Large electrical currents traverse growing pollen tubes

Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.     

Ionic currents and ion fluxes in Neurospora crassa hyphae

Voltage dependence of ionic currents and ion fluxes in a walled, turgor-regulating cell were measured in Neurospora crassa. The hyphal morphology of the model organism Neurospora simplifies cable analysis of ionic currents to determine current density for quantitative comparisons with ion fluxes. The ion fluxes were measured directly and non-invasively with self-referencing ion-selective microelectrodes. Four ions (H(+), Ca(2+), K(+), and Cl(-)) were examined. H(+) net uptake and Ca(2+) net release were small (10.2 nmol m(-2) s(-1) and 1.1 nmol m(-2) s(-1), respectively) and voltage independent. K(+) and Cl(-) fluxes were larger and voltage dependent. Maximal K(+) net release ( approximately 1440 nmol m(-2) s(-1)) was observed at positive voltages (+15 mV), while maximal Cl(-) net release ( approximately 905 nmol m(-2) s(-1)) was observed at negative voltage (-210 mV). A possible function of the net outward K(+) and Cl(-) fluxes is regulation of the plasma membrane potential. Total ion fluxes were 37-58% of the total ionic current density (about +/-244 mA m(-2), equivalent to +/-2500 nmol m(-2) s(-1), at 0 mV and -200 mV) so other ions must contribute significantly to the ionic currents.     

Endogenous ion currents traverse growing roots and root hairs of Trifolium repens

Abstract The patterns of naturally growing ion currents associated with horizontally growing roots of Trifolium repens L. seedlings were measured in a simple low-salt bathing medium using a highly sensitive vibrating electrode. Current consistently enters the main elongation zone of the root and leaves from mature, elongated tissue. This current enters and leaves with densities of ca. 4.0–11.0 mA m^?2 and 4.0 mA m^?2, respectively. Current was also delected entering the zone of emerging root hairs and also the root-hair tips themselves. These results are a further example of the involvement of self-generated electrical fields in plant developmental processes. Possible, secondary rhizosphere-associated effects of the extracellular loop of the developmental current are also suggested.     

Structure and differentiation of the cell wall of Phytophthora palmivora: Cysts,hyphae and sporangia

Summary Walls from cysts, hyphae and sporangia of Phytophthora palmivora consist chiefly (ca. 90% dry wt) of -glucans with 1,3-, 1,4- and 1,6-links. The glucans are predominatly -1,3-linked but there are significant differences in the relative proportion of 1,3-, 1,6- and 1,4-linked glucosyl residues among the three wall types. There are also differences in protein content, susceptibility to degradation by various -glucanases, and surface texture. The isolated cyst wall consists solely of a thin fabric of long, tightly interwoven, randomly oriented microfibrils. Both inner and outer surfaces of the cyst wall are distinctly microfibrillar. The hyphal wall has two different textures; the internal surface is distinctly microfibrillar while the external surface is non-fibrillar. In a germinated cyst, there is a zone of demarcation where the microfibrils of the cyst wall disappear into the smooth outer texture of the germ tube wall. An exo--1,3-glucanase preferentially removed the amorphous material of the outer surface of the germ tube leaving exposed a continuous microfibrillar fabric from cyst to hyphal tube. Conceivably, the textural and structural differentiation of the cell wall may play a decisive role in cellular morphogenesis.     

