An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa.

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenkörper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.

Heavy metals have different effects on mycelial morphology of Achlya bisexualis as determined by fractal geometry.

The morphological response, as measured by changes to mycelial area, radial extension and border fractal dimension, of the oomycete Achlya bisexualis to Cu, Co, Hg, Zn and Cd at concentrations of between 0.05 and 3 mM is described. All of the metals decreased mycelial area and radial extension. Border fractal dimension increased in the presence of Cu, Co and Hg with individual hyphae extending out beyond the mycelial margin. In the presence of 3 mM Hg these hyphae displayed spiral growth. Zn and Cd had no effect on border fractal dimension. We suggest that all of the metals slow growth and that Cu, Co and Hg may also disrupt the relationship between tip growth and branching at the edge of the mycelium.

Contributions of Na+/H+ exchanger isoforms to preimplantation development of the mouse.

Previous work provided evidence of Na+/H+ exchanger activity in the apical domain of mouse trophectodermal plasma membranes that provides a route for entry of extracellular Na+ (Manejwala et al., 1989). This activity was hypothesized to contribute to the trans-trophectodermal Na+ flux that is required for blastocoel expansion. In the present work, we have used reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry to identify members of the Na+/H+ exchanger (NHE) family that are likely to participate in this process. When cDNA preparations from ovulated oocytes and several stages of preimplantation development were tested with PCR primers specific for the NHE-1, -2, -3, and -4 isoforms of the exchanger, only amplicons representing the NHE-1 and NHE-3 isoforms were detected. The identity of these amplicons was confirmed by direct sequencing. NHE-1 mRNA is present in oocytes and in all preimplantation stages, increasing threefold on a per embryo basis between the 4-cell and blastocyst stages. NHE-3 mRNA, on the other hand, was only detected in oocytes. Immunocytochemical analysis of blastocysts revealed that NHE-1 is localized in the basolateral domain of the trophectoderm, whereas NHE-3 is localized in the apical domain, a situation like that in epithelia of adult organs. We conclude that NHE-3, an oogenetic product that persists into the blastocyst stage, is the Na+/H+ exchanger isoform most likely to be involved in blastocoel expansion.

Pathways for the permeation of Na+ and Cl- into protoplasts derived from the cortex of wheat roots.

Sodium permeation into cortex cells of wheat roots was examined under conditions of high external NaCI and low Ca(2+). Two types of K(+) inward rectifier were observed in some cells. The time-dependent K(+) inward rectifier was Ca(2+)-sensitive, increasing in magnitude as external Ca(2+) was decreased from 10 mM to 0.1 mM, but did not show significant permeability to Na(+). However, the spiky inward rectifier showed significant Na+ permeation at Ca(2+) concentrations of 1 and 10 mM. In cells that initially did not show K(+) inward rectifier channels, fast and sometimes slowly activating whole-cell inward currents were induced at membrane potentials negative of zero with high external Na(+) and low Ca(2+) concentrations. With 1 mM Ca(2+) in the external solution, large inward currents were carried by Rb(+), Cs(+), K(+), Li(+), and Na(+). The permeability sequence shows that K(+), Rb(+) and Cs(+) are all more permeant than Na(+), which is about equally as permeant as Li(+). When some K(+) was present with high concentrations of Na(+) the inward currents were larger than with K(+) or Na(+) alone. About 60% of the inward current was reversibly blocked when the external Ca(2+) activity was increased from 0.03 mM to 2.7 mM (half inhibition at 0.31 mM Ca(2+) activity). Changes in the characteristics of the current noise indicated that increased Ca(2+) reduced the apparent single channel amplitude. In outside-out patches inward currents were observed at membrane potentials more positive than the equilibrium potentials for K(+) and Cl(-) when the external Na(+) concentration was high. These channels were difficult to analyse but three analysis methods yielded similar conductances of about 30 pS.

Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri.

Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halphytic angiosperm Zostera muelleri. In wholecell preparations, both outward and inward rectifying currents that activate in a time- and voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K+. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K+ than to Na+. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K+. In addition, the plasma membrane contains a much larger K+ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl- channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd3+, an effect that is reversible upon washout.

Pump and K+ inward rectifiers in the plasmalemma of wheat root protoplasts.

An electrogenic pump, a slowly activating K+ inward rectifier and an intermittent, "spiky," K+ inward rectifier, have been identified in the plasmalemma of whole protoplasts from root cortical cells of wheat (Triticum) by the use of patch clamping techniques. Even with high external concentrations of K+ of 100 mM, the pump can maintain the membrane potential difference (PD) down to -180mV, more negative than the electrochemical equilibrium potentials of the various ions in the system. The slowly activating K+ inward rectifier, apparent in about 23% of protoplasts, allows inward current flow when the membrane PD becomes more negative than the electrochemical equilibrium potential for K+ by about 50 mV. The current usually consists of two exponentially rising components, the time constant of one about 10 times greater than the other. The longer time constant is voltage dependent, while the smaller time constant shows little voltage dependence. The rectifier deactivates, on return of the PD to less negative levels, with a single exponential time course, whose time constant is strongly voltage dependent. The spiky K+ inward rectifier, present in about 68% of protoplasts, allows intermittent current, of considerable magnitude, through the plasmalemma at PDs usually more negative than about -140mV. Patch clamp experiments on detached outside-out patches show that a possibly multi-state K+ channel, with maximum conductance greater than 400 pS, may constitute this rectifier. The paper also considers the role of the pump and the K+ inward rectifiers in physiological processes in the cell.

Ion channel activity and tip growth: tip-localized stretch-activated channels generate an essential Ca2+ gradient in the oomycete Saprolegnia ferax.

The plasma membrane of tip-growing hyphae of the oomycete Saprolegnia ferax contains stretch-activated (SA) Ca(2+)-permeable and Ca(2+)-activated K+ ion channels. Patch clamp measurements on protoplasts derived from specific regions of hyphae demonstrated that SA channels were most abundant in the tip. Gadolinium (Gd3+) inhibited SA channel activity and stopped tip growth. The Ca(2+)-sensitive fluorochrome INDO 1 revealed a tip-high gradient of free cytoplasmic Ca2+ in growing hyphae. This gradient could be dissipated with the addition of Gd3+. The calcium gradient returned and growth resumed when Gd3+ was washed out. This implies a fundamental requirement for growth for Ca2+ influx through the SA channels. Ca(2+)-activated K+ channels were distributed evenly along the hyphae. These channels were inhibited by tetraethylammonium concentrations which caused a rapid but transient decrease in growth. We suggest that the SA channels at the apex act as feedback sensors, responding to membrane stretching at the tip. They are an obligate requirement for tip growth. The Ca(2+)-activated K+ channels may act to maintain turgor pressure, but are not obligatory for growth.

Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals.

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K+ ion channels (2-6 pA) using the patch-clamp technique when the seals are less than 1 G omega (about 100 M omega). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K+ flux is due solely to concentration differences. Although conductances are in the range expected for K+ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.