Statoliths and microfilaments in plant cells

Microfilaments have been demonstrated in rhizoids of Chara fragilis Desvaux by labelling of actin with rhodamine-conjugated phalloidin. Each rhizoid contains thick microfilament-bundles arranged longitudinally in the basal region. In the subapical and apical regions, much thinner bundles exist which contact the statoliths and encircle them in the form of a dense envelope. In root statocytes from Lepidium sativum L. the presence of an actin network is indicated by the fact that application of cytochalasin B (25 g·ml^-1 for 4 h) results in an approximately threefold increase in the rate of statolith (amyloplast) sedimentation relative to controls. It is concluded that in gravity-perceiving plant cells statoliths may trigger the transduction mechanism via actin filaments.Abbreviation CB cytochalasin B - ER endoplasmic reticulum - MF microfilament     

Micromanipulation of statoliths in gravity-sensing Chara rhizoids by optical tweezers

Infrared laser traps (optical tweezers) were used to micromanipulate statoliths in gravity-sensing rhizoids of the green alga Chara vulgaris Vail. We were able to hold and move statoliths with high accuracy and to observe directly the effects of statolith position on cell growth in horizontally positioned rhizoids. The first step in gravitropism, namely the physical action of gravity on statoliths, can be simulated by optical tweezers. The direct laser microirradiation of the rhizoid apex did not cause any visible damage to the cells. Through lateral positioning of statoliths a differential growth of the opposite flank of the cell wall could be induced, corresponding to bending growth in gravitropism. The acropetal displacement of the statolith complex into the extreme apex of the rhizoid caused a temporary decrease in cell growth rate. The rhizoids regained normal growth after remigration of the statoliths to their initial position 10–30 m basal to the rhizoid apex. During basipetal displacement of statoliths, cell growth continued and the statoliths remigrated towards the rhizoid tip after release from the optical trap. The resistance to statolith displacement increased towards the nucleus. The basipetal displacement of the whole complex of statoliths for a long distance (>100 m) caused an increase in cell diameter and a subsequent regaining of normal growth after the statoliths reappeared in the rhizoid apex. We conclude that the statolith displacement interferes with the mechanism of tip growth, i.e. with the transport of Golgi vesicles, either directly by mechanically blocking their flow and/or, indirectly, by disturbing the actomyosin system. In the presence of the actin inhibitor cytochalasin B the optical forces required for acropetal and basipetal displacement of statoliths were significantly reduced to a similar level. The lateral displacement of statoliths was not changed by cytochalasin B. The results indicate: (i) the viscous resistance to optical displacement of statoliths depends mainly on actin, (ii) the lateral displacement of statoliths is not impeded by actin filaments, (iii) the axially directed actin-mediated forces against optical displacement of statoliths (for a distance of 10 m) are stronger in the basipetal than in the acropetal direction, (iv) the forces acting on single statoliths by axially oriented actin filaments are estimated to be in the range of 11–110 pN for acropetal and of 18–180 pN for basipetal statolith displacements.Abbreviation CB cytochalasin B This work was supported by the Bundesminister für Forschung und Technologie, and by Fonds der Chemischen Industrie. We thank Professor Dr. A. Sievers (Botanisches Institut, Universität Bonn, Germany) for helpful discussions.     

Statoliths pull on microfilaments

Summary Previous videomicroscopy ofChara rhizoids during parabolic flights of rockets showed that the weightless statoliths moved basipetally. A hypothesis was offered that the removal of gravity force disturbed the initial balance between this force and the basipetally acting forces generated in a dynamic interaction of statoliths with microfilaments (MFs). The prediction of this hypothesis that the statoliths would not be displaced basipetally during the microgravity phase (MG-phase) after disorganizing the MFs was tested by videomicroscopy of a rhizoid treated with cytochalasin D (CD) immediately before the flight. The prediction was fully supported by the flight experiment. Additionally, by chemical fixation of many rhizoids at the end of the MG-phase it was shown that all rhizoids treated with CD before the flight had statoliths at the same location, i.e., sedimented on the apical cell wall, while all untreated rhizoids had statoliths considerably displaced basipetally from their normal position. Thus, a dynamical interaction involving shearing forces between MFs and statoliths appears highly probable.Abbreviations CD cytochalasin D - g gravitational acceleration - MF microfilament - MG-phase microgravity phase - TEXUS technological experiments under reduced gravity Dedicated to Hilton H. Mollenhauer on the occasion of his retirement     

