Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth

Abstract

In growing Arabidopsis root hairs, the nucleus locates at a fixed distance from the apex, migrates to a random position during growth arrest, and moves from branch to branch in a mutant with branched hairs. Consistently, an artificial increase of the distance between the nucleus and the apex, achieved by entrapment of the nucleus in a laser beam, stops cell growth. Drug studies show that microtubules are not involved in the positioning of the nucleus but that subapical fine F-actin between the nucleus and the hair apex is required to maintain the nuclear position with respect to the growing apex. Injection of an antibody against plant villin, an actin filament-bundling protein, leads to actin filament unbundling and movement of the nucleus closer to the apex. Thus, the bundled actin at the tip side of the nucleus prevents the nucleus from approaching the apex. In addition, we show that the basipetal movement of the nucleus at root hair growth arrest requires protein synthesis and a functional actin cytoskeleton in the root hair tube.

Figure 1.

Hoffman Modulation Contrast Images from a Time-Lapse Recording of a Growing Arabidopsis Root Hair. Photographs were taken every 4 min. The nucleus (N) is indicated. The sequential images demonstrate that the distance between the nucleus and the tip in growing root hairs is more-or-less fixed. Bar = 10 μm.

Figure 2.

Distribution of the Distance between the Apex and the Nucleus in Growing and Fully Grown Root Hairs of Arabidopsis. The bars represent the frequency distribution, and the lines represent a fitted normal distribution curve.

Figure 3.

Sequential Images from a Time-Lapse Recording of Growing cow1-2 Root Hairs That Develop Branches. One photograph was taken every 15 min. The nuclei (N) are indicated. During this time lapse, both tips of the left root hair were growing, and the nucleus was moving between the two branches. In the right root hair, only one tip was growing. The nucleus was at a fixed distance from this tip. The branches of the root hairs are marked with arrows, and in the root hair in which only one tip was growing, the growing tip is marked with an arrowhead. Bar = 25 μm.

Figure 4.

The Microtubule Cytoskeleton in Arabidopsis Root Hairs. (A) Microtubules formed a shallow helix in Arabidopsis root hairs and were located cortically. (B) Application of 1 μM oryzalin led to the depolymerization of all microtubules within 5 min. Bars = 10 μm.

Figure 5.

The Actin Cytoskeleton in Arabidopsis Root Hairs. (A) Full projection of a freeze-fixed root hair labeled with anti-actin antibody. A flat projection of a z-series of confocal images shows net-axially oriented, thick bundles of actin filaments in the root hair base that branch into a subapical network of thinner bundles of actin filaments. In the extreme apex of growing root hairs, an area was present in which actin filament bundles could not be detected. Bar = 10 μm. (B) and (C) Partial projections (depth of 2 μm) of confocal images of an EAC-fixed and fluorescently labeled phallacidin-stained growing root hair show thin bundles of actin filaments in the cortical plane (B) and the median plane (C) of the subapical cytoplasm. (D) A fully grown root hair stained with fluorescently labeled phallacidin shows thick bundles that loop through the tip. Fine subapical actin filament bundles were absent. (E) A living root hair injected with fluorescently labeled phalloidin at 5 min after injection. (F) Five minutes after the application of 5 μM CD, the bundles of actin filaments in the root hair tube were intact, but the subapical fine F-actin had disappeared. The photograph shows a freeze-fixed root hair labeled with anti-actin antibody. (G) The application of 1 μM LA led to the depolymerization of all of the filamentous actin within 1 min. The photograph shows a freeze-fixed root hair labeled with anti-actin antibody.

Figure 6.

Positioning of the Nucleus in Arabidopsis Root Hairs after the Application of 5 μM CD to the Medium. After CD application, the distance between the nucleus and the apex increased, whereas root hairs terminated growth. The bars represent standard deviations. n ≥ 15 per time point.

Figure 7.

The Nucleus Does Not Migrate Basipetally during or after Growth Termination if Inhibition of Gene Transcription or Protein Synthesis Starts during Cell Growth, but It Migrates if Inhibition Starts after Growth Has Stopped. In root hairs that had terminated growth before the inhibition of gene transcription or translation, the basipetal movement of nuclei was not inhibited. (A) A root hair treated for 4 h with 10 μg/mL of the translation blocker cycloheximide. The nucleus (N) was still located at the fixed distance from the tip. (B) A root hair treated for 4 h with 20 μM actinomycin D, an inhibitor of gene transcription. (C) and (D) Micrographs of a growing root hair immediately after (C) and 2 h after (D) the application of 20 μM actinomycin D. The nucleus was still located at the same distance from the tip. (E) and (F) Micrographs of a fully grown root hair immediately after (E) and 2 h after (F) the application of 20 μM actinomycin D. In fully grown root hairs, the nucleus continued to migrate when the formation of new proteins was blocked. During protein synthesis inhibition in growing root hairs, we observed a slight flattening of the root hair tip that we cannot explain.

Figure 8.

Protein Gel Blot of an Arabidopsis Root Extract Labeled with Lily Anti-Villin Antibody. On Arabidopsis root extract, the antibody gave a band of ∼135 kD (lane 3), comparable to the mass of lily villin. Lane 1, silver-stained nitrocellulose membrane; lane 2, labeled with rabbit preimmune serum.

Figure 9.

Injection of Lily Anti-Villin Antibody Causes an Increase in the Number of Cytoplasmic Strands. During the first minutes after injection, newly formed cytoplasmic strands emerged through the vacuolated area of the root hair as a result of the unbundling of the bundles of actin filaments. Three minutes after injection, the nucleus moved toward the apex, until it stopped approaching the apex after 6 to 8 min (see Figure 10). Nuclei (N) are indicated. Bar = 10 μm.

Figure 10.

Injection of Lily Anti-Villin Antibody Leads to a Decrease in the Distance between the Nucleus and the Tip. The distance between the nucleus and the apex after injection of a 1:10 dilution of anti-villin antibody into growing Arabidopsis root hairs (n = 6). As a control, a 1:10 dilution of rabbit preimmune serum was used (n = 4). The distance between the nucleus and the tip was standardized by setting the distance before injection to 100 in all observed root hairs. Error bars indicate standard errors.

Figure 11.

Effects of Unbundling of the Actin Filaments. Actin cytoskeleton configuration and the location of the nucleus (n) in control root hairs (top) and after the injection of anti-villin antibody (bottom). V, vacuole.