKEN Center, Waco) for sharing T-DNA lines, and M. Bayer and F. El Kasmi for critical reading. This work was funded by the Deutsche Forschungsgemeinschaft (DFG JU 179/24-1 to G.J.). Additional DFG funding numbers: Leica TCS-SP8, INST 37/819-1 FUGG; TEM Electron microscope, INST 37/900-1 FUGG.

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Authors and Affiliations

  1. ZMBP, Developmental Genetics, University of Tübingen, Tübingen, Germany

    Misoon Park, Ulrike Mayer & Gerd Jürgens

  2. ZMBP, Microscopy, University of Tübingen, Tübingen, Germany

    Sandra Richter

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Contributions

M.P. and G.J. conceptualized the project and wrote the original draft of the manuscript. M.P. and U.M. devised the methodology and conducted the investigation. S.R. conducted the electron microscopy analysis. M.P., U.M., S.R. and G.J. reviewed and edited the manuscript. G.J. acquired the funding, provided the resources and supervised the project.

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Correspondence to Gerd Jürgens.

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Nature Plants thanks Panagiotis Moschou, Yiqun Bao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sequence alignment and predicted protein structures.

(a) Alignment of C-terminal sequences of αSNAP-related proteins. Arabidopsis thaliana αSNAP1 (AT3G56450) and αSNAP2 (AT3G56190) were aligned with αSNAP from human (Homo sapiens, GenBank NP_003818), cattle (Bos taurus, GenBank AAB25812) and yeast (Saccharomyces cerevisiae, GenBank NP_009503) in the CLC Main Workbench program. Note that αSNAP1 is larger than the other αSNAPs and less similar to bovine αSNAP (E value 1e-29 vs. 2e-71 for αSNAP2). Asterisk marks the conserved leucine residue L288 that was mutated to alanine in this study. Numbers indicate protein lengths (amino acid residues). (b-e) Structural models of NSF and αSNAP2. Predicted structures of wild type NSF (grey, b and c) and αSNAP2 (grey, d and e) are superimposed with those of dominant-negative NSFEQ (green, b and c) and αSNAP2LA (blue, d and e), respectively. The substituted residues are highlighted in red (b-e). (c and e) Boxed areas in (b and d) at higher magnification.

Extended Data Fig. 2 Schematic of NSF and αSNAP2 and impact of their dominant-negative variants on Arabidopsis embryo development.

(a) Schematic of NSF and αSNAP2. NSF domains indicated; N domain, interaction with αSNAP2. Numbers, amino acid positions. Residues altered in dominant-negative variants highlighted in red: E326 (NSF), L288 (αSNAP2). (b-m) Embryo abnormalities caused by RPS5A (R)»UAS two-component expression of NSFEQ:G (GFP-tagged dominant-negative NSFE326Q, b-g) or R:αS2LA (RFP-tagged dominant-negative αSNAP2L188A, h-m). Non-transgenic wild-type control embryo (n-p); (b, e, h, k, n) cell-wall staining with Renaissance blue 2200; (c, f, i, l, o) FP, fluorescent fusion proteins (c and f, GFP; i and l, RFP; o, no FP fluorescence); (d, g, j, m, p) bright-field Images; scale bars, 10 µm. Note that the mutants were clearly discernible around the heart stage, developing severe abnormalities thereafter (e-g, k-m). Number of embryos analysed, ≥300.

Source data

Extended Data Fig. 3 Interaction of NSF with αSNAP2 and impact of their dominant-negative variants on Arabidopsis seedling development.

(a-d) Seedling abnormalities caused by estradiol (EST)-induced expression of NSFEQ:G (a) or R:αS2LA (b) were analyzed 5 days after germination (5DAG) on EST plates. Below: Boxed areas at higher magnification. C, non-transformed wild-type; two transgenic lines (1, 2) each for the wild-type (NSF:G, R:αS2) and dominant-negative (NSFEQ:G, R:αS2LA) constructs. (c, d) Control seedlings grown in estradiol solvent DMSO for 5 days. Scale bars, 1 cm. Number of seedlings analysed, ≥200 (a-d). (e-f) Expression of transgene-encoded proteins. Protein extracts from seedlings of two transgenic lines (1, 2) incubated with EST (inducer) or DMSO (solvent control) for 24 hours were separated of SDS-PAGE gels and immunoblotted (IB) with anti-GFP (e) and anti-RFP (f) antibodies. C, non-transformed wild-type; two transgenic lines (1, 2) each for the wild-type (NSF:G in e, R:αS2 in f) and dominant-negative (NSFEQ:G in e, R:αS2LA in f) constructs. Molecular marker size on the left (kDa, kilodalton); Pon S, Ponceau S-stained membrane as loading control. (g) Co-immunoprecipitation of NSF:G and R:αS2. T, total extract; U, unbound; IP, immunoprecipitate (IP-GFP, anti-GFP beads). IB, immunoblot with antibody indicated (right); Molecular marker size on the left (kDa, kilodalton) NSF:G/R:αS2, doubly transgenic; NSF:G or R:αS2, singly transgenic.

