Low concentration of LatB dramatically changes the microtubule organization and the timing of vegetative nucleus/generative cell entrance in tobacco pollen tubes

Abstract

Low concentration of LatB inhibits not only the actin polymerization, but also induces profound alteration of MT distribution in pollen tubes of Nicotiana tabacum. The short randomly oriented MTs in the apical and subapical regions, became organized as bundles forming subapical rings or basket-like structures, surrounding the apex. Moreover, the depolymerization of AFs in the cortical regions of the apex and subapical region affects the timing of entrance of the vegetative nucleus and generative cell into the pollen tube.

After reaching a compatible stigma, pollen grains hydrate and translate external stimuli into specific actin filament (AF) distribution and secretion patterns, to allow pollen tube growth.^1-3 On the way to reach the ovary, the interaction with the female tissues largely depends on the lipid/protein composition of PM and cell wall.^4-7 Control of exocytosis to and endocytosis from the PM, largely modulated by the cytoskeletal apparatus,⁸ enables precise regulation of the interaction between the cell and its environment and allows the efficiency of vegetative nucleus (VN) and generative cell (GC) movement. Actin filaments (AFs) and microtubules (MTs) form the cytoskeleton of pollen tubes, running parallel in subapical regions of the cell to form an interconnected cortical system with the plasma membrane (PM) and elements of the endoplasmic reticulum.^9-11 Close association of MTs with actin filaments has also been shown by immunogold labeling, suggesting that MTs may act as guide elements for actin filaments or vice versa.¹¹ We performed experiments of AFs inhibition, to assess the effect of localized actin polymerization on endocytosis and endosome trafficking¹² and parallely, we have checked the integrity of MTs. Surprisingly, the MT apparatus was dramatically affected by 5nM LatB, suggesting that AFs could be involved directly or indirectly in MT organization in tobacco pollen tube.

Our recent paper showed that 5nM Lat B inhibits the actin polymerization in the tip and in the cortical subapical region of the tube without disturbing AFs in central part of the tube.¹² In order to observe whether drugs disturbing cortical AFs also affect the distribution of MTs, immunolocalization experiments, using an anti-tubulin monoclonal antibody, were performed in control cells and in pollen tubes incubated for 15 and 45 min with 5 nM LatB. In control cells, Mts are organized in longitudinal bundles in the shank of the tubes (up to 30–40 min from the tip) (Fig. 1a, yellow arrows), whereas in the apical dome, Mts appeared as short, randomly oriented strands (SS) (Fig. 1a, blue arrows). A second type of control experiment, using DMSO did not affect the MT distribution in the shank (Fig. 1b, yellow arrows), whereas the extent of the apical region characterized by SS MTs, appeared to be less extended respect to the control (Fig. 1b, blue arrows), suggesting that DMSO stabilizes the MT apparatus. Surprisingly, the distribution of MTs in the apical dome (30–40 min from the tip PM) was dramatically affected by 5 nM LatB, since after 15 and 45 min of incubation the short MT fragments changed into long strands often oriented transverse to the long axis of the tube (Fig. 1c, d, white arrows). In regions closer to the apex (5–10 min from the tip) the Mt bundles either exhibited orientation transverse to the main axis (subapical rings, SR) of the tube or were organized in a basket-like structure, surrounding the apex (apical network, AN) (Fig. 1c, d red arrows). A fourth array of MTs was observed in the apical dome of tubes incubated with 5nM LatB, consisting of medium MT strands (MS) (see Figure 1c, green arrow). In the shank the alteration of the MT apparatus was less evident: long longitudinally oriented MTs were bridged together, however the MT bundles appeared much thicker respect to those seen in the control and DMSO treated pollen tubes (Fig. 1c, d, yellow arrows), further supporting the idea that 5nM LatB. induce stabilization of MTs.