Centrifugation causes adaptation of microfilaments Studies on the transport of statoliths in gravity sensingChara rhizoids

Summary The actin cytoskeleton is involved in the positioning of statoliths in tip growingChara rhizoids. The balance between the acropetally acting gravity force and the basipetally acting net out-come of cytoskeletal force results in the dynamically stable position of the statoliths 10–30 m above the cell tip. A change of the direction and/or the amount of one of these forces in a vertically growing rhizoid results in a dislocation of statoliths. Centrifugation was used as a tool to study the characteristics of the interaction between statoliths and microfilaments (MFs). Acropetal and basipetal accelerations up to 6.5 g were applied with the newly constructed slow-rotating-centrifuge-microscope (NIZEMI). Higher accelerations were applied by means of a conventional centrifuge, namely acropetally 10–200 g and basipetally 10–70 g. During acropetal accelerations (1.4–6 g), statoliths were displaced to a new stable position nearer to the cell vertex (12–6.5 m distance to the apical cell wall, respectively), but they did not sediment on the apical cell wall. The original position of the statoliths was reestablished within 30 s after centrifugation. Sedimentation of statoliths and reduction of the growth rates of the rhizoids were observed during acropetal accelerations higher than 50 g. When not only the amount but also the direction of the acceleration were changed in comparison to the natural condition, i.e., during basipetal accelerations (1.0–6.5 g), statoliths were displaced into the subapical zone (up to 90 m distance to the apical cell wall); after 15–20 min the retransport of statoliths to the apex against the direction of acceleration started. Finally, the natural position in the tip was reestablished against the direction of continuous centrifugation. Retransport was observed during accelerations up to 70 g. Under the 1 g condition that followed the retransported statoliths showed an up to 5-fold increase in sedimentation time onto the lateral cell wall when placed horizontally. During basipetal centrifugations 70 g all statoliths entered the basal vacuolar part of the rhizoid where they were cotransported in the streaming cytoplasm. It is concluded that the MF system is able to adapt to higher mass accelerations and that the MF system of the polarly growing rhizoid is polarly organized.Abbreviations g gravitational acceleration (9.81 m/s²) - MF microfilament - NIZEMI Niedergeschwindigkeits-Zentrifugen-Mikroskop (slow-rotating-centrifuge-microscope)     

Regulation of the position of statoliths inChara rhizoids

Summary The behavior of statoliths in rhizoids differently oriented with respect to the gravity vector indicates that there are cytoskeleton elements which exert forces on the statoliths, mostly in the longitudinal directions. Compared to the sum of the forces acting on a statolith, the gravitational force is a relatively small component,i.e., less than 1/5 of the cytoskeleton force. The balance is disturbed by displacing the rhizoid from the normal vertical orientation. It is also reversibly disturbed by cytochalasin B such that some statoliths move against the gravity force. Phalloidin stabilizes the position of the statoliths against cytochalasin B. We infer that microfilaments are involved in controlling the position of statoliths, and that there is a considerable tension on these microfilaments. The vibration frequency of the microfilaments corresponding to this tension is in the ultrasonic range.Visiting Professor on a grant from Deutsche Forschungsgemeinschaft.     

