Actin is involved in auxin-dependent patterning

Abstract

Polar transport of auxin has been identified as a central element of pattern formation. The polarity of auxin transport is linked to the cycling of pin-formed proteins, a process that is related to actomyosin-dependent vesicle traffic. To get insight into the role of actin for auxin transport, we used patterned cell division to monitor the polarity of auxin fluxes. We show that cell division in the tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line is partially synchronized and that this synchrony can be perturbed by inhibition of auxin transport by 1-N-naphthylphthalamic acid. To address the role of actin in this synchrony, we induced a bundled configuration of actin by overexpressing mouse talin. The bundling of actin impairs the synchrony of cell division and increases the sensitivity to 1-N-naphthylphthalamic acid. Addition of the polarly transported auxins indole-3-acetic acid and 1-naphthyl acetic acid (but not 2,4-dichlorophenoxyacetic acid) restored both the normal organization of actin and the synchrony of cell division. This study suggests that auxin controls its own transport by changing the state of actin filaments.

Figure 1.

Cell division in nontransformed BY-2 cells follows a pattern that depends on polar auxin fluxes. A, Cell density (white symbols) and mitotic indices (black symbols) over time after subcultivation of nontransformed BY-2 cells (wild type, squares, solid curves) and BY-2 cells overexpressing YFP-mT (triangles, dotted curves). Each point represents the mean from 1,000 scored cells. B to E, Frequency distribution over cell number per file in nontransformed BY-2 cells at day 4 after inoculation in the absence of NPA (B) or in presence of 3 (C), 12 (D), or 30 (E) μm NPA. Error bars indicate se. F and G, Effect of NPA on cell division (F1, F2) and cell elongation (G1) in nontransformed BY-2 cells (white bars) versus cells overexpressing YFP-mT (black bars) at day 4 after inoculation. Each distribution is based on 3,000 individual cells from three independent experimental series. Cell division is plotted as mean number of cells per file (F1) and as cell density (F2) and cell elongation as ratio of cell length over cell width (G1). Error bars indicate se. Asterisks, YFP-mT cells died at 30 μm NPA such that a measurement of length to width ratio was not meaningful. G2, Morphology of nontransformed BY-2 cells after treatment with 30 μm NPA. Bar = 20 μm.

Figure 2.

Colocalization of YFP-mT and of rhodamine-phalloidin in BY-2 cells overexpressing YFP-mT in the absence (A–C) or in presence (D–F) of 2 μm IAA. YFP-mT fluorescence (A and D). Rhodamine-phalloidin fluorescence (B and E). C and F, Merged fluorescences of A and B or D and E, respectively. Orange color indicates areas where the images overlap and where the two markers colocalize. Bars = 20 μm.

Figure 3.

Actin filaments in nontransformed BY-2 cells (wild type, A–D) visualized by TRITC-phalloidin in comparison with cells overexpressing YFP-mT (E–J) in the absence (A, B, E, and F) and the presence (C, D, G, and H) of 2 μm IAA or in the presence of additional 2 μm 2,4-D (I and J). For each cell, a focal section in the cortical (A, C, E, G, and I) and in the central (B, D, F, H, and J) region is shown. Bars = 20 μm.

Figure 4.

Frequency distribution over cell number per file in BY-2 cells overexpressing YFP-mT at day 4 after inoculation in the absence of NPA (A), in the presence of 3 (B) or 12 μm NPA (C), or upon supplementation with 2 μm IAA (D) or additional 2 μm 2,4-D (G). As a control, the response of nontransformed (wild type) cells to 2 μm IAA (E) is shown in comparison to the distribution in the absence of IAA (F). Each distribution is based on 3,000 individual cells from three independent experimental series. Error bars indicate se.

Figure 5.

Manipulation of synchrony by 12 μm TIBA (compared to the untreated sample; A), a combined treatment with 3 μm NPA and 2 μm IAA (compared to the effect of 12 μm NPA and the untreated sample; B), and 2 μm NAA (compared to the effect of 2 μm IAA and the untreated sample; C). Frequency distribution over cell number per file is shown for nontransformed BY-2 cells (A and B) and BY-2 cells overexpressing YFP-mT (C) at day 4 after inoculation. Each distribution is based on 3,000 individual cells from three independent experimental series. Error bars indicate se.