Figure 1. Effect of 5 nM LatB on MT organization. (A) In control cells, MTs form cortical bundles in the shank of the tube (yellow arrows) and short MT segments (SS) are seen in the apical and subapical regions up to 40 min from the tip(blue arrows). (B) In the presence of DMSO the MT bundles are more extended toward the apex (blue arrow). (C, D) After 15 and 45 min of incubation with LatB the arrangement of MTs in the apical and subapical regions changed radically: the SS of MT became long bundles lying transverse to the main axis of the tube (white and red arrows). Bar = 10 min. (E) MT arrays observed in the presence of 5nM LatB were compared with those in control and DMSO treated cells. The percentage of pollen tubes showing medium strands (MS) and/or subapical rings-apical network (SR-AN) in the apical dome gradually increases after 15 and 45 min incubation with LatB respect to control and DMSO where SS are mostly observed. The x axis shows time points at which samples were taken. Error bars indicate 68% confidence intervals (100 tubes for each time point in three replicate experiments).

To quantify the effect of 5nM LatB on the MTs organization in the apical dome, the different MT arrays were counted in two independent experiments of immunolocalization with and without LatB. The results of these assays showed that whereas in control and DMSO treated tubes SS were present in the tip region of almost all cells (Fig. 1e), the percentage of tubes showing SS decreased dramatically after 15 and 45 min incubation with LatB. Moreover, a parallel increase of MS and subapical rings-apical networks (SR-AN) was observed in the apical dome (Fig. 1e).

These data showed that the destabilization of AFs in the cortex of the apical and subapical regions of tobacco pollen tubes induces the development of new MT arrays characterized by longer polymers, presumably organized as bundles, that substitute the short, randomly oriented MT segments. Changes in the organization of MTs have been previously observed in other species; in pollen tubes of Papaver rhoeas in the presence of 10 nM LatB long, transversely oriented MTs were observed in the proximal region of LatB treated tubes instead than short MT strands normally present in the control.¹³ At higher concentration LatB induced complete depolymerization of both AFs and MTs,¹⁴ suggesting that one filamentous system supports the organization of the other in Papaver rhoeas pollen tubes. Our findings showed that also in Nicotiana tabacum pollen tubes the integrity of the actin cytoskeleton is a critical condition for MT organization in the distal region of the tube. A tight relation between the two systems was also suggested by experiments using taxol on high speed supernatants of tobacco pollen tubes. These experiments allowed to isolate MT bundles together with thin filaments that were identified as AFs after staining with rhodamine-phalloidine.¹⁵ Physical interactions between AFs and MTs have been shown recently, by in vitro binding experiments, to be due to metabolic enzymes.¹⁶ It remains to be established what is the mechanism that links the organization of AFs to that of MTs. The nucleation of MTs in pollen tubes was not clarified, several studies suggested that MT-nucleating centers could be associated with the apical-subapical PM.¹⁷ However, how the inhibition of AF polymerization could affect MT nucleation/organization remains obscure at the moment. Moreover, although LatB has a profound effect on endocytosis in the subapical region of the tube.¹² the inhibition of PM internalization could not be due to changes in MT arrangement since drugs destabilizing MTs do not inhibit internalization of FM4–64 or positively charged nanogold particles (work in progress).

