Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

Abstract

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.

Figure 1

Sterol composition specifies KNOLLE syntaxin localization at the end of cytokinesis. (A–R) KNOLLE whole-mount immunofluorescence localization (red) in late cytokinetic cells from root tips of 5-day-old Arabidopsis seedlings. DAPI staining of DNA (blue). (A) Wild-type Landsberg erecta (Ler). (B) cpi1-1 mutant. (C) Wild-type Columbia-0 (Col-0) without inhibitor treatment (−fen). (D) Cell from Col-0 seedling grown on a growth-agar plate including 200 μg/ml fen (+fen). (E) smt1^orc. (F) smt1^cph-G213. (G–R) KNOLLE fluorescence co-labelling with fluorescent-protein fusions of other cell-plate proteins (green). (G–I, M–O) Wild type (wt). (J–L, P–R) cpi1-1. (H, K) YFP-RAB-A2a. (I) Merged image of (G, H) and DAPI. (L) Merged image of (J, K) and DAPI. (N, Q) DRP1A-GFP. (O) Merged image of (M, N) and DAPI. (R) Merged image of (P, Q) and DAPI. Scale bars are 5 μm. Immunolabelling experiments were performed at least three independent times using a total of 25–30 roots per genotype or treatment.

Figure 2

Sterols and KNOLLE co-localize and act in a common pathway. (A–C) Phenotypes of genotyped 5-day-old seedlings from the progeny of a cpi1-1/+;knolle^X37-2/+ heterozygote plant (see Supplementary data). (A) Wild-type (Ler), knolle^X37-2 (kn^X37-2) and cpi1-1. (B) Close up of kn^X37-2 seedling from (A). (C) cpi1-1;kn^X37-2 double mutant. (D–O) Co-detection of filipin–sterol fluorescence (fil; red) and anti-KNOLLE immunofluorescence labelling or fluorescent-protein fusions to different endomembrane markers (green) in cells at the end of cytokinesis. (D, G, J, M) Filipin–sterol fluorescence (fil). (E) Anti-KNOLLE (KNOLLE). (H) pRAB-A2a:YFP-RAB-A2a (YFP-RAB-A2a). (K) pARF1:ARF1-EGFP (ARF1-GFP). (N) pRAB-F1:RAB-F1-GFP (RAB-F1-GFP). (F, I, L, O) Merged images of the two images to the left, respectively. (P) Quantitative co-localization analysis between KNOLLE-immunofluorescence or fluorescent-protein-labelled endomembrane compartments co-labelled with filipin–sterol fluorescence in cytokinetic cells expressed as the percentage (%) of compartments co-localizing with sterols. Data is derived from CLSM images such as in (D–O) scoring co-localization of the geometrical centres of the two labelled objects within the resolution limit of the microscope objective. Data are mean±s.d. from 14 cells per marker (n=14). Markers were anti-KNOLLE (KNOLLE), pARF1:ARF1-EGFP (ARF1), pRAB-A2a:YFP-RAB-A2a (RAB-A2a), pVHA-a1:VHA-a1-GFP (VHA-a1), pRAB-F1:RAB-F1-GFP (RAB-F1), p35S:RAB-F2b-GFP (RAB-F2b), p35S:N-ST-YFP (N-ST), p35S:NAG1-EGFP (NAG1), p35S:ER-mGFP5-HDEL (HDEL). Preferential subcellular localization is indicated and colour coded.

Figure 3

Ultrastructural detection of filipin–sterol complexes at forming and fused cell plates, TGN and MVBs. (A–J) TEM images of 80 nm ultrathin sections from cytokinetic epidermal cells of chemically fixed Arabidopsis roots embedded in Spurr epoxy-resin (A–D, I) Filipin-labelled (+fil). (E–H, J) DMSO solvent without filipin (−fil). (A–C) Note, 20–30 nm filipin–sterol complex deformations of trans-Golgi network (tgn), unfused cell-plate membranes/vesicles (cp) and fused cell-plate membranes in the plane of cell division (cdp), compared with (E–G) smooth membranes in samples not treated with filipin. (D) Filipin–sterol complex deformations at tgn compared with (H) smooth Golgi (g) membranes in samples not treated with filipin. (I) Filipin–sterol complexes at multi-vesicular body (mvb) membranes and plasma membrane (pm). (H, J) Smooth tgn and mvb membranes in sample not treated with filipin. (A–J) Endoplasmic reticulum (er), ER-body (erb), microtubule (mt), nucleus (nu), microtubule (mt), mitochondrion (mi), vacuole (v). Scale bars are 500 nm.

