Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth

Abstract

Formins are present in all eukaryotes and are essential for the creation of actin-based structures responsible for diverse cellular processes. Because multicellular organisms contain large formin gene families, establishing the physiological functions of formin isoforms has been difficult. Using RNAi, we analyzed the function of all 9 formin genes within the moss Physcomitrella patens. We show that plants lacking class II formins (For2) are severely stunted and composed of spherical cells with disrupted actin organization. In contrast, silencing of all other formins results in normal elongated cell morphology and actin organization. Consistent with a role in polarized growth, For2 are apically localized in growing cells. We show that an N-terminal phosphatase tensin (PTEN)-like domain mediates apical localization. The PTEN-like domain is followed by a conserved formin homology (FH)1-FH2 domain, known to promote actin polymerization. To determine whether apical localization of any FH1-FH2 domain mediates polarized growth, we performed domain swapping. We found that only the class II FH1-FH2, in combination with the PTEN-like domain, rescues polarized growth, because it cannot be replaced with a similar domain from a For1. We used in vitro polymerization assays to dissect the functional differences between these FH1-FH2 domains. We found that both the FH1 and the FH2 domains from For2 are required to mediate exceptionally rapid rates of actin filament elongation, much faster than any other known formin. Thus, our data demonstrate that rapid rates of actin elongation are critical for driving the formation of apical filamentous actin necessary for polarized growth.

Fig. 1.

For2 are required for polarized growth. (A) Gene models for the 3 classes of formins present in Physcomitrella. SP, signal peptide (orange), TM, transmembrane domain (purple), FH1 (green), FH2 (red), PTEN (blue), RhoGAP-Rho GTPase activating domain (brown). (B) Representative micrographs of chlorophyll autofluorescence from 1-week-old moss plants, transformed with the indicated RNAi constructs. (Scale bar, 100 μm.) (C) Quantification of plant area (gray) and circularity (black). Area is determined from the area of chlorophyll autofluorescence, and is represented as a fraction of plants transformed with the control RNAi construct. Circularity is 4πArea/Perimeter². Error bars represent SEM. Numbers above the bars represent the total number of plants measured for each condition. Statistical analyses of these data are presented in Table S1. (D) Real-time RT-PCR analysis of formin expression in 1-week-old moss plants. Expression levels were determined with respect to the Ubiquitin10 gene in control (green), For1A-F + 3 (blue) and For2AB (red) RNAi plants. Expression levels are represented as a percentage of total formin gene expression, with all For1 comprising 100% expression and all For2 comprising 100% expression. The For3 is not included, because it was not detected in plants of this age. (Inset) Magnification of the graph for For1A, B, C, and E.

Fig. 2.

For2 are required for proper actin organization. Representative images of fluorescent-phalloidin labeling of the F-actin cytoskeleton in cells from 1-week-old plants transformed with the indicated constructs. (Scale bar, 10 μm.)

Fig. 3.

Complementation analysis of formin-RNAi plants. Complementation of formin RNAi phenotype by coexpression of For2A coding sequence construct (For2Acds). Quantification of plant area (gray circles) and circularity (black circles) as a function of the amount in micrograms of For2Acds. Error bars represent SEM. Numbers above the symbols in the graph represent the total number of plants measured for each condition.

Fig. 4.

For2A-3XmEGFP and PTEN-GFP are localized to the apical region of growing moss cells. Two representative fluorescence micrographs of transgenic moss plants containing the indicated constructs. (Scale bar, 10 μm.)

Fig. 5.

Class II FH1-FH2 domains are critical for polarized growth. (A) Schematic representation of constructs used for complementation studies. PTEN-like domain (blue); class II FH1 domain (green); class I FH1 domain (yellow); class II FH2 domain (red); class I FH2 domain (orange). (B) Fluorescence micrographs of 1-week-old moss plants transformed with the indicated constructs. Panels labeled with + “Construct” are all transformed with For2AB-5′ UTR in addition to the construct indicated. (Scale bar, 100 μm.) (C) Quantification of plant area (gray) and circularity (black). Error bars represent SEM. Numbers above the bars represent the total number of plants measured for each condition. Statistical analyses of these data are presented in Table S2.

Fig. 6.

The FH1-FH2 domains from For2A, For1D, Chim2-1, and Chim1-2 have distinct actin elongating activity. Direct visualization by TIRF microscopy of the effect of FH1-FH2 domains of For2A, For1D, Chim2-1, or Chim1-2 on actin filament elongation by profilin/actin complex. The following concentrations were used: 1.2 μM actin-alexa-532, 3.6 μM moss profilin, 2 nM For2A, or 100 nM For1D; 0.8 μM actin-Alexa-568, 2.4 μM profilin, 10 nM Chim2-1, or 5 nM Chim1-2. Frames were taken at the indicated time during polymerization. Arrowheads mark the end of actin filaments growing at the rate consistent with the presence of a formin. Triangles mark actin filaments growing at the rate of a free barbed end (see yellow arrows in Movies S1–S8). Rates of elongation for control and formin associated filaments come from a measure of example filaments such as the ones marked with triangles and arrowheads, respectively, for each formin construct tested. Quantification of the data is presented in the graph. Error bars represent SD. (Scale bar, 5 μm.)