Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K⁺ and Ca²⁺ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.     

Confocal Microscopy of Microtubule Cytoskeleton in the Pollen Protoplast of Narcissus

Pollen protoplasts were isolated from the mature pollen grains of Narcissus cyclamineus using cellulase Onozuka'R-10 and pectinase in Bs medium. The microtubule cytoskeleton in the pollen protoplasts was studied using immunofluorescence and confocal microscopy. In the cortical region there was a very complex microtubule network. The network contained numerous whirl-like arrays. The microtubule bundles in the whirl-like arrays were connected with each other by smaller bundles indicating that the arrangement of the whirl-like bundles were quite well organized and not at random. From the cortex to the centre of the protoplast another microtubule network having a structure different from the one in the cortical region was present. This network was much loosely packed than the cortical network. The arrangement of the microtubule bundles near the vegetative nucleus was again different. Numerous granules appeared outside the nuclear membrane. From these granules microtubule bundles radiated towards the cytoplasm. The arrangement of the microtubule network around the generative cell showed no specialized features. But inside the cell three types of microtubule arrays were present. 1. parallel arrays, 2. network, and 3. a mixture of the two. In the bursted pollen protoplast (as a result of osmotic shock treatment )some microtubule bundles could still be found attached to the ghost. The microtubule bundles associated with the ghost were much fragmented. But some still retained their branches and junctions. In the dry cleaved samples,a number of organelles still remained attached to the membrane and they included : microtubules, microfilaments, coated vesicles, endoplasmic reticulum and numerous honey-comb-like apparatus. The honey-comb-like apparatus was named as coated pits by Traas (1984). But we feel that it is more appropriate to call this organelle the honey-comb apparatus and we also believe that this organelle may be involved in microtubule and/or microfilament organization.     

Distribution of cortical microtubules in tobacco protoplasts. An immunofluorescence microscopic and ultrastructural study

Summary The arrangement of cortical microtubules in tobacco protoplasts is described using the following techniques: 1. Transmission electron microscopy (TEM) of thin sections of whole protoplasts, 2. TEM of negatively stained protoplast ghosts, and 3. Indirect immunofluorescence microscopy of protoplast ghosts. Ghosts were prepared by attaching freshly isolated protoplasts to glass coverslips or formvar/carbon-coated grids with poly-L-lysine and then bursting them either osmotically or by detergent treatment in the presence of a microtubule stabilizing buffer. Osmotic bursting of protoplasts yielded large pieces of plasma membrane with attached microtubules. These preparations proved very useful for measuring the density and length of cortical microtubules. Detergent treatment dissolved the plasma membrane and altered the distribution of cortical microtubules.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.     

Conifer somatic embryogenesis for studies of plant cell biology

Summary Embryogenic cultures have been produced for a wide range of conifers and current methods developed for spruce permit the maturation of high quality embryos that can be desiccated and then germinated to form plantlets. Embryogenic suspensions consisting of immature embryos are an excellent source of regenerable protoplasts. This review considers examples of applications of embryogenic suspension cultures for basic studies in three areas of plant cell biology. a) Immunofluorescence studies of microtubules in mitotic spruce cells reveal focused spindle poles at prophase and anphase, suggesting the presence of microtubule organizing centers (MTOCs). Antibodies known to recognize animal MTOCs do not stain the polar regions but do stain developing kinetochores. b) Embryo-derived protoplasts regenerate directly to somatic embryos. Fluorescence studies of the cytoskeleton in freshly derived protoplasts reveal random cortical microtubules and a fine network of actin filaments. During culture, protoplasts change shape and develop transverse cortical microtubule arrays. Embryonal cells of newly formed embryos possess distinctive arrays of cortical microtubules and networks of fine actin filaments while suspensor cells are characterized by transverse cortical microtubules and longitudinal actin cables. c) Transmission electron microscope studies of endocytosis in spruce protoplasts reveal an endocytotic pathway similar to that described previously for soybean. Uptake results are confirmed using high pressure freeze fixation instead of conventional chemical fixation. Presented in the Session-in-Depth Morphogenesis: Plant Cell and Tissue Differentiation at the 1994 Congress on Cell and Tissue Culture, Research Triangle Park, NC, June 4–7, 1994.     

