Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro

Summary. Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5–15 μm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes. Correspondence: Shao-ling Zhang, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China.     

Actin filaments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio

Summary Actin filaments in the microridges on the surface of the fish oral mucosa taken from Cyprinus carpio were examined by electron microscopy after detergent extraction and decoration with myosin subfragment 1. After extraction with saponin, an irregular and densely packed meshwork of actin filaments was observed in the bases of the microridges, just lateral to the tight junctions with their fibrous undercoats. Actin filaments formed cores in the microridges and numerous linkages were seen between the filaments and the plasma membrane. Extraction with Triton X-100 and decoration with myosin subfragment 1 showed the ends of the actin filaments to be associated with the plasma membrane of the microridges, and in the bases of microridges the filament ends were anchored to intermediate filaments. Some actin filaments interconnected with the fibrous undercoats of the tight junctions. On the basis of these observations, the mechanism of the formation of microridges, including their pattern, is discussed.     

Organization of actin filaments in regenerating and outgrowing subprotoplasts from pollen tubes ofNicotiana tabacum L.

The dynamics of actin-filament organization in pollen-tube subprotoplasts ofNicotiana tabacum L. cv. Samsun during regeneration and outgrowth was examined using phalloidin probes and a non-fixation method. A succession of actin arrays was examined during subprotoplast regeneration that strongly resembled the actin dynamics described for developing microspores by Van Lammeren et al. (1989, Planta178, 531–539) and activated pollen by Tiwari and Polito (1988, Protoplasma147, 5–15). At the end of the succession the actin filaments often became extended between two opposite polar foci. The ordering of the cortical actin filaments reflected a polarity in the subprotoplasts which determined the plane of outgrowth. The site of outgrowth was often marked by a ring of actin filaments. As growth proceeded and tube-like structures were formed, the arrangement of cortical actin filaments was found to be transverse to the elongation axis. Since the patterns of actin distribution were identical in both caryoplasts and cytoplasts, it was concluded that the pollen-tube cytoplasm has the intrinsic capacity of reorganizing actin filaments and imposing polarity on the spherical subprotoplasts.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Changes in the relationship between actin filaments and the plasma membrane in culturedZinnia cells during tracheary element differentiation investigated by using plasma membrane ghosts

Aggregates of actin filaments appear immediately before secondary wall thickening during tracheary element differentiation in isolatedZinnia cells. An analysis of plasma membrane ghosts revealed that the aggregates were bound to the plasma membrane. The properties of the binding of actin filaments to the plasma membrane were investigated in this system. Present address and for correspondence: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate, 024 Japan.     

The cytoskeleton of spreadingDictyostelium amoebae

Summary The cytoarchitectural elements ofDictyostelium discoideum amoeba have been visualized by light and electron microscopy in cells prepared with mixtures of glutaraldehyde and Triton-X-100. After negative staining, the peripheral regions of spreading amoebae show a complex meshwork of actin filaments, the majority of which were less than 0.25 microns in length. Multiple branch points, end to side abutments and cross-overs were characteristic features of the actin meshworks. Filopodia extending from the cell periphery consisted of bundles of actin filaments that penetrated into and merged with the actin meshworks in the spreading lamellae. Microtubules emanating from the nucleus associated body penetrated to differing extents into the actin meshworks, sometimes extending close to the cell periphery.Dictyostelium cytoskeletons preparted as described here should prove useful for further studies on the locomotory mechanism.     

Actin filaments line up acrossTradescantia epidermal cells,anticipating wound-induced divison planes

Summary This paper describes the role of actin filaments in setting up the phragmosome — the transvacuolar device that anticipates the division plane — and in forming a supracellular system that seems to override cell boundaries. Tradescantia leaf epidermal cells were induced to divide by wounding the leaf. New division planes formed parallel to slits, and encircled puncture wounds — the new division planes lining up across cells, instead of the joints being off-set as in normal, unwounded tissue. Within 30 min after wounding, rhodamine phalloidin staining showed that a belt of fine, cortical actin filaments formed parallel to the wound. In the next stage, migration of nuclei to a wall adjacent to the wound, involved pronounced association of actin filaments with the nucleus. Migration could be inhibited with cytochalasin D, confirming the role of actin in traumatotaxis. Later still, actin strands were seen to line up from cell to cell, parallel to the wound, anticipating the future division plane. Next, actin filaments accumulated in this anticlinal plane, throughout the depth of the cell, thereby contributing to the formation of the phragmosome. The phragmosome has been shown in previous work (Flanders et al. 1990) to contain microtubules that bridge nucleus to cortex, and is now found to contain actin filaments. Actin filaments are therefore involved in the key stages of nuclear migration and division plane alignment. The supracellular basis of actin alignment is discussed.Dedicated to the memory of Professor Oswald Kiermayer     