Actomyosin-mediated statolith positioning in gravisensing plant cells studied in microgravity

The positioning and gravity-induced sedimentation of statoliths is crucial for gravisensing in most higher and lower plants. In positively gravitropic rhizoids and, for the first time, in negatively gravitropic protonemata of characean green algae, statolith positioning by actomyosin forces was investigated in microgravity (<10(-4) g) during parabolic flights of rockets (TEXUS/MAXUS) and during the Space-Shuttle flight STS 65. In both cell types, the natural position of statoliths is the result of actomyosin forces which compensate the statoliths' weight in this position. When this balance of forces was disturbed in microgravity or on the fast-rotating clinostat (FRC), a basipetal displacement of the statoliths was observed in rhizoids. After several hours in microgravity, the statoliths were loosely arranged over an area whose apical border was in the same range as in 1 g, whereas the basal border had increased its distance from the tip. In protonemata, the actomyosin forces act net-acropetally. Thus, statoliths were transported towards the tip when protonemata were exposed to microgravity or rotated on the FRC. In preinverted protonemata, statoliths were transported away from the tip to a dynamically stable resting position. Experiments in microgravity and on the FRC gave similar results and allowed us to distinguish between active and passive forces acting on statoliths. The results indicate that actomyosin forces act differently on statoliths in the different regions of both cell types in order to keep the statoliths in a position where they function as susceptors and initiate gravitropic reorientation, even in cells that had never experienced gravity during their growth and development.     

Immunolocalization of myosin in rhizoids ofChara globularis Thuill

Summary Myosin-related proteins have been localized immunocytochemically in gravity-sensing rhizoids of the green algaChara globularis using a monoclonal antibody against the heavy chain of myosin from mouse 3T3 cells and a polyclonal antibody to bovine skeletal and smooth muscle myosin. In the basal zone of the rhizoids which contain a large vacuole, streaming endoplasm and stationary cortical cytoplasm, the monoclonal antibody stained myosin-related proteins as diffusely fluorescing endoplasmic strands. This pattern is similar to the arrangement of subcortical actin filament bundles. In the apical zone which contains an aggregation of ER membranes and secretory vesicles for tip growth, diffuse immunofluorescence was detected; the intensity of the signal increasing towards the apical cell wall. The most prominent myosin-staining was associated with the surface of statoliths in the apical zone. The polyclonal antibody produced a punctate staining pattern in the basal zone, caused by myosin-related proteins associated with the surface of drganelles in the streaming endoplasm and the periphery of the nucleus. In the apical zone, this antibody revealed myosin-immunofluorescence on the surface of statoliths in methacrylate-embedded rhizoids. Neither antibody revealed myosin-immunofluorescence on the surface of organelles and vesicles in the relatively stationary cytoplasm of the subapical zone. These results indicate (i) that different classes of myosin are involved in the various transport processes inChara rhizoids; (ii) that cytoplasmic streaming in rhizoids is driven by actomyosin, corresponding to the findings onChara internodal cells; (iii) that actindependent control of statolith position and active movement is mediated by myosin-related proteins associated with the statolith surfaces; and (iv) that myosin-related proteins are involved in the process of tip growth.     

轮藻假根中的平衡石在回转器水平回转时的运动

利用回转器重现了在ＴＥＸＵＳ火箭抛物线飞行的微重力实验中轮藻假根内平衡石和假根基部方向的运动。在快速回转器上回转１５ｍｉｎ时,假根中的平衡石复合体中心离假根顶端的距离比在原来沿重力方向生长的假根中的距离增加了６０％。细胞松弛素Ｄ的实验证实平衡石的这种运动是和肌动蛋白丝相关,而且在重力场中作用于平衡石的向基力也是肌动蛋白丝产生的。因此回转器和细胞松弛素Ｄ的实验证实了在地球上,平衡石的位置取决于作用方     

