Localization of profilin- and actin-like immunoreactivity in in vitro-germinated tobacco pollen tubes by electron microscopy after special water-free fixation techniques

Double immunogold labeling of profilin and actin was performed on ultrathin sections of in vitro germinated tobacco pollen using different anti-profilin and anti-actin antibodies. Since profilin, besides its role as an actin-binding protein, is known as an allergen, water-free fixation in p-formaldehyde vapor was used. Profilin labeling occurs throughout the cytoplasm of the pollen tube. There is no profilin in the pollen tube wall. Actin reactivity is found in the cytoplasm and extracellularly in the pollen tube wall where three out of four different anti-actin antibodies give a positive signal. This labeling of the pollen tube wall may result from a wall-bound actin, an isoform of actin not yet described or from the presence of a molecule immunologically indistinguishable from actin.     

Profilin revealed in pollen nuclei: immuno-electron microscopy of high-pressure frozen Ledebouria socialis Roth (Hyacinthaceae)

 Cryoimmobilization by high-pressure freezing, combined with cryosubstitution and resin embedding, allowed accurate retention in situ of the small (12–15 kDa) water-soluble protein, profilin, in anthers of Ledebouria socialis Roth (Hyacinthaceae). The subcellular distribution of profilin was investigated by using post-embedding immunogold labelling with rabbit antisera raised against recombinant birch profilin (RP2) or birch COOH-terminal profilin peptide (RP3). The patterns observed in mature pollen grains are novel to eukaryotic organisms: profilin was consistently demonstrated within both the vegetative and generative nuclei, an addition to its well-known presence in the cytoplasm. Methodological and immunological aspects, as well as possible biological implications, of this finding are considered. Received: 17 March 1997 / Revision accepted: 10 July 1997     

Reversal of profilin inhibition of actin polymerization invitro by erythrocyte cytochalasin-binding complexes and cross-linked actin nuclei

Actin polymerization in 2 mM MgCl₂ is known to be inhibited by profilin. We found that small amounts of cytochalasin-binding complexes from human red cell membranes or actin nuclei cross-linked by $\text{p}\text{-}\text{NN′}$-phenylenebismaleimide can reverse the inhibitory action of profilin, leading to the rapid polymerization of the actin. This type of polymerization is inhibited by low concentrations of cytochalasin B. These results indicate that (a) the complexes and nuclei promote actin polymerization in the presence of profilin by providing sites onto which actin monomers can be added, and (b) profilin and cytochalasin B affect two distinct steps (i.e. nucleus formation and filament elongation, respectively) in the polymerization reaction.     

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the musselMytilus galloprovincialis

Summary Cell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesiles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cystoskeleton inMytilus galloprovincialis haemocytes are discussed.     

Profilin promotes barbed-end actin filament assembly without lowering the critical concentration

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1.Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin.Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin.Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4.Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4.Actin and ushers actin as a Profilin.Actin complex onto growing barbed filament ends.     

黄蝉和姜花生殖细胞内肌动蛋白微丝的定位

用荧光标记的鬼笔碱染色,对离体的黄蝉和姜花的生殖细胞内肌动蛋白微丝的分布进行了研究,结果证明两种植物的生殖细胞内部都存在一个微丝网络,黄蝉生殖细胞的比姜花的简单,微丝束较粗。但姜花生殖细胞的网络微丝束比黄蝉的更紧密地环绕着核。用免疫荧光技术在黄蝉生殖细胞的分裂前期和中期,可以观察到一些微丝束的存在,但在分裂后期和末期细胞内的肌动蛋白则变为颗粒状。     

The Cytoskeletal Proteins Actin and Tubulin in the Spermatozoa of Four Decapod Crabs (Crustacea,Decapoda)