Identification of a birefringent structure which appears and disappears in accordance with the shuttle streaming inPhysarum plasmodia

Summary R-HMM (rhodamine-heavy meromyosin) stained the birefringent fibrous structure which appears and disappears cyclically in parallel with the periodic shuttle streaming in the plasmodium ofPhysarum polycephalum. In addition, 0.6 M KI readily made the birefringent fibrils fade away. These results clearly show that the birefringent fibrils are composed of actin filaments and prove the possibility of actin filaments to alter in the aggregation state during the cyclic production of the motive force responsible for the cytoplasmic streaming.     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

Actin filaments predominate in morphogenic cell stages, whereas plaques predominate in non-morphogenic cell stages in Peronosporomycetes

We investigated the structural distribution of both types of actin arrays, filaments and plaques, in a soil-borne phytopathogenic peronosporomycete (oomycete), Aphanomyces cochlioides, under standardized host-free bioassays. The phenomenon was monitored during progression through all the asexual developmental processes of the organism. It was noted that the filamentous-form of actin was predominant during the morphogenic (morphologically active) stages of development. Conversely, during non-morphogenic (morphologically quiescent) stages, plaques dominated. From these analyses, we proposed a criterion that predominance of an actin form relates to, and precedes the morphological behaviour of a cellular stage in Peronosporomycetes. A decrease in the quantity of plaques in the encysted zoospore (non-morphogenic stage) during its developmental progression into morphogenic stages, both in germination and regeneration processes, asserted the notion that plaques function as the organization centres and are related to the reorganization of cell structure and the transition of the cell into a new stage. Furthermore, polymerization of filamentous-form during emergence stages in zoospore regeneration process revealed that filaments render motility to a developing zoospore. This unprecedented function of filaments in the developing zoospores was demonstrated using nicotinamide (0.8 × 10⁻⁶ m), which did not cause actin disruption, but could induce zoospore encystment, and its further replacement with water triggered the zoospore emergence process. Additionally, by using latrunculin B, an actin polymerization inhibitor, we also demonstrated the functional necessity of actin during various developmental processes in Aphanomyces.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.     

Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules

Summary. Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B. Correspondence: Department of Cytophysiology, University of Łódź, Pilarskiego 14, 90-231 Łódź, Poland.     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole     

Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erythrocephala (Insecta: Diptera)

Summary Calliphora erythrocephala has cross-striated cardiac muscle cells with A, I and Z-bands. The diameters of the myosin and actin filaments are 200–250 Å and 85 Å respectively and the length of the myosin filaments (A-band) is approximately 1.5 . Usually 8–10 actin filaments surround each myosin filament.The myocardial cells show a well-developed membrane system and interior couplings. A perforated sheet of SR envelopes the myofibrils at the A-band, dilates into flattened cisternae at both A-I band levels before it merges into a three-dimensional net-work between the actin filaments of the I-bands and between the dense bodies of the discontinuous Z-discs. The T-system consists of broad flattened tubules running between the myofibrils at the A-I band levels forming dyads with the SR-cisternae. Longitudinal connections between the transverse (T-) tubules often occur.It is suggested that this well-developed SR may be an adaptation to facilitate a rapid contraction/relaxation frequency by an effective Ca²⁺ uptake.     

Three-dimensional organization and dynamic changes of the actin cytoskeleton in embryo sacs ofZea mays andTorenia fournieri

Summary Actin organization was observed inm-maleimidobenzoic acid N-hydroxysuccinimide ester(MBS)-treated maize embryo sacs by confocal laser scanning microscopy. The results revealed that dynamic changes of actin occur not only in the degenerating synergid, but also in the egg during fertilization. The actin filaments distribute randomly in the chalazal part of the synergid before fertilization; they later become organized into numerous aggregates in the chalazal end after pollination. The accumulation of actin at this region is intensified after the pollen tube discharges its contents. Concurrently, actin patches have also been found in the cytoplasm of the egg cell and later they accumulate in the cortical region. To compare with MBS-treated maize embryo sacs, we have performed phalloidin microinjection to label the actin cytoskeleton in living embryo sacs ofTorenia fournieri. The results have extended the previous observations on the three-dimensional organization of the actin arrays in the cells of the female germ unit and confirm the occurrence of the actin coronas in the embryo sac during fertilization. We have found that there is an actin cap occurring near the filiform apparatus after anthesis. In addition, phalloidin microinjection into the Torenia embryo sac has proved the presence of intercellular actin between the cells of the female germ unit and thus confirms the occurrence of the actin coronas in the embryo sac during fertilization. Moreover, actin dynamic changes also take place in the egg and the central cell, accomplished with the interaction between the male and female gametes. The actin filaments initially organize into a distinct actin network in the cortex of the central cell after anthesis; they become fragmented in the micropylar end of the cell after pollination. Similar to maize, actin patches have also been observed in the egg cortex after pollination. This is the first report of actin dynamics in the living embryo sac. The results suggest that the actin cytoskeleton may play an essential role in the reception of the pollen tube, migration of the male gametes, and even gametic fusion.     

