De novo TANGLED1 recruitment from the phragmoplast to aberrant cell plate fusion sites in maize

Abstract

Division plane positioning is crucial for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site-localized proteins, which remain at the division site after the PPB disassembles. Here, we show that the division site-localized protein TANGLED1 (TAN1) is recruited independently of the PPB to the cell cortex by the plant cytokinetic machinery, the phragmoplast, from experiments using both the PPB-defective mutant discordia1 (dcd1) and chemical treatments that disrupt the phragmoplast in maize. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and the myosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site-localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis.

Keywords: Cytoskeleton; Division; Maize; Mitosis; Phragmoplast; Preprophase band.

Fig. 1.

PPB formation and TAN1–YFP recruitment is defective in dcd1 mutants. (A,B) Model of (A) wild-type (WT) or (B) dcd1 mutant subsidiary cell divisions. Cell walls (black), microtubule structures (green), and TAN1–YFP (magenta) are shown. Below are representative images with CFP–TUBULIN labeling microtubules (green) and TAN1–YFP (magenta) labeling the division site (>) and sometimes the nucleolus indicated with a diamond (♦). (C) Observed TAN1–YFP accumulation patterns. Darker and lighter shades of magenta represent higher and lower TAN1–YFP intensities reflecting greater or less accumulation, respectively. Below, stacked bar plot comparing wild-type and dcd1 mutant cells that exhibit various TAN1–YFP patterns represented by the schematic models above. Numbers above bars represent cells examined. ***P<0.001 (Fisher's exact test). N=19 wild-type plants and 7 dcd1 mutant plants. Scale bars: 10 μm.

Fig. 2.

Defective preprophase bands and TAN1 localization result in misoriented divisions. Time-lapses of subsidiary cell divisions expressing CFP-TUBULIN and TAN1-YFP in (A) wild-type (WT) cells and (B–D) dcd1 mutant cells. Left-most columns show TAN1–YFP localization at t=0 [merge shows both TAN1–YFP (magenta) and microtubules (green)]. The last column overlays the PPB in the first frame (cyan) and final division frame (magenta). Carets (>) mark the division site. Time stamps are in hours:minutes. Scale bars: 10 µm. (E) Comparative TAN1–YFP and PPB intensity from time-lapses of dcd1 mutant cells. ‘Oriented’ describes phragmoplasts that return to the division site and ‘misoriented’ describes cell plate insertion at atypical locations. n=85 cells, N=4 plants. (F) Histogram displaying the mean TAN1–YFP fluorescence intensity of cell division sites in dcd1 mutant cells colored by division orientation at the first timepoint for time-lapses that start after prophase. n=112 cells. N=4 plants. Dotted line represents the visible detection limit or the point at which TAN1–YFP fluorescence is distinguishable over background. For E and F, blue, oriented, magenta, misoriented. AU, arbitrary units.

Fig. 3.

Cell plate insertion sites accumulate de novo TAN1–YFP. (A–D) CFP–TUBULIN (green) and TAN1–YFP (magenta) in various dividing cells. Carets (>) mark the division site and asterisks (*) mark de novo TAN1–YFP. (A) dcd1 mutant subsidiary mother cell with de novo cortex-localized TAN1–YFP indicated with asterisks. (B) Time-lapse of a dcd1 mutant cell cortex during phragmoplast expansion. Daggers (†) marks the edge of TAN1–YFP previously recruited in prophase and the triangle (▾) marks movement of the phragmoplast. Time stamps are in hours:minutes. (C) Z-projection and cortex views of wild-type and dcd1 add1 mutant embryos in telophase. Yellow dotted lines outline the cell. (D) Representative Z-projections of subsidiary mother cell phragmoplasts from CIPC and DMSO control treated samples. Asterisks mark de novo TAN1–YFP; carets mark the expected division site. (E) Bar plots of de novo TAN1–YFP cell cortex accumulation in dcd1 or dcd1 add1 mutants, or DMSO and CIPC treated wild-type plants. Numbers above bars represent total cell numbers. N≥3 plants or kernels of each genotype or treatment. ***P<0.001 (Fisher's exact test). Scale bars: 10 µm.

Fig. 4.

Actin and myosin XI motor protein OPAQUE1 increase TAN1 accumulation at de novo cell plate insertion sites. (A) Subsidiary cell divisions in the opaque1 (o1-n) mutant and wild-type (WT) siblings. (B) Boxplot of TAN1–YFP intensities at telophase in oriented and misoriented divisions in wild type and o1-n mutant cells. P=1.02×10^–12 (one-way ANOVA followed by Tukey's HSD, letters mark significant differences between groups). (C) TAN1–YFP accumulation in control and 25 µM LatB-treated dcd1 mutant cells. Bracket and asterisk indicate diffuse TAN1–YFP observed in LatB treatments. (D) Boxplot of TAN1–YFP intensity at misoriented divisions of dcd1 mutant in DMSO control (n=23 cells, N=2 plants) and 25 µM LatB (n=9 cells, N=2 plants) treatments. P=0.0417 (Wilcoxon rank sum test). (E) Time-lapse of dcd1 mutant cells in control and LatB treatments. Panels display two examples each of cells at the beginning and end of control or LatB treatment: (i,ii) Sharp TAN1 accumulation in control treatment, (iii,iv) reduced TAN1 accumulation in LatB treatment, and (v,vi) lack of TAN1 maintenance with Lat B treatment. Carets (>) mark the division site and asterisks (*) mark de novo TAN1–YFP. Images in E are representative of n=15 and 17 cells in i,ii; n=13 and 18 cells in iii,iv; and n=5 and 18 cells in v,vi all from four plants. For boxplots, the box represents the 25–75th percentiles, and the median is indicated. Whiskers are for 1.5× the interquartile range from the quartile 1 and quartile 3 boundaries. AU, arbitrary units.