Other observation performed during our experiments with 5nM LatB convinced us that actin dynamic was also involved in regulating VN/GC movement along the tube. Particularly, the use of LatB accelerated the entrance and the transport of the male germ unit to the tip region. Three independent experiments of AF depolymerisation by 5nM LatB, showed that the percentage of cells in which the male germ unit was already within the tube increased significantly after 45 and 75 min incubation with 5nM LatB, respect both to the control and DMSO treated cells (Fig. 2A a, and 2B a-f) (p = 0,003583 and p = 0,022452 respectively, LatB vs. DMSO). Since 5nM LatB also profoundly affected the MT apparatus, in order to determine whether the timing of the entrance of male germ unit in the pollen tube was due to changes in MT organization, pollen tubes were treated with 5M Nocodazole or 0.05% DMSO. In the presence of DMSO, that was shown to stabilize pollen tube MTs, the percentage samples having the male germ unit into the tube was slightly lower respect to the control (Fig. 2A, b). On the other hand, the presence of 5M Nocodazole, that affects MT polymerization in the apical and subapical region of the tube (data in progress) did not influence VN/GC movements respect to DMSO. Therefore, these data led to hypothesize that alteration in the movement of the male germ unit could not be attributed to changes in MTs organization. Studies of immunolocalization using antibodies raised to the myosin heavy chain have revealed that a related polypeptide occurs in association with the GC and VN in pollen tubes^18,19 leading to hypothesize that the acto-myosin system contributes to transport the male germ unit. Our data further support this idea, suggesting that cortical AFs rather than AFs in the central part of the cytoplasm are involved in the VN/GC movement.

Figure 2. Effect of 5 nM LatB and 5 M Nocodazole on the movement of the VN/GC. Graphs showing the percentage of cells having the VN/GC within the pollen tube in control conditions and in the presence of 5nM LatB/DMSO (A) and in the presence of 5M Nocodazole/ DMSO (B). Control pollen tubes stained with DAPI were compared with pollen tube treated with DMSO or 5nM LatB. Pollen tubes at t⁰ do not have VN/CG in the tube (A). After 15–45 min incubation with LatB some of the tubes already show the male germ unit (D, E) In the presence of LatB for 75 min, a higher number of pollen tube contained the VN/GC (F) respect to control (B) and DMSO treated pollen tubes (C).

Materials and Methods

DAPI staining

Pollen of Nicotiana tabacum (L.) was allowed to germinate in BK medium for one hour (t⁰) and then 5 nM LatB (BIOMOL International, NY, USA) or a correspondent amount of DMSO was added to the culture. Parallel experiments were performed incubating pollen tubes with 5M Nocodazole (Sigma) or the corresponding amount of DMSO. Samples of control, LatB/Nocodazole and DMSO treated tubes were taken after 15 and 45 and 75 min. and fixed as reported below. Pollen tube were then incubated with 2M DAPI (Invitrogen) for 1 h at room temperature. To quantify the percentage of cells having the male germ unit within the tube one hundred tubes were considered for each sample in three (LatB) and two (Nocodazole) independent experiments and the cells showing the VN/GC in the tube at each time point were counted with a LEICA DM-RD light/epifluorescence microscope equipped with a Ploe mopack 1 System and a mercury lamp (OSRAM HBO 100W) with a UV-1A filter (365nm excitation filter, 400nm barrier filter). (Microsystems Heidelberg GmbH, Germany). Data were analyzed by the Excel Program.

MT immunolabelling

For MT labeling, tobacco pollen tubes were incubated with the fixing buffer pH 7.0 (100 mM Pipes, 5 mM MgSO₄, 0.5 mM CaCl₂, 10% (w/v) sucrose, 0.1% (w/v) MBS, 3.7%(v/v) formaldehyde) for 30 min. The cell wall of pollen tubes was partially digested by incubating cells with 2% (w/v) cellulysin for 2 min in the dark and then the cells were incubated for 3 min with cold methanol at -20°C. Mts were stained by indirect immunofluorescence using an anti- tubulin monoclonal antibody (clone B-5–1-2, purchased by SIGMA) (1:200 final dilution) and a secondary goat anti-mouse IgG FITC-conjugated polyclonal antibody (Molecular Probes) (1:200 final dilution). Optical sections (1 min) and three-dimensional projections of specimens were obtained by CLSM (Leica Microsystems, Heidelberg GmbH, Germany). A 40X objective and BHS filter set were used for imaging. All images were recorded using a stepper motor to make Z-series. To quantify changes of MT organization in the presence of 5nM LatB 50 tubes were considered for each sample in two independent experiments. Data were analyzed by the Excel Program.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.