Martin W. Steer

Evidence for the role of Ca2+ ions in tip extension in pollen tubes

SummaryPollen tube growth is shown to be inhibited by both suboptimal (<10−4 M) and supraoptimal (>10−3 M) Ca2+ ion concentrations. Tip extension rates are independent of Ca2+ ion concentration over the optimal range. Changes in the structure of the tips of pollen tubes after transfer to inhibitory Ca2+ ion conditions provide evidence in support of our proposed mechanism of pollen tube tip growth (J. theor. Biol.98, 15–20, 1982). It is clearly demonstrated that tip extension is controlled, not only by the rate of vesicle fusion, but also by the state of plasticity of the tip; these processes appear to be sensitive to changes in Ca2+ ion concentration.

A model for the mechanism of tip extension in pollen tubes

Abstract Three main mechanisms are proposed to account for the tip growth of pollen tubes. (1) The tip region is supported against the internal osmotic pressure of the cell by a fibrillar network, composed mainly of microfilaments, that is stabilized by calcium ions. Tip extension is promoted by a lowering of the local cytoplasmic calcium ion concentration, through uptake by the mitochondria and/or endoplasmic reticulum, which leads to a weakening of the fibrillar network. (2) Vesicles, derived from dictyosomes in the main body of the tube, fuse with the apical plasma membrane, providing new membrane and further carbohydrate for the wall. The rate of fusion is proportional to the rate of diffusion of calcium ion across the plasma membrane at the tip. (3) The callose lining present in the pollen tube wall, except at the tip, renders the wall impermeable and restricts entry of calcium ions to the apical plasma membrane. This restriction limits the rate of vesicle fusion, and tube growth, to the tip. This model is discussed in the light of previous observations on the growth and structure of pollen tubes under normal and experimental conditions.

The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport, vesicle fusion and tip extension in pollen tubes

The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine, all antagonists of Ca2+ function in cells, have been studied in growing pollen tubes of Tradescantia virginiana. All four drugs inhibit pollen-tube growth but bring about different ultrastructural changes at the growing tips and within the cytoplasm. The results strongly support the hypothesis that Ca2+ plays a vital role in the mechanism of pollen-tube tip growth. The effect of ruthenium red provides evidence that sequestration of Ca2+ by mitochondria critically adjusts the concentration of these ions at tube tips. Fluorescein isothiocyanate appears to be a potent inhibitor of vesicle fusion at the plasma membrane, with vesicles accumulating in the tip at rates equivalent to those determined previously for their production. Both vesicle fusion and tip extension are regulated by Ca2+ but appear to be independently controlled processes.