Transcellular ion currents and extension ofNeurospora crassa hyphae

Summary Hyphae ofNeurospora crassa, like many other tipgrowing organisms, drive endogenous electric currents through themselves such that positive charges flow into the apical region and exit from the trunk. In order to identify the ions that carry the current, the complete growth medium was replaced by media lacking various constituents. Omission of K⁺ or of phosphate diminished the zone of inward current, effectively shifting the current pattern towards the apex. Omission of glucose markedly reduced both inward and outward currents; addition of sodium azide virtually abolished the flow of electric current. Growing hyphae also generate a longitudinal pH gradient: the medium surrounding the apex is slightly more alkaline than the bulk phase, while medium adjacent to the trunk turns acid. The results suggest thatNeurospora hyphae generate a proton current; protons are expelled distally by the H⁺-ATPase and return into the apical region by a number of pathways, including the symport of protons with phosphate and potassium ions. Calcium influx may also contribute to the electric current that enters the apical region. There seems to be no simple obligatory linkage between the intensity of the transcellular electric current and the rate of hyphal extension. Calcium ions, however, are required in micromolar concentrations for extensions and morphogenesis of hyphal tips.     

Potassium gradients in the growing hyphae of Neurospora crassa

X-ray electron probe microanalysis of a Neurospora crassa hyphal preparation revealed a significant heterogeneity of the potassium content in the hyphae. Low-potassium areas were found in a close proximity of the growing tip and in the distal part of the hyphae in the submembrane area of about 1 microm width. The intracellular potassium concentration was shown to change more than 3-5 times from apical to distal regions of the leading hyphae, the potassium contents in the apical part of the hyphae usually being minimal. We suggest that the content of intracellular potassium is determined by the volume occupied by free intracellular water, i.e., by the size of the space available for diffusion of substances, including inorganic ions.     

Ultrastructure of chitin in hyphae ofCandida albicans and other dimorphic and mycelial fungi

Summary Chitin microfibrils exposed by chemical extraction of hyphal walls ofCandida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidiodes brasiliensis, Coprinus cinereus andMucor mucedo were of variable morphology but gave identical infrared spectra and behaved as pure chitin in chromatographic analyses. The microfibrils of the four dimorphic fungi studied were shorter than those in the mouldsC. cinereus andM. mucedo but were similar to those reported for the yeastSaccharomyces cerevisiae. InC. albicans the microfibrils in the septal plates of hyphae were predominantly tangentially orientated and were longer than those in the lateral walls. Microfibrils produced by chitin synthasein vitro were very much longer than any observed from hyphal preparations.     

Large naturally-produced electric currents and voltage traverse damaged mammalian spinal cord

Background  

Immediately after damage to the nervous system, a cascade of physical, physiological, and anatomical events lead to the collapse of neuronal function and often death. This progression of injury processes is called "secondary injury." In the spinal cord and brain, this loss in function and anatomy is largely irreversible, except at the earliest stages. We investigated the most ignored and earliest component of secondary injury. Large bioelectric currents immediately enter damaged cells and tissues of guinea pig spinal cords. The driving force behind these currents is the potential difference of adjacent intact cell membranes. For perhaps days, it is the biophysical events caused by trauma that predominate in the early biology of neurotrauma.     

The dynamic behavior of cytoplasmic F-actin in growing hyphae

Summary A dynamic population of cytoplasmic F-actin was observed with electroporated rhodamine phalloidin (RP) staining in growing hyphae ofSaprolegnia ferax. This central actin population was distinct from the fibrillar peripheral network previously described in chemically fixed hyphae in that it was diffuse, pervaded the entire cytoplasm and was most concentrated in the central cytoplasm 8.4 m from the tip. The peripheral network did not stain with electroporated RP. The apical concentration of central cytoplasmic actin was only present in growing hyphae and developed prior to tip extension. It co-localized with the polarized distribution of mitochondria and endoplasmic reticulum in the tip, suggesting that it functions in positioning these organelles during tip growth. Within the central actin there was a consistent apical cleft which only occurred in growing hyphae and whose position predicted the direction of tip growth. This cleft was coincident with the known accumulation of apical wall vesicles, suggesting that it is either established by vesicle exclusion of the central actin network or is permeated by a portion of the in vivo unstained peripheral network. Photobleaching studies showed that in both growing and non-growing hyphae, cytoplasmic actin continually and rapidly moved from subapical regions to the tip where it accumulated. It mostly moved forward at the rate of tip growth, while some also left the tip, presumably to populate subapical regions.Abbreviations RP rhodamine phalloidin - F-actin filamentous actin - DIC Nomarski differential interference contrast - FITC fluorescein isothiocyanate     