The mature spermatozoa of four species of European decapod crabs (Clibanarius erythropus, Maja squinado, Cancer pagurusand Potamon fluviatile)have been investigated using indirect immuno-fluorescence techniques for the presence of the cytoskeletal proteins actin and tubulin. Indirect immunofluorescence labelling with monoclonal anti-actin antibody and three different monoclonal anti-tubulin antibodies indicate that actin is present in the spermatozoa of all four species, but tubulins are restricted to the two species with microtubular arms, Clibanariusand Maja.The pattern of actin fluorescence varies between the spermatozoa of the four species, with Majaand Cancershowing intense fluorescence in the acrosome vesicle and in elements of the sperm cell involved in the acrosome reaction. The spermatozoon of each species is described ultrastructurally using transmission electron microscopy and correlations made between observed patterns of fluorescence and the cellular components described. No obvious filamentous actin (F-actin) is visible in the electron micrographs of the spermatozoa of any of the species. In most cases the fluorescence is sufficiently specific to indicate in which region of the mature sperm cell the actin and tubulin occurs. Actin is acrosomal in Maja, Cancerand Potamonbut appears to be cytoplasmic in Clibanarius, while the tubulins appear only to be present in the cytoplasm of Clibanarius, Majaand Cancer.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band     

Microinjection of profilins from different sources into the green algaMicrasterias causes transient inhibition of cell growth

Summary Recombinant profilins from different sources (Betula verrucosa, Schizosaccharomyces pombe, Acanthamoeba castellani, or man) cause marked effects on cell growth and morphogenesis when microinjected into growing cells of the green algaMicrasterias denticulata. Whereas control injections with -lactoglobulin only result in a slight delay of cell growth, when profilin is injected cell differentiation ceases and only resumes about 1 to 2 h after the injection, depending on the dose. The resulting cell does not show any malformations, but is reduced in size and retarded in differentiation compared to controls. As a consequence of the profilin microinjection the pattern of cytoplasmic streaming and cytoplasmic structure are also altered. Gelsolin, injected for comparison, leads to minor retardation of cell development but produces less marked effects than profilin. Microinjection of fluorescently labeled profilin shows even distribution throughout the cytoplasm and more intense fluorescence in the nucleus. Electron microscopical investigations of cells fixed immediately after profilin injection show a normal distribution of dictyosomes, ER cisternae, microtubules, and secretory vesicles compared to noninjected controls at the same developmental stage. Our results indicate that disturbance of the natural actin turnover by the injection of actin-binding proteins strongly affects development ofMicrasterias, corroborating a key role of actin in the morphogenetic process.     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期(即生殖细胞在形成后从靠壁部位移向中央部位的阶段)的水稻(Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程.在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成.微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状.尔后,中心微丝不断延长,最终在细胞中央的胞质内形成一个非常复杂的类似多个纺锤体结合在一起的网络结构.这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质(或称周质)的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构.未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系.     

Nitration of profilin effects its interaction with poly (L-proline) and actin

Profilin from bovine spleen was nitrated with peroxynitrite; immunoblotting and spectrophotometric quantitation of nitrotyrosine residues suggested nitration of a single tyrosine residue in profilin with a stoichiometry of 0.6 mol of nitrotyrosine/mole of profilin. A decrease in the nitrotyrosine immunoreactivity of nitroprofilin during digestion with carboxypeptidase Y indicated that nitrotyrosine is located at the C-terminus of profilin. Nitroprofilin interaction with ligands such as phosphatidylinositol 4,5-bisphosphate, actin and poly (l-proline) was analyzed by monitoring the tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no significant difference in affinity of nitroprofilin to phosphatidylinositol 4,5-bisphosphate (K(d) of 4.8 +/- 0.5 muM for profilin, and K(d) of 5.7 +/- 0.6 muM for nitroprofilin), while poly (l-proline) binding studies revealed a twenty-fold increase in the affinity of profilin to poly (l-proline) upon nitration (K(d) of 21.8 +/- 1.7 muM for profilin, and K(d) of 1.1 +/- 0.1 muM for nitroprofilin). Actin polymerization studies involving pyrene-labeled actin indicated that profilin nitration inhibits the actin sequestering property of profilin. The critical actin monomer concentration (C(c)) was 150 and 250 nM in the presence of nitroprofilin and profilin, respectively. Thus, nitric oxide and free radicals produced under different conditions could alter the functions of profilin through nitration, such as its interaction with actin and poly (l-proline).     