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: I. The effect of cadmium

Summary. The aim of the study was to elucidate the effect of cadmium ions on the arrangement of the actin and tubulin cytoskeleton, as well as the relationships between cytoskeletal changes and growth processes in the green filamentous alga Spirogyra decimina. Batch cultures of algae were carried out under defined conditions in the presence of various cadmium concentrations. In control cells, the cytoskeleton appeared to be a transversely oriented pattern of both microtubules and actin filaments of various thickness in the cell cortex; colocalization of cortical microtubules and actin filaments was apparent. Microtubules were very sensitive to the presence of cadmium ions. Depending on the cadmium concentration and the time of exposure, microtubules disintegrated into short rod-shaped fragments or they completely disappeared. A steep increase in cell width and a decrease in growth rate accompanied (and probably ensued) a very rapid disintegration of microtubules. Actin filaments were more stable because they were disturbed several hours later than microtubules at any cadmium concentration used. When cadmium ions were washed out, the actin cytoskeleton was rebuilt even in cells in which actin filaments were completely disintegrated at higher cadmium concentrations (40 or 100 μM). The much more sensitive microtubules were regenerated after treatment with lower cadmium concentrations (10 or 15 μM) only. Correspondence and reprints: Centre of Phycology, Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, 379 82 Třeboň, Czech Republic.     

Localization of actin in the electrocyte of Electrophorus electricus L.

Summary The cytoplasm of the electrocyte of Electrophorus electricus possesses a meshwork of 7-nm thick filaments distributed throughout the cell. Observation of stereopairs of transmission electron micrographs shows association of the filaments with the plasma membrane and the membranes of cytoplasmic organelles. Intense fluorescence, indicative of the presence of actin, was observed in the cytoplasm of electrocytes incubated in the presence of NBD-phallacidin or anti-actin antibodies.     

Dynamic changes in the actin cytoskeleton during the high-fluence rate response of theMougeotia chloroplast

Summary Since photo-induced orientation movement of a single, ribbon-shaped chloroplast in each cell of the filamentous green algaMougeotia is inhibited in the presence of cytochalasin B, actin is thought to be involved in the process of chloroplast movements. However, this possibility remains to be proved. A specific class of cytoplasmic filaments, which emerge from the advancing front of the moving chloroplast, can be seen by differential interference contrast (DIC) microscopy. However, no one has yet succeeded in defining the nature of these filaments. We have been able to stain the actin filaments (AFs) associated with the moving chloroplast with fluorescein-conjugated phalloidin (FP) after pre-treatment withm-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). No filamentous structures were observed in cells that had been pre-irradiated with low-fluence rate red light. However, transversely oriented fluorescent filaments appeared at the front edge of the moving chloroplast when it began to rotate under irradiation with high-fluence rate white light. These filaments disappeared after completion of the orientation movement, suggesting the simultaneous appearance of AFs and the orientation movement of the chloroplast. Thick cytoplasmic strands connecting the edge of the chloroplast with the parietal cytoplasm were often seen by DIC microscopy before and after completion of the high-fluence rate orientation movement. These thick cytoplasmic strands could not be stained by FP, but were often stained by 3,3-dihexyloxacarbocyanine iodide (DiOC₆(3)), suggesting that they are transvacuolar strands that include endoplasmic reticulum.     

Alterations in flightin phosphorylation inDrosophila flight muscles are associated with myofibrillar defects engendered by actin and myosin heavy-chain mutant alleles

Flightin is a 20-kD myofibrillar protein found in the stretch-activated flight muscles ofDrosophila melanogaster. Nine of the eleven isoelectric variants of flightin are generatedin vivo by multiple phosphorylations. The accumulation of these isoelectric variants is affected differently by mutations that eliminate thick filaments or thin filaments. Mutations in the myosin heavy-chain gene that prevent thick filament assembly block accumulation of all flightin variants except N1, the unphosphorylated precursor, which is present at much reduced levels. Mutations in the flight muscle-specific actin gene that block actin synthesis and prevent thin filament assembly disrupt the temporal regulation of flightin phosphorylation, resulting in premature phosphorylation and premature accumulation of flightin phosphovariants. Cellular fractionation of fibers that are devoid of thin filaments show that flightin remains associated with the thick filamentrich cytomatrix. These results suggest that flightin is a structural component of the thick filaments whose regulated phosphorylation is dependent upon the presence of thin filaments.This work was supported by National Science Foundation Grant IBN-9253045.