Visualization of actin arrays in growing hyphae of the fungusSaprolegnia ferax

Summary We have observed the distribution of filamentous actin in growing hyphae of the oomyceteSaprolegnia ferax. The actin was stained by electroporating intact hyphae in the presence of 4×10^–8 M rhodamine phalloidin. Hyphae quickly recovered from electroporation and showed an apical cap of densely packed actin filaments. The pores created by the electric shock resealed in 8–10min and within 1/2 h hyphae resumed growth and appeared normal. This technique allows us to observe actin arrays during growth and may prove to be a useful tool in determining the complex roles of actin in apical growth.Abbreviations RP rhodamine phalloidin - F-actin filamentous actin     

Ionic currents and secretion in the exocrine glands

Exocrine glands extrude both proteins and salt. Fluid secretion is related to a modification of the membrane permeability of secreting cells. This permeability change may be measured as an increase of labelled ion fluxes or as a rise of membrane conductance. It involves Na+, K+, Cl- and Ca2+ ions. Intracellular Ca2+ acts as "second messenger" in the development of the electrical response. Recent recordings using the "patch-clamp" technique have revealed three types of ion channel activated by secretory agents. These channels are sensitive to internal Ca2+ ions. They are respectively selective to K+, Cl- and positively charged monovalent ions. Two models suggesting possible roles for these channels in the secretion process are presented. However, evaluation of such models is presently restricted by numerous uncertainties on the function of secreting cells in vivo. Information is notably lacking concerning the exact composition of the secreted fluid, and the exchanges between exocrine glands and blood circulation.     

Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences

Video-enhanced light microscopy of the apical and subapical regions of growing hyphae of several fungal species revealed the existence of momentary synchronized motions of subcellular organelles. First discovered in a temperature-sensitive morphological mutant (ramosa-1) of Aspergillus niger, these seemingly spontaneous cytoplasmic contractions were also detected in wild-type hyphae of A. niger, Neurospora crassa, and Trichoderma atroviride. Cytoplasmic contractions in all fungi lasted about 1 s. Although the cytoplasm recovered its motility and appearance, the contraction usually led to drastic changes in Spitzenkörper (apical body) behavior and hyphal morphology, often both. Within 10 s after the contraction, the Spitzenkörper commonly became dislodged from its polar position; sometimes it disassembled into phase-dark and phase-light components; more commonly, it disappeared completely. Whether partial or complete, the dislocation of the Spitzenkörper was always accompanied by a sharp reduction or cessation of growth, and was usually followed by marked morphological changes that included bulbous hyphal tips, bulges in the hyphal profile, and formation of subapical and apical branches. The cytoplasmic contractions are vivid evidence that the most conspicuous cell organelles (membrane-bound) in living hyphae are interconnected via a contractile cytoskeletal network.     

Structural and functional organization of growing tips of Neurospora crassa hyphae

Data are presented on a variety of intracellular structures of the vegetative hyphae of the filamentous fungus Neurospora crassa and the involvement of these structures in the tip growth of the hyphae. Current ideas on the molecular and genetic mechanisms of tip growth and regulation of this process are considered. On the basis of comparison of data on behaviors of mitochondria and microtubules and data on the electrical heterogeneity of the hyphal apex, a hypothesis is proposed about a possible supervisory role of the longitudinal electric field in the structural and functional organization of growing tips of the N. crassa hyphae.     