Cytoplasmic streaming inChara rhizoids

Summary In-vivo videomicroscopy ofChara rhizoids under 10^–4g demonstrated that gravity affected the velocities of cytoplasmic streaming. Both, the acropetal and basipetal streaming velocities increased on the change to microgravity. The endogenous difference in the velocities of the oppositely directed cytoplasmic streams was maintained under microgravity, yet the difference was diminished as the basipetal streaming velocity increased more than the acropetal streaming velocity. Direction and structure of microfilaments labeled by rhodamine-phalloidin had not changed after 6 min of microgravity.Abbreviations g gravitational acceleration - Nizemi slow rotating centrifuge microscope - Texus technological experiments under reduced gravity     

轮藻假根中的平衡石在回转器水平回转时的运动(英文)

利用回转器重现了在TEXUS火箭抛物线飞行的微重力实验中轮藻假根内平衡石向假根基部方向的运动。在快速回转器上回转15 min时,假根中的平衡石复合体中心离假根顶端的距离比在原来沿重力方向生长的假根中的距离增加了60%。细胞松弛素D的实验证实平衡石的这种运动是和肌动蛋白丝相关,而且在重力场中作用于平衡石的向基力也是肌动蛋白丝产生的。因此回转器和细胞松弛素D的实验证实了在地球上,平衡石的位置取决于作用方向相反的重力和肌动蛋白丝作用力的动态平衡的假说。然后在快速回转器上,平衡石中心在一个新的位置上维持了30 min左右的稳定,也就是出现了一个新的动态平衡状态。这一新的状态是在原先的向着假根顶端的重力和向着假根基部的肌动蛋白丝作用力的平衡在回转器上被打破后再经约有15 min时达到的。更进一步的快速回转器实验还展示了可能因平衡石位置的这一变化而启动的肌动蛋白丝的再组织和由此产生的平衡石向假根顶端方向再转运的过程。快速和慢速回转器实验在这里的结果有差异,推测是和回转器上颗粒的振幅随回转器转速的增加而减小有关。加之,轮藻假根的单细胞性质,因此在假根处于回转轴上时,快速回转器是更适合这项模拟失重的研究。总之,在失重条件下平衡石和肌动蛋白丝的关系是可以利用回转器来研究的。     

How to activate a plant gravireceptor. Early mechanisms of gravity sensing studied in characean rhizoids during parabolic flights

Early processes underlying plant gravity sensing were investigated in rhizoids of Chara globularis under microgravity conditions provided by parabolic flights of the A300-Zero-G aircraft and of sounding rockets. By applying centrifugal forces during the microgravity phases of sounding rocket flights, lateral accelerations of 0.14 g, but not of 0.05 g, resulted in a displacement of statoliths. Settling of statoliths onto the subapical plasma membrane initiated the gravitropic response. Since actin controls the positioning of statoliths and restricts sedimentation of statoliths in these cells, it can be calculated that lateral actomyosin forces in a range of 2 x 10(-14) n act on statoliths to keep them in place. These forces represent the threshold value that has to be exceeded by any lateral acceleration stimulus for statolith sedimentation and gravisensing to occur. When rhizoids were gravistimulated during parabolic plane flights, the curvature angles of the flight samples, whose sedimented statoliths became weightless for 22 s during the 31 microgravity phases, were not different from those of in-flight 1g controls. However, in ground control experiments, curvature responses were drastically reduced when the contact of statoliths with the plasma membrane was intermittently interrupted by inverting gravistimulated cells for less than 10 s. Increasing the weight of sedimented statoliths by lateral centrifugation did not enhance the gravitropic response. These results provide evidence that graviperception in characean rhizoids requires contact of statoliths with membrane-bound receptor molecules rather than pressure or tension exerted by the weight of statoliths.     