The Effects of ADF/Cofilin and Profilin on the Conformation of the ATP-Binding Cleft of Monomeric Actin

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer''s ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound ɛ-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound ɛ-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound ɛ-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.     

Cell motility: proline-rich proteins promote protrusions

Many proline-rich proteins participate in delivering actin monomers to specific cellular locations where actin-rich membrane protrusions, such as ruffles, filopodia and microspikes, are formed. These protrusions are necessary for cell motility. Actin monomer is usually delivered to the site of polymerization in the form of profilactin - a complex of G-actin with a polyproline-binding protein, profilin. Here, we describe proline-rich proteins involved in regulating actin polymerization and classify them according to their role in recruiting profilin to the membrane.     

Profilin connects actin assembly with microtubule dynamics

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期（即生殖细胞在形成后从靠壁部位移向中央部位的阶段）的水稻（Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程。在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成。微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状。尔后,中心微丝不断瞎长,最终在细胞中央的胞质内形成一个非常 类似多个纺锤体结合在一起的网络结构。这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质（或称周质）的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构。未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系。     

The spermatogenous body cell of the coniferPicea abies (Norway spruce) contains actin microfilaments

Summary In conifer pollen, the generative cell divides into a sterile stalk cell and a body cell, which subsequently divides to produce two sperm. InPicea abies (Norway spruce, Pinaceae) this spermatogenous body cell contains actin microfilaments. Microfilament bundles follow the spherical contour of the body cell within the cell cortex, and also traverse the cytoplasm and enmesh amyloplasts and other organelles. In addition, microfilaments are associated with the surface of the body cell nucleus. The sterile stalk cell also contains microfilament bundles in the cytoplasm, around organelles, and along the nuclear surface. Within the pollen grain, microfilament bundles traverse the vegetative-cell cytoplasm and are enriched in a webbed cage which surrounds the body cell. Microfilaments were identified with rhodamine-phalloidin and with indirect immunofluo-rescence using a monoclonal antibody to actin. The majority of evidence in literature suggests that the spermatogenous generative cell in angiosperms does not contain actin microfilaments, so the presence of microfilaments within the spermatogenous body cell inP. abies appears to be a fundamental difference in sexual reproduction between conifers and angiosperms.     

Cyclical transformations of the actin cytoskeleton of hyacinth pollen subjected to recurrent vapour-phase hydration and dehydration

Summary— Transformations of the actin cytoskeleton of the pollen of Hyacinthus orientalis during cycles of vapour-phase hydration and dehydration have been examined using a non-fixation, DMSO permeabilisation method for TRITC-phalloidin staining, coupled with microwave stabilisation. In freshly shed pollen actin appears: a) at the plasmalemma in the form of extended, thin fibrils co-oriented with the cellulosic microfibrils of the contiguous intine; b) as a sheath investing the generative cell; c) as spicules around the vegetative nucleus; and d) in scattered spicules in the cytoplasm. During hydration in 85–95% relative humidity (RH), actin from all of these sites is progressively translated into a system of extended fibrils in the vegetative cell, concurrently with the onset of movement in the cytoplasm. Dehydration at 5–7% RH reverses this process, actin accumulating in rodlets, spicules or larger fusiform bodies in close association with the generative cell and vegetative nucleus, and also in the cytoplasm. The fibril system initially present at the plasmalemma is not restored. After ten cycles of hydration and dehydration 3.6% of the grains remained germinable. The ecological significance of the findings is noted, and the possibility that the observed transformations result from variation in the Ca²⁺ concentration in the cytosol as the water content of the cytosol changes is considered.     

Phosphorylation of Amoeba G-actin and its effect on actin polymerization

Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.