Figure 4

KNOLLE is not enriched in DRM fractions. Western-blot analysis of proteins in DRM fractions extracted from an Arabidopsis cell suspension culture. Equal amounts of membrane protein (4 μg) were loaded from total membrane (TM) fraction, the floating fraction mock extracted at Triton-X-100 (TX-100) detergent/protein ratio 0 (R0), DRM fractions after extraction at ratio 4 (R4) and ratio 8 (R8). Antibodies were directed against KNOLLE, the DRM-enriched marker protein(s) PM-H⁺ATPase (DRM), the DRM-depleted (non-DRM) proteins BiP and SMT1. Similar results were obtained in three independent experiments.

Figure 5

KNOLLE lateral diffusion from the cell-division plane is not altered in the cpi1-1 mutant. (A–N) FRAP analyses combined with (D–K) FLIP analyses of GFP-KNOLLE fluorescence. (A, B, D, E, G, I, J, L, M) Pre-bleach (pre), post-bleach (post) and subsequent post-bleach images acquired at time points in minutes. Boxes indicate bleach ROIs. (A–C) FRAP analyses after bleaching of a 2 μm ROI in the plane of cell division in (A) wild type (wt) or in (B) wild type after 45 min pre-incubation with energy inhibitors (-e), 0.02% sodium azide, 50 mM 2-deoxy-D-glucose (wt -e). (C) Quantitative analyses of experiments such as in (A, B) showing normalized values of pre- and post-bleach fluorescence intensities at recovery time points in 30 s post-bleach image acquisition intervals. Data are mean±s.d. from 14 cells (n=14). (D, E) Bleaching of GFP-KNOLLE at the end of cytokinesis, except for fluorescence in the plane of cell division, in phenotypically wild-type (wt, +/+ and cpi1-1/+) offspring from a cpi1-1/+;GFP-KNOLLE plant. (D) 45 min, 50 μM CHX (wt CHX). (E) 45 min, 50 μM CHX,-e (wt CHX -e). (F) Quantitative analyses of experiments such as in (D, E) for FLIP at membranes in the cell-division plane (CP) and FRAP at the outer plasma membrane (PM) under CHX or CHX,-e application, respectively. Data are mean±s.d. from 10 cells (n=10). (G) as E, but in cpi1-1;GFP-KNOLLE background (cpi1-1 CHX -e). (H) Quantitative comparison of experiments such as in (E, G) for FLIP at the CP and FRAP at the PM. Data are mean±s.d. from 10 cells (n=10). (I, J) Bleaching of GFP-KNOLLE at the plane of cell division in (I) wild type and (J) cpi1-1 mutant both pre-treated 45 min, 50 μM CHX,-e (wt CHX -e). (K) Quantitative comparison of experiments such as in (I, J) for FLIP at the PM and FRAP at the CP. Data are mean±s.d. from seven cells (n=7). (L, M) GFP-KNOLLE FRAP in fully bleached (L) cpi1-1/+ or +/+ and (M) cpi1-1 cells. (N) Quantitative comparison of FRAP experiments such as in (L, M). Data are mean±s.d. from 10 cells (n=10). (O–Q) KNOLLE immunofluorescence localization (red) in late cytokinetic cells of Ler seedling roots with or without inhibitor treatment. DAPI DNA staining (blue). (O) Ler. (P) Ler, 30 min, 50 μM CHX. (Q) Ler, 30 min, 50 μM CHX -e. All scale bars are 5 μm.