Confocal Microscopy Observations on Actin Cytoskeleton in the Pollen and Pollen Protoplast of Narcissus

Actin cytoskeleton was localized in the pollen and pollen protoplast of Narcissus cyclamineus using fluorescence labelled phalloidin andconfocal microscopy. In the hydrated pollen (before germination) actin filamem bundles were arranged in a parallel array and at right angles to the long axis of the pollen grain in the cortex. But at the germination pore region(or fur row) the actin filament bundles formed a reticulate network. In the centre of the grain there was also an actin filament network which was more open and had less bundles associated with it than the network underneath the furrow. When the pollen grain started to produce pollen tube, most(if not all) of the actin filament bundles in the pollen grain rearranged into a parallel array pointing towards the tube. The bundles in the array later elongated and extended into the pollen tube. In the pollen protoplast a very tightly-packed actin bundle network was present. Numerous branches and jonts of actin filament bundles could be seen in the network. If the protoplasts were fixed before staining, the bundles aggregated and the branches and joints became less obvious indicating that fixation had affected the nature and arrangement of the actin filament bundles. If the pollen protoplasts were bursted (using the osmotic shock technique) or extracted (using Triton X-100), fragments of actin filament bundles could still be found associated with the membrane ghost indicating that some of the actin filament bundles in the cortex were tightly attached to the membrane. Using a double staining technique, actin filaments and microtubules were co-localized in the pollen protoplast. The co-alignment of some of the actin filament bundles with the microtubule bundles suggested that the actin cytoskeleton and the microtubule cytoskeleton were not distributed at random but in a well organized and orchestrated manner [possibly under the control of a yet undiscovered structure(s). The actin filament cytoskeleton in the generative cells failed to stain either in pollen or pollen tube, but they became stained in the pollen protoplast. The actin cytoskeleton in the generative cell appeared as a loosely organized network made up of short and long actin filament bundles.     

Isolation of cortical MTs from tobacco BY-2 cells

We isolated the cortical microtubules (CMTs) from tobacco BY-2 cells to identify their components. By centrifugation of protoplasts homogenized in the presence of taxol, a MT-stabilizing reagent, in a density gradient of Percoll, we obtained membranous vesicles to which MTs forming a sheet-like bundle were attached. Rhodamine-conjugated Ricinus communis agglutinin I (RCA-I), a lectin that bound to the surface of protoplasts, stained these vesicles, indicating that they were plasma membrane (PM) vesicles that retained CMTs. CMTs were released by solubilization of PM vesicles with Triton X-100. A sheet-like array of CMTs was retained even after solubilization of PM vesicles. Immunoblot analysis of the isolated CMTs demonstrated the presence of tubulin, actin, the 65 kDa microtubule-associated protein (MAP) and a 130 kDa RCA-I binding protein. Purification of the isolated CMTs by the temperature dependent disassembly-reassembly cycling method revealed four polypeptides, 190, 120, 85 and 65 kDa, co-assembling with CMTs.     

Cortical Actin Filaments Potentially Interact with Cortical Microtubules in Regulating Polarity of Cell Expansion in Primary Roots of Maize (Zea mays L.)

Evidence is accumulating implicating cortical microtubules in the directional control of cell expansion. However, the role of actin filaments in this process is still uncertain. To determine the involvement of actin in cell elongation, the organization of actin filaments in primary roots of maize (Zea mays L.) was examined by use of an improved fluorochrome-conjugated phalloidin-labeling method. With this method, a previously undetected state of actin organization was revealed in the elongation and maturation zone of maize roots. Fine transversely oriented cortical actin was observed in all cells of the elongation zone, including the epidermis, cortex, and vascular tissues. The orientation of cortical actin shifted from a predominantly transverse orientation to oblique, longitudinal, and/or random arrangements as the cells matured. The reorientation of cortical actin in maturing root cells mimics the behavior of cortical microtubules reported in other studies. Furthermore, roots treated with the microtubule-stabilizing drug taxol improved the quality of actin preservation as evidenced by the thicker bundles of cortical actin. This suggested that taxol was also capable of stabilizing the cortical actin networks. The elongation of roots exposed to 1 micromole Latrunculin B, an actin-disrupting drug, was inhibited, and after 24 h the roots exhibited moderate swelling particularly along the elongation zone. Latrunculin B also caused microtubules to reorient from transverse to oblique arrays. The results from this study provide evidence that cortical microtubules and actin filaments respond in a coordinated way to environmental signals and may well depend on both elements of the cytoskeleton.     