The application of laser light scattering to the study of biological motion

Physical Principles of Light Scattering.- Structure and Movement in Cells.- Applications of Laser Light Scattering to Biological Systems.- Techniques and Instrumentation.- Correlation Function Profile Analysis in Laser Light Scattering I. General Review on Methods of Data Analysis.- Correlation Function Profile Analysis in Laser Light Scattering II. A Hybrid Photon Correlation Spectrometer.- Electrophoretic Light Scattering: Modern Methods and Recent Applications to Biological Membranes and Polyelectrolytes.- Laser Doppler Velocimetry in a Biological Context.- Implementation of Two Different Techniques for Measuring Lateral Diffusion and Optimisation of the Fluorescence Correlation Spectroscopy Concept.- Laser Doppler Microscopy: Especially as a Method for Studying Brownian Motion and Flow in the Sieve Tubes of Plants.- Studies of Neurotransmitter Receptor Interactions Using Quantitative Video Intensification Microscopy (V.I.M.).- Macromolecules and Gels.- Analysis of Diffusion of Biological Materials by Quasielastic Light Scattering.- The Diffusion of Compact Macromolecules Through Biological Gels.- Correlation Spectroscopy and Structural Properties of Macromolecular Solutions.- Depolarized Rayleigh Spectra of DNA in Solution.- Double Scattering in a Structured System of Particles.- Membranes and Amphiphilic Systems.- Fluorescence Techniques for the Study of Biological Motion.- Light Scattering by Model Membranes.- The Movement of Molecules Across Membranes: the Thermodynamic Analysis of the Dependence on Structure, Pressure and Temperature.- Light Scattering and Phase Transitions in GMO Membranes.- Photon Correlatior Studies of Phase Transitions in Lipid Monolayers.- Light Scattering from Micellar Solutions - Proposal for a Light Scattering Standard.- Laser Light Scattering Study of the Fractionation of Casein Micelles in Skim Milk by Controlled Pore Glass Chromotography.- Structural Studies on Bovine Casein Micelles by Laser Light Scattering.- Piological Applications.- Vesicles.- Structure and Dynamics of Disk Membrane Vesicles.- Vesicle Dynamics in Pollen Tubes.- A Preliminary Rheological Investigation of Living Physarum Endoplasm.- Muscles and Muscle Proteins.- The Application of Quasi-Elastic Light Scattering to the Study of Muscular Contraction.- Dynamic Light Scattering Study of Muscle F-Actin in Solution.- Further Evidence of Cross-Bridge Motion in Limulus Thick Myofilament Suspensions.- Stretch-Induced Transparency Change Associated With Cross-Bridge Deformation in Active Frogs Muscle.- Actin Polymerization in Cell Cytoplasm.- Ctyoplasmic Streaming.- Dynamic Cellular Phenomena in Physarum Possibly Accessible to Laser Techniques.- Amoeboid Movement in Chaoscarolinensis.- Cytoplasmic Streaming in Plant Cells and Its Relation to the Cytoskeleton.- The Rotation Model for Filament Sliding as Applied to the Cytoplasmic Streaming.- Motility.- Motility of Living Cells and Micro-organisms.- Chemotaxis and Band Formation of Escherichia coli Studied by Light Scattering.- A Comparison of Models Used in the Analysis of Quasi-Elastic Light Scattering Data from Two Motile Systems: Spermatozoa and Chlamydomonasreinhardtii.- Light Scattering Studies of Biological Populations and Biological Structures.- Systematic Assessment of Sperm Motility.- The Application of Laser Light Scattering to the Study of Photo-Responses of Unicellular Motile Algae.- Concluding Statements.- A Biologist Sums Up.- Epilogue.- Participants.

Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining

SummaryStandard methods for visualising microfilament (MF) arrays in pollen tubes using rhodamine phalloidin (RP) involve treatment of living tubes with a variety of stabilising, permeabilising, and fixation agents. Video differential interference contrast (DIC) microscopy has been used to investigate the effect of these agents on normalNicotiana pollen tube structure and activity. Most of these agents were found to induce extensive axial translocations generally starting with tipward contractions. These movements were less extensive in the apex compared to more distal regions, however, tips often suffered swelling damage. RP staining patterns of the actin cytoskeleton were highly variable within fixation treatments. In addition to investigating agents used by other authors on pollen tubes, we investigated the efficacy of pretreatment withm-maleimido-benzyol N-hydroxysuccinimide ester (MBS). This resulted in less disruption to the pollen tubes, especially when used alone in growth medium. It also gave better HP-labelling than that achieved in standard aldehyde-fixed specimens. We conclude that standard preparation methods do not faithfully preserve in vivo cytoplasmic integrity in pollen tubes so that subsequent images of MF distribution may be misleading.

Isolation, properties, and structure of fraction I protein from Avena sativa L.

Summary1.A method is described for the extraction and purification of Fraction I protein from Avena sativa L. leaves.2.The protein possesses ribulose diphosphate carboxylase activity. Chromatography on gels of Sephadex G-200 separates phosphoribulokinase and ribose phosphate isomerase from the carboxylase.3.The S°20w was calculated to be 18.2, the Stokes radius (determined by gel filtration on a cabibrated column) 74 Å, the molecular weight 5.7×105, and the frictional ratio 1.35.4.An amino acid analysis is presented.5.Electron microscope observations of negatively-stained Avena Fraction I protein molecules are compatible with the suggestion that they consist of 24 protomers disposed on the surface of an octahedral shell with 4:3:2 symmetry, and of diameter approximately 105 Å.