Figure 6

At the end of cytokinesis KNOLLE internalizes through early endosomes, but is more retained at the plane of cell division in cpi1-1. (A, D, G) GFP-KNOLLE fluorescence and (B, E, H) FM4-64 fluorescence in wild type overlaid in (C, F, I). (A–C) Cells pre-treated 45 min with 50 μM CHX, pulsed with 25 μM FM4-64 for 5 min and incubated were examined for FM4-64 internalization (D–F) after 6 min and (G–I) 60 min of incubation with 50 μM CHX, 50 μM BFA. Note co-localization in endosomes between GFP-KNOLLE and FM4-64 (arrowheads in C, F, I). (J–M) anti-KNOLLE immunolocalization (red) in late cytokinetic cells with DAPI staining for DNA (blue). (J) KNOLLE after 45 min of pre-treatment with 50 μM CHX before BFA application (0 min) (K) becomes internalized into BFA compartments and disappears from the cell plate after prolonged incubation for 60 min (60′) with 50 μM CHX, 10 μM BFA. (L) cpi1-1 mutant cells after 45 min of pre-treatment with 50 μM CHX and (M) prolonged incubation for 60 min (60′) with 50 μM CHX, 10 μM BFA. Note, KNOLLE in plane of cell division in (M) compared with (K). (N) Quantification of KNOLLE immunofluorescence intensity at the cell-division plane (CDP) versus internal compartments (IC) from experiments in (J–M). The normalized fluorescence intensity ratio Rn_CDP/IC (normalized to time point 0) is given per cell. This was obtained by calculating R_CDP/IC=[mean amplitude relative pixel intensity of KNOLLE at CDP: length CDP (μm)]: [mean amplitude relative pixel intensity of KNOLLE label/area IC (μm²)] per cell and normalizing R_CDP/IC by dividing it through the average Rav_CDP/IC of the analysed cell population at 0 min (start intensity ratio) for wild type and cpi1-1, respectively. Exact P-values obtained from non-parametric, two-tailed Mann–Whitney U-test were wild type at 0 min (n=47) versus wild type at 60 min (n=30), P<2e-06; cpi1-1 at 0 min (n=50) versus cpi1-1 at 60 min (n=49) P<2e-06, and wild type at 60 min (n=30) versus cpi1-1 at 60 min (n=49) P=0.0277 with a significance threshold at P<0.05; n indicates the numbers of epidermal cells at the end of cytokinesis analysed and obtained from 18 to 24 roots per genotype or treatment. Scale bars are 5 μm.

Figure 7

ARF1, clathrin and dynamin-dependent endocytosis restricts KNOLLE to the cell division plane. (A–S) KNOLLE immunofluorescence labelling in late cytokinetic cells. (A–L) KNOLLE immunofluorescence (cyan) co-labelling with filipin–sterol fluorescence (red) and fluorescent fusion proteins (green). (A–L) Heat-shock-treated plants expressing the following ARF1-fluorescent-protein fusions (A–D) pARF1:ARF1-GFP (green), (E–H) pHSP:ARF1-GFP (green), (I–L) pHSP:arf1^T31N-CFP (green). (A, E, I) filipin–sterol (B, F, J) anti-KNOLLE. (D, H, L) Merged image of (A–C), (E–G) and (I, J), respectively. (M–S) anti-KNOLLE immunofluorescence (red) co-labelling with DAPI staining of DNA (blue) of wild-type Ler treated with (M) 1 h, DMSO at 0.2%, (N) 1 h, 50 μM Tyrphostin A23 (TyrA23), (O) 1 h, 50 μM Tyrphostin A51 (TyrA51), (P) 1 h, 50 μM TyrA23 followed by 1 h wash out (wash). (Q) Wild-type Col-0, (R) drp1A^rsw9, (S) drp1A^SALK_060977 (drp1A⁶⁰⁹⁷⁷). (T) Quantification of the percentage of cells in late cytokinesis displaying lateral KNOLLE mis-localization in roots of indicated genotypes or treatments. Data are mean±s.d. from 12 to 18 independent roots (n=12–18) with 84–145 cells analysed per genotype or treatment. Scale bars are 5 μm.

Figure 8

Synergistic genetic interaction between DRP1A and CPI1. (A) Five-day-old seedlings of wild-type Landsberg erecta (Ler), drp1a^rsw9, cpi1-1 and a genotyped drp1a^rsw9;cpi1-1 double mutant. (B–D) Five-day-old drp1a^rsw9;cpi1-1 double mutant seedlings identified by PCR-based genotyping with molecular markers (see Material and methods). (B) Higher magnification stereomicroscopy image of seedling in (A). Scale bars are 1 mm.