Microtubular organization in protoplasts and cells of somatic embryo-regenerating and non-regenerating cultures of Larix

Protoplasts were isolated from both somatic embryo-regenerating and non-regenerating cultures of hybrid larch ( Larix x eurolepis Henry) and fractionated on a discontinuous Percoll density gradient, whereby a highly embryogenic protoplast fraction could be enriched. This fraction was cultured for 14 days, and the differentiating protoplasts, cells, proetmbryos and embryo-like cell clusters sampled at days 3, 5 and 14. Immunofluorescence studies showed that the microtubules became organized into parallel and helical arrays in protoplast-derived cells of the embryogenic tissue as early as day 3 in culture, at which time the protoplast-derived cells started to elongate. In most of the protoplasts from non-regenerating tissue the microtubules retained a random orientation for a longer period. Preprophase bands were observed in both lines. Mitotic spindles consisted mainly of kinetochore-associated microtubules and displayed broad polar regions at metaphase. The spindle poles contracted at anaphase, giving the spindles a pointed appearance. A difference between the two tissue lines was observed at telophase, when the phragmoplast in the non-regenerating tissue had a normal appearance, while a proportion of the phragmoplasts from the embryo-regenerating line were branched or Y-shaped. Y-shaped phragmoplasts resulted in two nucleated cells and one enucleated cell after fusion of the cell plate with the plasma membrane. The early rearrangement of cortical microtubules is an indication that organized growth is occurring but, as this phenomenon has been observed also in regenerating non-emhryogenic cells, it appears to be a doubtful indicator of the distinction between emhryogenic and non-embryogenic development.     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

On the alignment of cellulose microfibrils by cortical microtubules: A review and a model

Summary The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae,Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Plasma membrane-adjacent actin filaments, but not microtubules, are essential for both polarization and hyphal tip morphogenesis in Saprolegnia ferax and Neurospora crassa

The organization and roles of F-actin and microtubules in the maintenance and initiation of hyphal tip growth have been analyzed in Saprolegnia ferax and Neurospora crassa. In hyphae of both species, the apex is depleted of microtubules relative to subapical regions and near-normal morphogenesis occurs in concentrations of nocodazole or MBC which remove microtubules, slow growth, and disrupt nuclear positioning. In contrast, each species contains characteristic tip-high arrays of plasma membrane-adjacent F-actin, whose organization is largely unaltered by the loss of microtubules but disruption of which by latrunculin B disrupts tip morphology. Hyphal initiation and subsequent normal morphogenesis from protoplasts of both species and spores of S. ferax are independent of microtubules, but at least in S. ferax obligatorily involve the formation of F-actin caps adjacent to the hyphal tip plasma membrane. These observations indicate an obligatory role for F-actin in hyphal polarization and tip morphogenesis and only an indirect role for microtubules.     

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     

The perinuclear microtubule system in the green algaAcetabularia: anchor or motility device?

Summary Migrating secondary nuclei inAcetabularia are tightly associated with actin bundles and possess a comet-like tail composed of microtubules. When secondary nuclei begin to settle in preparation for cyst morphogenesis, the tails expand into radially symmetrical arrays of microtubules. Concomitantly, nuclei become gradually dissociated from the actin bundles and eventually stop moving, even though the actin bundles remain intact and persist through this stage. If, however, the radial perinuclear microtubule arrays are destroyed by inhibitors, the nuclei reassociate with the actin bundles and regain their motile activity. Because this movement is sensitive to Cytochalasin D, we propose that actin is required for nuclear movements, whereas microtubules most likely function as a trailing anchor that begins to act as a braking device above a certain threshold in the number and length of perinuclear microtubules.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Microtubules at wound sites of Nitella internodal cells passively co-align with actin bundles when exposed to hydrodynamic forces generated by cytoplasmic streaming

The organization of cortical microtubules at wound sites in Nitella pseudoflabellata(A. Br. & Nordst.) em. R.D.W. and N. flexilis(L.) Ag. internodal cells was examined in relation to the regeneration of actin filament bundles in order to identify the mechanisms by which microtubules are oriented. Actin bundle regrowth occurs prior to that of microtubules, so it was considered possible that microtubule alignment is actin-dependent, perhaps mediated by cross-linking proteins. In all types of wounds investigated, subcortical actin bundles regenerated parallel to the direction of cytoplasmic streaming. Microtubule orientation patterns, however, varied according to the nature of wound formation and the type of wound wall eventually produced. In chloroplast-free windows induced by blue light irradiation, microtubule orientation varied according to the size of the window. Microtubules were randomized in 10- to 30-μm-wide windows where exposure to cytoplasmic flow is minimal, but were aligned more or less parallel to regenerated actin bundles in 80- to 100-μm-wide windows. Where co-alignment between microtubules and actin bundles was obvious after fluorescence labelling, electron micrographs revealed that microtubules and actin bundles were too widely spaced to account for any cross-linkages. Furthermore, treatments that inhibited or reduced cytoplasmic streaming without altering the direction of actin bundles caused randomization of microtubules previously oriented in the streaming direction, even in the presence of taxol. When evenly flat wound walls were induced by 10⁻⁴ M chlortetracycline, microtubules were co-aligned with nearby actin bundles at the surface of the wound wall. At wounds induced by treatment with 5 × 10⁻² M CaCl₂, however, microtubules were randomly oriented and preferentially located in the narrow clefts between the wound-wall protuberances, up to several micrometers away from the actin bundles near the wound-wall tips. These results indicate that microtubules regenerated in wounds are merely co-aligned with actin filament bundles because they are passively aligned by the hydrodynamic forces created by cytoplasmic flow. Received: 4 August 1998 / Accepted: 30 January 1999     

Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis

Summary In accordance with former observations of Hoffman (1962a), ghost populations obtained by hypotonic hemolysis and subsequent restoration of isotonicity by the addition of alkali salts, were found to be composed of 3 types of ghosts. For our purposes it was useful to distinguish between: (1) ghosts which reseal immediately after hemolysis (type I); these ghosts are incapable of incorporating alkali ions which are added after hemolysis; (2) ghosts which reseal after the addition of alkali ions (type II); salt added to the hemolysate becomes trapped inside these ghosts in the course of the resealing process at temperatures above 0°C; and (3) ghosts which remain leaky regardless of the experimental condition (type III). The discrimination between the various types of ghosts was partly achieved by a kinetic method first devised by Hoffman (1962a), and partly by sucrose density gradient centrifugation.The relative sizes of the 3 fractions depend on the temperature at which hemolysis took place and on the time interval which elapsed between hemolysis and the addition of salt. At 37°C the resealing process is fast. Many of the ghosts reseal before salt can be added to the hemolysate. Hence, the fraction of type I ghosts is high after hemolysis at that temperature. At 0°C resealing is extremely slow. Hence, salt which has been added to the hemolysate at that temperature will enter the ghosts and become trapped during subsequent incubation at 37°C. There are no ghosts of type I and many ghosts of type II (about 60%). Regardless of the temperature at hemolysis, there are always ghosts which do not reseal even after prolonged incubation at 37°C. A method has been designed which permits the preparation of homogeneous populations of type II ghosts.Complexing agents (ATP, EDTA, 2,3-DPG) may prevent the resealing of the ghost membrane. However, they exert this effect only at elevated temperatures and when present in the medium at the instant of hemolysis. At 0°C, the presence of complexing agents in the medium at the instant of hemolysis has no effect on the subsequent resealing at 37°C. The recovery of the ghost membrane takes place in spite of the continued presence of the agents and eventually leads to trapping of these agents inside the resealed ghosts.The experiments support the contention that the complexing agents interact with a membrane constituent which is neither accessible from the inner nor from the outer surface of the cell membrane but becomes exposed during the hemolytic event when the complexing agents penetrate across the membrane. Apparently, at low tempertrures membrane ligands are more successful in competing with the added complexing agents for this constituent than at higher temperatures.Extending former observations of Hoffman, we found that not only Mg⁺⁺ but also Ca⁺⁺ facilitates the resealing process. Perhaps one or the other of the two alkaline earth ions is the membrane constituent which normally participates in the maintenance of the integrity of the red blood cell membrane.     

Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton.

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.     

The initiation and organization of microtubules in germinating pear (Pyrus communis L.) pollen

To study microtubule organization in germinating pear (Pyrus communis L., cv., Bartlett) pollen, we removed the pollen wall by freeze-fracturing before treating the resultant pollen protoplasts by conventional immunofluorescence procedures. Results reveal that axial bundles of microtubules are present in the generative cell of both inactivated and activated pollen grains. Microtubules are not present in the vegetative cells of inactivated pollen, but they are present in the vegetative cells of activated pollen grains. Microtubule nucleation occurs in the vegetative cell cortex. Subsequently, the microtubules grow as branching arrays through most of the vegetative cell cortex except at the apertures where they form localized converging or criss-cross patterns. Eventually, in a germinated pollen grain, the microtubules form network-like arrays through most of the pollen grain and a collar of short arrays at the base of the pollen tube. It is suggested that the role of vegetative cell microtubules in pollen germination is indirect through their mediation of the conformational changes in actin organization that are essential for pollen germination.