Cell biology and genetics of root hair formation inArabidopsis thaliana

SummaryIn this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants ofArabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca2+ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation, to control of tip diameter and extent and polarity of elongation.

Dictyosome vesicle production and plasma membrane turnover in auxin-stimulated outer epidermal cells of coleoptile segments fromAvena sativa (L.)

SummaryDark grown coleoptile segments were floated on solutions of IAA alone and of IAA and the secretion inhibitors cytochalasin and monensin. The secretion inhibitors prevented normal elongation of the tissue segments, the monensin inhibition being virtually complete while cytochalasin gave a 40% reduction over the first six hours with little further further elongation in the following 18 hours. Vesicle production was assessed in outer epidermal cells after 6 hours of IAA-stimulated elongation using the vesicle accumulation method following a cytochalasin-block of vesicle transport. The results were compared with the area of plasma membrane required to enable cell elongation to proceed at the observed rate. The area of vesicle membrane delivered to the cell surface exceeded this requirement to such an extent that at least 65% of the delivered membrane must be recycled back into the cytoplasm. Expressed in terms of the whole cell, the plasma membrane turnover rate was found to be once every 200 minutes. It is concluded that limitation of elongation by secretion inhibitors is more likely to reflect a requirement for the vesicle contents than the vesicle membrane. These results are compared with those obtained from other secretory systems using a similar approach.

Observations on tubules derived from the endoplasmic reticulum in leaf glands ofPhaseolus vulgaris

SummaryTubular systems present in bean leaf glands have been studied electron microscopically. Ordered arrays of small tubules (290 Å in diameter) arise from the endoplasmic reticulum in early stages of gland development and remain connected to it. Subsequently larger tubules (560–660 Å in diameter) appear among the smaller tubules and gradually replace many of them. The large tubules are not connected to the endoplasmic reticulum. They contain an electron dense material and their walls exhibit a patterned substructure. In older gland cells the bundles of large tubules run randomly through the cytoplasm. The relationship of the two types of gland tubules to conventional microtubules has been examined morphologically and experimentally. The small tubules have larger diameters and thicker walls than microtubules. Neither type of gland tubule is affected by low temperature or colchicine, or, in thin sections, by pepsin digestion. This suggests that these tubules are not closely related chemically to either cytoplasmic or ciliary microtubules. The two systems of tubules are closely associated with prominent protein vacuoles in the gland cells, but are not directly connected to them.

Endocytotic uptake of fluorescent dextrans by pollen tubes grown in vitro

SummaryPollen tubes grow by tip growth, with high levels of exocytosis at the apex. The commercial availability of FITC labelled α-linked dextrans provides a source of biologically inert tracers for endocytotic activity in pollen tubes. Growing tubes ofNicotiana andTradescantia were transferred to media containing 1% FD-4 for varying period of time before washing in control media and observation in a fluorescence microscope. Fluorescent material appeared to enter the pollen tubes only at the tip region, and to accumulate in vacuoles, starting with smaller vacuoles near the tip and spreading to the main vacuolated part of the tube. Mature tubes, with callose plugs, were only labelled up to the first complete plug from the tip, younger tubes without plugs were labelled into the pollen grain vacuole. The fluorescent material within the pollen tubes was shown to represent uptake of intact high molecular weight dextran by the following criteria: (i) free FITC and low molecular weight dextrans could not be detected in any of the media or pollen tubes using thin layer chromatography and (ii) pollen tube growth rates were unaffected by the fluorescent dextran, but were severely inhibited by low levels of free FITC. It was concluded that the dextrans entered the tubes by endocytosis, possibly in the tip region, and were then transferred to the vacuole system of the pollen tube.