Source data

Extended Data Fig. 4 Subcellular localization of NSF and αSNAP2 in seedling roots.

(a-b) Live imaging of wild type NSF:GFP (a) and dominant-negative NSFEQ:GFP (b). (c-e) Live imaging of NSF:GFP (NSF:G) and wild type RFP:αSNAP2 (R:αS2) in seedling roots. (g-n) Localization of NSF at TGN/early endosomes. Live imaging of NSF:GFP (g-i) and NSFEQ:GFP (k-m) against TGN marker a1-RFP. Arrowheads indicate merged punctate signals of NSF:GFP (i) or NSFEQ:GFP (m) with a1-RFP. (f, j, n) Signal intensities of the scanned lines in (e, i, m). f, NSF:G (green) to R:αS2 (red); j, NSF:G (green) to a1-RFP (red); n, NSFEQ:G (green) to a1-RFP (red). Arrowheads indicate merged signals (e, i, m). Double arrowheads indicate the cell division plane positively labeled with NSF:G (e and i) or NSFEQ:G (b). Scale bars, 5 µm (a, b, e, i, m). Images were taken after 24 hours of EST induction. Numbers of seedlings analysed: >100 (a and b); 12 (c-e); 22 (g-i); 23 (k-m).

Extended Data Fig. 5 Effect of dominant-negative NSF on endocytosis.

(a-l) Live imaging of NSF:G (a-c) or NSFEQ:G (d-f) and endocytic tracer SynaptoRedTM C2 in seedling roots after 24 hours of EST induction. (g-l) Imaging of NSF:G (g-i) or NSFEQ:G (j-l) and SynaptoRedTM C2 upon brefeldin A (BFA) application. (m) Cytosolic intensity quantification of SynaptoRedTM C2 signals. Cytosolic area in a single cell was selected for quantification. The P value was calculated using two-tailed Welch’s t-test by comparing NSF:G to NSFEQ:G. The center line of the box plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. Data points are shown as jitter. n, number of counted cells.

Source data

Extended Data Fig. 6 Effect of dominant-negative NSF on vacuolar traffic.

(a-f) Live imaging of NSF:G (a-c) or NSFEQ:G (d-f) and vacuolar soluble cargo marker AFVY:RFP in seedling roots after 24 hours of EST induction. (g) Intensity quantification of AFVY:RFP. Vacuolar area in each single cell was selected for quantification. The P value was calculated using unpaired Student’s t-test (two-tailed) by comparing wild type NSF:G to NSFEQ:G. The center line of the box plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. Data points are shown as jitter. n, the number of counted cells. Scale bars, 5 µm (c, f).

Source data

Extended Data Fig. 7 Semi-quantitative analysis of coimmunoprecipitation assay.

(a-b) Group box plots of semi-quantitative analysis. Signal intensities of the immunoprecipitate (IP) fractions with anti-GFP (a) and anti-RFP (b) beads were measured from the two or three immunoblots that were probed with Q-SNARE antisera as in Fig. 1i,j: SYP132, SYP121, SYP61, SYP31, SYP21, SYP81. Band intensity values were normalized with NSF:G and R:αS2 set at 100; A.U, arbitrary units. For simplicity, IP fractions from the non-transformed wild type extract were not shown. The P values were calculated using unpaired Student’s t-test (two-tailed) by comparing the respective wild type to its dominant negative mutant. The center line of the box plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. n, number of the counted immunoblots. Note that P value was not calculated (n.c) for n≤2.

Source data

Extended Data Fig. 8 Cytokinesis defects caused by expression of dominant-negative αSNAP2 dominant negative variant.

Dividing cells from embryos (a-f) or seedling roots (h-m) expressing wild type R:αS2 (a-c, h-j) or dominant-negative R:αS2LA (d-f, k-m) counterstained with anti-KNOLLE (KN) (a-f) and anti-callose (h-m) antisera. (a, d, h, k) RFP; (b, e, i, l) KNOLLE; (j, m) callose; (c, f) merged images and nuclei stained with DAPI (blue); (j, m) Insets, merged images of GFP, anti-callose (cyan) and anti-KNOLLE (green); double arrowheads, cell division plane region (a, d, h, k); scale bars, 5 µm (c, f, j, m). (a-f) RPS5A (R)::GAL4»UAS expression; (h-m) estradiol induction. Double arrowheads, plane of cell division region (a, d, h, k). (g, n) Width quantification of KNOLLE (g) or callose (n) label in the cell division region. The P values were calculated using two-tailed Welch’s t-test by comparing wild type R:αS2 (pale green, g; pale blue, n) to dominant-negative R:αS2LA (green, g; blue, n). The center line of the box plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. Data points are shown as jitter. n, number of counted cells.