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m^–2 · s^–1) to a low PPFD (65 ± 15 mol · m^–2 · s^–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m^–2 · s^–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m^–2 · s^–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat substrate-saturated turnover rate of fully carbamylated enzyme - PPFD photosynthetically active photon flux density (400–700 nm) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate     

Synthesis of mono- and disaccharide amino-acid derivatives for use in solid phase peptide synthesis

N-Fluorenylmethyloxycarbonyl-protected serine and threonine derivatives, carryingO-glycosidically - or -linked peracetylated -d-Galp-(1–3)-d-GalNAcp carbohydrate chains, were prepared. These derivatives are intended for use in solid phase glycopeptide synthesis. Suitably protected mono- and disaccharide thioglycosides were used as carbohydrate intermediates. These were activated by treatment with bromine to give the glycosyl bromides, which were then used in silver triflate-promoted glycosidations ofN-fluorenylmethyloxycarbonyl amino-acid phenacyl esters. Removal of the phenacyl esters with zinc gave the target free acids.     

Central root cap cells are depleted of endoplasmic microtubules and actin microfilament bundles: implications for their role as gravity-sensing statocytes

Summary Indirect immunofluorescence, using monoclonal antibodies to actin and tubulin, applied to sections of root tips ofLepidium, Lycopersicon, Phleum, andZea, revealed features of the cytoskeleton that were unique to the statocytes of their root caps. Although the cortical microtubules (CMTs) lay in dense arrays against the periphery of the statocytes, these same cells showed depleted complements of endoplasmic microtubules (EMTs) and of actin microfilament (AMF) bundles, both of which are characteristic of the cytoskeleton of other post-mitotic cells in the proximal portion of the root apex. The scarcity of the usual cytoskeletal components within the statocytes is considered responsible for the exclusion of the larger organelles (e.g., nucleus, plastids, ER elements) from the interior of the cell and for the absence of cytoplasmic streaming. Furthermore, the depletion of dense EMT networks and AMF bundles in statocyte cytoplasm is suggested as being closely related to the elevated cytoplasmic calcium content of these cells which, in turn, may also favour the formation of the large sedimentable amyloplasts by not permitting plastid divisions. These latter organelles are proposed to act as statoliths due to their dynamic interactions with very fine and highly unstable AMFs which enmesh the statoliths and merge into peripheral AMFs-CMTs-ER-plasma membrane complexes. Rather indirect evidence for these interactions was provided by showing enhanced rates of statolith sedimentation after chemically-induced disintegration of CMTs. All these unique properties of the root cap statocytes are supposed to effectively enhance the gravity-perceptive function of these highly specialized cells.Dedicated to Prof. Dr. Benno Parthier on the occasion of his retirement     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Root system architecture and gravity perception of a mangrove plant,Sonneratia alba J. Smith

We describe the features of the root system and the gravitropism of roots produced bySonneratia alba. The root system consists of four root types with different growth directions: (a) Pneumatophores, which are negatively orthogravitropic and their statocytes are very large (922 μm²) and the statolith is located near the proximal wall, (b) Cable roots and (c) Feeding roots which are both diagravitropic and their statoliths are settled along the longitudinal wall, and (d) Anchor roots which are positively orthogravitropic. The statocyte cells are the smallest (420 μm²) and statoliths settled at the distal wall. We found that all roots with marked gravitropism have statoliths that settle along different walls of the statocyte. This implies that the statoliths sensing of gravity is done by gravity on mass, and that they are denser than surrounding cytoplasm and this position is related to root growth direction. This finding matches the statoliths sediment under the effect of gravity. Irrespective of statolith, position and direction of growth may be stable.     

Bioelectric and biosynthetic aspects of cell polarity inAllomyces macrogynus

Summary In contrast to all filamentous fungi examined to date, vegetative hyphae ofAllomyces macrogynus, whether extending or not, produced an outward flow of positive electrical current, at a maximum of 0.16 A cm^–2 around 40 m behind the apex, as measured with a vibrating probe. Inward currents of up to 0.55 A cm^–2 were recorded around the rhizoids. Increases in outward current were observed in hyphae pre-grown under oxygen deficiency and then allowed to widen backwards to the hyphal base in sufficient oxygen. When spores were germinated in an applied electrical field they produced rhizoids predominantly towards the anode. Hyphae were produced initially towards the cathode but later bent around towards the anode. Experiments with a range of chemicals provided no evidence for the involvement of calcium in vegetative growth and development inA. macrogynus. Polyoxin and nikkomycin, inhibitors of chitin synthesis, had no effect on swimming zoospores, but inhibited wall formation of cysts, rhizoids and forward and backward growing hyphae.     

