De Novo TANGLED1 Recruitment to Aberrant Cell Plate Fusion Sites in Maize

Abstract

Division plane positioning is critical 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 a division site localized protein, TANGLED1 (TAN1), is recruited independently of the PPB to the cell cortex at sites, by the plant cytokinetic machinery, the phragmoplast. 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: Preprophase band; cytoskeleton; division; maize; mitosis; phragmoplast.

Figure 1.. Both PPB formation and TAN1-YFP recruitment is defective in the dcd1 mutant.

(A-B) Model of (A) wild-type or (B) dcd1 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) Possible TAN1-YFP accumulation patterns. Darker and lighter shades of magenta represent higher and lower TAN1-YFP intensities reflecting more or less accumulation respectively. Below, stacked barplot comparing wild-type and dcd1 cells that exhibit various TAN1-YFP patterns represented by the schematic models above. Numbers above bars represent cells examined. N = 19 wild-type plants and 7 dcd1 plants. Scale bars = 10 μm.

Figure 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 cells and (B-D) dcd1 cells. Left-most columns show TAN1-YFP localization at t = 0 (TAN1-YFP is magenta and microtubules are green in the merge). The last column overlays the the first (cyan) and last frames (magenta) of the microtubule channel showing the final division in relation to the preprophase band. Carets (>) mark the division site. Scale bars are 10 µm. (E) Comparative TAN1-YFP and PPB intensity from time lapses of dcd1 cells. “Oriented” is when the phragmoplast returned to the expected division site and “misoriented” is when the cell plate inserted at atypical locations. n = 85 cells, N = 4 plants. (F) TAN1-YFP fluorescence intensity histograms colored by division orientation outcome (blue = oriented and magenta = misoriented) for time lapses that start after prophase in dcd1. The dotted line represents the visible detection limit. n = 112 cells. N = 4 plants.

Figure 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 subsidiary mother cell with de novo cortex-localized TAN1-YFP indicated with asterisks. (B) Time lapse of a dcd1 cell cortex during phragmoplast expansion. Dagger (✝) 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 while carets mark the expected division site. (E) Barplots of de novo TAN1-YFP cell cortex accumulation in the dcd1 mutant, dcd1 add1 double mutant, 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. Asterisks indicate significant differences by Fisher’s Exact Test, P < 0.001. Scale bars = 10 µm.

Figure 4.. Actin and myosin XI motor protein OPAQUE1 increase TAN1 accumulation at de novo cell plate insertion sites.

A) Subsidiary cell divisions in the o1 mutant and wild-type siblings. B) Boxplot of TAN1-YFP intensities at telophase in oriented and misoriented divisions in wild type and o1 mutant cells. P = 1.02e-12, One-way ANOVA followed by Tukey’s HSD, letters mark significant differences between groups. (C) TAN1-YFP accumulation in control and 25 µM Lat B treated dcd1 cells. (D) Boxplot of TAN1-YFP intensity at misoriented divisions of dcd1 in DMSO control (n = 23 cells, N = 2 plants) and 25 µM Lat B (n = 9 cells, N = 2 plants) treatments. P = 0.0417, Wilcoxon rank sum sum test. (E) Timelapse of dcd1 cells in control and Lat B treatments. Carets (>) mark the division site and asterisks (*) mark de novo TAN1-YFP. Boxplot horizontal lines represent the quartiles and median. Whiskers are 1.5*IQR.