Source data

Extended Data Fig. 9 Semi-quantitative analysis of coimmunoprecipitation assay, interaction of NSF and KNOLLE and chromatography analysis of NSF and KNOLLE complexes.

(a-b) Group box plots of semi-quantitative analysis. Signal intensities of the immunoprecipitate (IP) fractions with anti-GFP (a) and anti-RFP (b) beads were measured from two or three immunoblots probed with SNARE antisera as in Fig. 3a,b: KNOLLE, NPSN11, SYP33, SYP71, VAMP721/722 (VAMP721). Band intensity values were normalized with wild type NSF and αSNAP2 set at 100; A.U, arbitrary units. For simplicity, immunoprecipitate from the non-transformed wild type extract was not included. The P values were calculated using unpaired Student’s t-test (two-tailed) by comparing the respective wild type to its dominant negative mutant. The center line of the whisker plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. n, number of the counted immunoblots. (c-d) Coimmunoprecipitation of wild type NSF:G or dominant-negative NSFEQ:G with Myc:KNOLLE (Myc:KN). Extracts from non-transformed wild-type (C) or transgenic seedlings coexpressing Myc:KN and NSF:G or NSFEQ:G were immunoprecipitated (IP) with anti-Myc beads. Protein blots were immunoblotted (IB) with antibodies indicated on the right. Myc:KN, immunoprecipitate of extract from transgenic seedlings expressing only Myc:KNOLLE. Band intensity values were normalized with wild type NSF:G in IP fraction set at 100%. Molecular marker size on the left (kDa, kilodalton); Total, total extract; IP, immunoprecipitate. (d) Semi-quantitative analysis of (c). Signal intensities of the immunoprecipitate (IP) were measured from two immunoblots. Band intensity values were normalized with wild type NSF:G in IP fraction set at 100; A.U, arbitrary units. For simplicity, immunoprecipitate from the non-transformed wild type extract was not included. The center line of the box plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. n, number of the counted immunoblots. Note that P value was not calculated (n.c) for n≤2. (e) Immunoblots from size-exclusion chromatography of NSF and KNOLLE complexes shown in Fig. 3c. Continuous fractions of NSF:G and NSFEQ:G in the high molecular mass range were immunoblotted (IB) with the indicated antisera anti-GFP, anti-KNOLLE (KN), and anti-VAMP721/722 (V721). INPUT, filtered protein extracts before running size exclusion chromatography; C, non-transformed wild-type protein extracts. Peaks of the standard markers are indicated (669 kDa, thyroglobulin; 443 kDa, ferritin).

Source data

Extended Data Fig. 10 Interference with KEULE localization at the cell division plane by αSNAP2 dominant negative variant.

(a-h) KNOLLE (KN) and KEULE localization in dividing cells of seedling roots after estradiol induction of wild-type R:αS2 (red, a-d) or dominant-negative αS2LA (red, e-h). (b, f) anti-KN antiserum (blue); (c, g) vYFP (vY):KEULE (green); same images as shown in Fig. 3d–i; (a, e) RFP channels; (d, h) merged images; scale bars, 5 µm (d, h). Double arrowheads, cell division plane region (a, e). (i) Localization efficiency of KEULE at the cell division plane (CDP). The P value was calculated using two-tailed Welch’s t-test by comparing R:αS2 to R:αS2LA. The center line of the box plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. Data points are shown as jitter. n, number of images comprising 40 cytokinetic cells in R:αS2 and 119 cells in R:αS2LA that were analysed in total, respectively. Numbers of seedlings analysed: 24 (a-d); 40 (e-h).

Source data

Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Data 1

List of primers used for cloning.

Supplementary Table 1

Seed viability for Supplementary Table 1.

Source data

Source Data Fig. 1

Unprocessed immunoblots for Fig. 1.

Source Data Fig. 3

Unprocessed immunoblots for Fig. 3.

Source Data Extended Data Fig. 2

Width measurements of KNOLLE and callose for Extended Data Fig. 2g,n.

Source Data Extended Data Fig. 3

Unprocessed immunoblots for Extended Data Fig. 3.

Source Data Extended Data Fig. 5

Cytosolic signal intensity of SynaptoRed C2 for Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Signal intensity of AFVY–RFP for Extended Data Fig. 6.

Source Data Extended Data Fig. 7

Signal intensity of co-immunoprecipitation for Extended Data Fig. 7.

Source Data Extended Data Fig. 8

Width measurements of KNOLLE and callose for Extended Data Fig. 8g,n.

Source Data Extended Data Fig. 9

Unprocessed immunoblots for Extended Data Fig. 9.

Source Data Extended Data Fig. 9

Signal intensity of co-immunoprecipitation for Extended Data Fig. 9a,b,d.

Source Data Extended Data Fig. 10

Localization efficiency of KEULE at the cell division plane for Extended Data Fig. 10i.

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Park, M., Mayer, U., Richter, S. et al. NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis. Nat. Plants 9, 889–897 (2023). https://doi.org/10.1038/s41477-023-01427-8

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