Uptake of iron byGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from⁵⁹FeCl₃ showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentK _m = 3 m,V _max = 0.054 nmol · mg^–1 · min^–1). The maximal rate of Fe²⁺ uptake was higher than Fe³⁺ (V _max = 0.25 nmol · mg^–1 · min^–1) but theK _m was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (K _m = 2 M,V _max = 0.22 nmol · mg^–1 · min^–1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either⁵⁷FeCl₃ or⁵⁷FeCl₂ accumulated unknown ferric and ferrous binding metabolites.     

Regulation of aplanospore germination in Vaucheria

Germination of aplanospores in Vaucheria longicaulis Hoppaugh var. macounii Blum proceeds through three stages of development. Stage I begins with the initiation of germination and lasts approx. 2 h. During this stage germinating filaments grow at an accelerated rate (266 ± 12 m · h^–1). Stage II is characterized by a sharp decline in the growth rate of germinating filaments (96 ± 4 m · h^–1) and lasts 4 h. This is followed, during the next 4 h, by a recovery in the growth rate (168 ± 8 m · h^–1) of germinating filaments, stage III. Growth rates stabilize and remain unchanged during subsequent development (Oliveira and Fitch, 1988, J. Submicrosc. Cytol. Pathol. 20, 397–406). The Ca²⁺-influx modulators LaCl₃, nifedipine and methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4 (2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K-8644), the ionophore calcimycin (A23187), the intracellular Ca²⁺-release antagonist 8-N-N'-(diethylamino)-octyl-3,4, 5-trimethoxybenzoate (TMB-8), the Ca²⁺-uptake inhibitor ruthenium red and the phosphoinositide-cycle modulators LiCl and myo-inositol show that the events required for the initiation are distinct from those required for the completion of each stage of germination. These studies in conjunction with microinjection of germinating filaments with inositol 1,4,5-trisphosphate, the natural ligand for Ca²⁺ release from Ca-storing organelles (endoplasmic reticulum, vacuole), and treatment with chlorotetracycline (CTC), to visualize the distribution of membrane-bound Ca²⁺ reveal that both the initiation and completion of each stage of germination are controlled by Ca²⁺ signals which are restricted to well-defined time intervals and are modulated by the origin (source) of Ca²⁺.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - Bay K-8644 methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromethylphenyl)-pyridine-5-carboxylate - CTC chlorotetracycline - InsP₃ inositol 1,4,5-trisphosphate - RR ruthenium red - TMB-8 8-N-N-(diethylamino)-octyl-3,4,5-trimethoxybenzoate The author wishes to express his gratitude to the technical group of the Immunocytochemistry Unit for their help with the microinjection studies. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (grant A-7844).     

Shuttle-like movements of Golgi vesicles in the tip of growingChara rhizoids

Summary In tip-growingChara rhizoids, the in-vivo saltatory movements of Golgi vesicles were recorded. The movements in radial direction back and forth between the ER aggregate and the plasma membrane occurred three times more often than movements passing the ER aggregate tangentially. The mean velocity of the class of Golgi vesicles observed (0.4–1 m in diameter) was approx. 0.3 m/s. Higher speed of 1–1.5 m/s occurred only in radial directions. Possibly, the ER aggregate is involved in guidance of the Golgi vesicles.Abbreviations DIC differential interference contrast - ER endoplasmic reticulum - OsFeCN osmium tetroxide-potassium ferricyanide Dedicated to the memory of Professor O. Kiermayer