The Organization of mitochondria in growing hyphae of Neurospora crassa

Details of mitochondrial system organization and behavior in the tip of growing N. crassa hyphae and hyphal fragments were studied by means of cell-permeable mitochondria-selective probes in glucose- and sorbitol-containing media. It was shown that filamentous mitochondria concentrate in the 30-μm apical zone of growing hyphae and hyphal fragments independently of the carbon source. The mitochondrial assemblies propagate forward with elongation of hypha, split upon apical branching, and are formed de novo when branches are formed away from the growing tip. These activities resemble microtubule behavior. Co-staining with Mitotracker Red and Mitotracker Green revealed possible functional heterogeneity in subpopulations of mitochondria. It was proposed that the observed features are governed by the microtubular network, while the primary function of mitochondria was to sustain the growth rate. The organizing role of electrical gradients on the growing tip and their influence on mitochondrial and microtubular networks are discussed.     

Distribution and role of calmodulin in tip growing hyphae of Saprolegnia ferax

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Ultrastructural differences between wall apices of growing and non-growing hyphae ofSchizophyllum commune

Summary Newly synthesized chitin at the hyphal apex ofSchizophyllum commune was shown to be highly susceptible to chitinase degradation and solubilization by dilute mineral acid. With time this chitin became gradually more resistant to these treatments. With a combination of the shadow-cast technique and electron microscopic autoradiography it could be shown that this process occurred as the newly synthesized chitin moved into subapical parts of growing hyphae but also in non-growing apices which had ceased growth after incorporation of theN-acetyl[6-³H]glucosamine. These results are in agreement with a model which explains apical morphogenesis by assuming that the newly synthesized wall material at the apex is plastic due to the presence of individual polymer chains but becomes rigidified because of subsequent physical and chemical changes involving these polymers.Dedicated to Dr. A.Quispel, Professor of Botany at the University of Leiden, on occasion of his retirement.     

Branching is coordinated with mitosis in growing hyphae of Aspergillus nidulans

Filamentous fungi like Aspergillus nidulans can effectively colonize their surroundings by the formation of new branches along the existing hyphae. While growth conditions, chemical perturbations, and mutations affecting branch formation have received great attention during the last decades, the mechanisms that regulates branching is still poorly understood. In this study, a possible relation between cell cycle progression and branching was studied by testing the effect of a nuclei distribution mutation, cell cycle inhibitors, and conditional cell cycle mutations in combination with tip-growth inhibitors and varying substrate concentrations on branch initiation. Formation of branches was blocked after inhibition of nuclear division, which was not caused by a reduced growth rate. In hyphae of a nuclei distribution mutant branching was severely reduced in anucleated hyphae whereas the number of branches per hyphal length was linearly correlated to the concentration of nuclei, in the nucleated hyphae. In wild type cells, branching intensity was increased when the tip extension was reduced, and reduced when growing on poor substrates. In these situations, the hyphal concentration of nuclei was maintained and it is suggested that branching is correlated to cell cycle progression in order to maintain a minimum required cytoplasmic volume per nucleus and to avoid the formation of anucleated hyphae in the absence of nuclear divisions. The presented results further suggest the hyphal diameter as a key point through which the hyphal element regulates its branching intensity in response to the surrounding substrate concentrations.     

Protein synthesis during the differentiation of sporangia in the water mold Achlya

During the synchronous differentiation of sporangia in the absence of added nutrients, the water mold Achlya bisexualis (Coker and Couch) actively synthesized protein. Inhibition of protein synthesis at any time during the sporulation process completely inhibited further differentiation. Large changes in the rate of radioactive amino acid uptake resulted in changes in the specific activity of the cellular amino acid pool. The rate of protein synthesis was calculated from the amino acid pool specific activity and the incorporation of isotope into protein. During the 1st h after induction of the sporulation process, the rate of protein synthesis increased to two times the initial value. The amino acid precursors for this synthesis were supplied by the degradation of preexisting protein. Proteolytic enzyme activity assayed in vitro increased in proportion to the in vivo rates of protein synthesis and degradation. Differentiation was accompanied by a slight decline in dry weight of the mycelium as well as by a decrease in the protein content, whereas the relative size of the amino acid pools remained constant.