Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells.

The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.

Actin and pollen tube growth.

Actin microfilaments (MFs) are essential for the growth of the pollen tube. Although it is well known that MFs, together with myosin, deliver the vesicles required for cell elongation, it is becoming evident that the polymerization of new actin MFs, in a process that is independent of actomyosin-dependent vesicle translocation, is also necessary for cell elongation. Herein we review the recent literature that focuses on this subject, including brief discussions of the actin-binding proteins in pollen, and their possible role in regulating actin MF activity. We promote the view that polymerization of new actin MFs polarizes the cytoplasm at the apex of the tube. This process is regulated in part by the apical calcium gradient and by different actin-binding proteins. For example, profilin binds actin monomers and gives the cell control over the initiation of polymerization. A more recently discovered actin-binding protein, villin, stimulates the formation of unipolar bundles of MFs. Villin may also respond to the apical calcium gradient, fragmenting MFs, and thus locally facilitating actin remodeling. While much remains to be discovered, it is nevertheless apparent that actin MFs play a fundamental role in controlling apical cell growth in pollen tubes.

Polarized cell growth in higher plants.

Pollen tubes and root hairs are highly elongated, cylindrically shaped cells whose polarized growth permits them to explore the environment for the benefit of the entire plant. Root hairs create an enormous surface area for the uptake of water and nutrients, whereas pollen tubes deliver the sperm cells to the ovule for fertilization. These cells grow exclusively at the apex and at prodigious rates (in excess of 200 nm/s for pollen tubes). Underlying this rapid growth are polarized ion gradients and fluxes, turnover of cytoskeletal elements (actin microfilaments), and exocytosis and endocytosis of membrane vesicles. Intracellular gradients of calcium and protons are spatially localized at the growing apex; inward fluxes of these ions are apically directed. These gradients and fluxes oscillate with the same frequency as the oscillations in growth rate but not with the same phase. Actin microfilaments, which together with myosin generate reverse fountain streaming, undergo rapid turnover in the apical domain, possibly being regulated by key actin-binding proteins, e.g., profilin, villin, and ADF/cofilin, in concert with the ion gradients. Exocytosis of vesicles at the apex, also dependent on the ion gradients, provides precursor material for the continuously expanding cell wall of the growing cell. Elucidation of the interactions and of the dynamics of these different components is providing unique insight into the mechanisms of polarized growth.

Actin polymerization is essential for pollen tube growth.

Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.

Cellular oscillations and the regulation of growth: the pollen tube paradigm.

The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real-time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self-sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical-growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86-94, 2001.

Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip.

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube.

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (beta-D-glucosyl)3 Yariv phenyl-glycoside [(beta-D-Glc)3] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(beta-D-Glc)3 complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall.

Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum.

Pollen tubes show a rapid and dramatically polarized growth in which the actin cytoskeleton appears to play a central role. In order to understand the regulation of actin we characterized its associated protein, profilin, in pollen tubes of Lilium longiflorum. By using purified polyclonal antibodies prepared against bean root profilin [Vidali et al., 1995: Plant Physiol. 108:115-123] we detected in pollen grains and tubes two profilin polypeptides with molecular masses of 14.4 and 13.4 KDa, and an identical isoelectric point of 5.05. Profilin comprises approximately 0.47% of the total grain protein, with actin being approximately 1.4%. We were unable to detect a statistically significant profilin increase after germination, while the actin increased approximately 68%. We also spatially localized the distribution of profilin using immunocytochemistry of fixed cells at both the light and electron microscope level, and by fluorescent analog cytochemistry on live cells. The results show that profilin is evenly distributed throughout the cytoplasm and does not specifically associate with any cellular structure.

Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy.

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells. Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40-75 microns from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.

Tip-localized calcium entry fluctuates during pollen tube growth.

Studies have been conducted on the dynamics of Ca2+ entry in pollen tubes using ratiometric ion imaging to measure the intracellular gradient and an ion selective vibrating electrode to detect the extracellular influx. A steep tip-focused gradient occurs in all species examined, including Lilium longiflorum, Nicotiana sylvestris, and Tradescantia virginiana. Anlaysis of Lilium pollen tubes loaded with dextran conjugated fura-2 reveals that the gradient derives from Ca2+ entry that is restricted to a small area of plasma membrane at the extreme apex of the tube dome. Since the apical membrane is continually swept to the flanks during tube elongation, either Ca2+ channels are specifically retained at the extreme apex or, as seems more likely, the Ca2+ channels which were active at the tip rapidly inactivate, as new ones are inserted during vesicle fusion. Ratiometric imaging further indicates that the high point of the gradient fluctuates in magnitude from 0.75 to above 3 microM, during measuring intervals of 60 sec, with the elevated points being correlated with an increased rate of tube growth. Independent analysis of the growth at 2- to 3-sec intervals reveals that the rates can fluctuate more than threefold; tubes longer than 700 mu m exhibit oscillations with a period of 23 sec, while tubes shorter than 700 mu m display erratic fluctuations. Inhibition of pollen tube growth caused by mild temperature shock or caffeine (1.5 to 3.0 mM) is correlated with the dissipation of the tip-focused gradient and the Ca2+ influx. Recovery from both treatments is denoted by a global swelling of the pollen tube tip, concomitant with a high transient entry of Ca2+ in the tip. The location of the highest Ca2+ domain within the tip region defines the point from which normal cylindrical elongation will proceed.

Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata.

The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.

Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media.

Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.

Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes.

A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies. The epitopes of these antibodies were found to reside on the myosin heavy chain head and rod portion and were, therefore, designated anti-S-1 (myosin S-1) and anti-LMM (light meromyosin). On Western blots of the total soluble pollen tube proteins, both anti-S-1 and anti-LMM label a polypeptide of approximately 175,000 Mr. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the whole length of the tube, often with an enrichment in the tube tip. These fluorescent spots are thought to represent vesicles and/or organelles in the pollen tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope. The different staining patterns of the nucleus between anti-S-1 and anti-LMM may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and/or organelles.

Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining.

A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.