Microsporidian Spore Envelope Keratins Phosphorylate and Disassemble During Spore Activation

The microsporidian spore stage of the nerve parasite, Spraguea lophii, consists of outer envelope stabilized in part by keratins, including K4 and K13. The nonepidermal K4 and K13 keratins were found only in the spore envelope and were absent in the internal microsporidian sporoplasm. At the time of spore activation, the keratin-based outer spore envelope assemblage dissociated and became phosphorylated when the spores were placed in the presence of labeled ATP. Verapamil or lanthanum, agents which block S. lophii spore activation, also blocked spore envelope keratin disassembly and phosphorylation when the spores were incubated in activation medium with labeled ATP. However, after the removal of the verapamil or lanthanum, the spores regained the capacity to activate in discharge medium and the keratin analogues appeared to dissociate and phosphorylate.     

Spore Discharge in the Basidiomycetes: I. Periodicity of Spore Discharge by Trametes gibbosa Fr.

Variations in the rates of spore discharge from this fungushave been examined in controlled laboratory conditions, usingapparatus designed to make a continuous record of the numbersof spores discharged by a strip of hymenium measuring approximately4.5 cm. X o.1 cm. These records have been extended by a directmicroscopic study of the numbers of spores falling through asmall area below the hymenium during successive short periods. Analysis of the continuous record has shown that at any onetime the rate of spore discharge is not uniform over the wholehymenium, and that at any one site it ia not constant. The variationin the rate of discharge in any one site is rhythmical, withintervals of the order of 30 to 90 minutes between maxima. Thesmall linear areas of hymenium which have been examined haveshown nearly the same rhythm-overall, but the phase of thisrhythm has differed at different sites in the area.     

Light and Spore Discharge in Ascobolus

Spore discharge in Ascobolus crenulatus occurs both in the lightand in the dark. In a 12 h light: 12 h dark daily regime discharge-ratehas peaks in the dark periods, due apparently to light stimulationwith about half a day's interval between stimulus and response. Using a ‘spore clock’ the course of discharge hasbeen followed for a single apothecium on changing from darknessto light. Exposure to light (500 lux) of wave-lengths around400, 440 and 460 mµ immediately causes ’puffing‘,whilst light of longer wave-length (504 and 580 mµ) hasno effect. Change from darkness to white light has no immediateeffect, but there is a delayed stimulation with a marked increasein discharge-rate 10–14 h later. Simultaneous illuminationof an apothecium, which has been in darkness, by blue light(420 mµ, or 440 mµ, 500lux) and yellow light (580mµ, 500 lux) does not result in puffing. The yellow appearsto prevent the blue light from exerting its effects.     

Nuclear and Organelle DNA Replication during Spore Germination in Bryophytes and Equisetum

In contrast to ferns, the spore nuclei of the moss, Funaria hygrometrica, are in the G₂ phase. They do not incorporate the thymidine analogue BUdr before the first mitosis. When spores germinate in the presence of aphidicolin, an inhibitor of the (nuclear) DNA polymerase alpha, they give rise to two-celled, negatively phototropic protonemata, consisting of relatively long and thin cells. Determinations of nuclear DNA content further confirm the presence of G₂-nuclei in Funaria spores. Spores of Pogonatum urnigerum and of Equisetum hyemale likewise contain G₂-nuclei, as shown by BUdr incorporation or experiments with aphidicolin, respectively. In germinating Funaria spores, the organelle DNA is synthesized very early and independent of nuclear DNA. A gradient in replication of organelle DNA is found only in the long tip cells which are formed in the presence of aphidicolin.     

Periodicity of Spore Discharge in Daldinia

In Daldinia concentrica spore discharge under natural conditionsis periodic, most spores being discharged during the night andfew in the daytime. An experimental study has been made of dischargeunder controlled conditions of light and temperature. In continuousdarkness periodic discharge was maintained for 12 days, butthen, although spore output continued, it ceased to be periodic.Return to alternating light (12 hrs., 100 f.c.) and darkness(12 hrs.) at once re-established the periodicity. In continuouslight (100 f.c.) periodic discharge ceased after 2 to 3 days,but was immediately re-established in alternating light (12hrs.) and darkness (12 hrs.). When the fungus was placed underconditions of alternating light and darkness each of 6 hours'duration, two peaks of spore-output were soon developed in the24-hour period. The experiments suggest that the natural periodicityis determined by the alternation of day and night.     

Short-Chain Phosphoinositide Partitioning into Plasma Membrane Models

Phosphoinositides are vital for many cellular signaling processes, and therefore a number of approaches to manipulating phosphoinositide levels in cells or excised patches of cell membranes have been developed. Among the most common is the use of “short-chain” phosphoinositides, usually dioctanoyl phosphoinositol phosphates. We use isothermal titration calorimetry to determine partitioning of the most abundant phosphoinositol phosphates, PI(4)P and PI(4,5)P₂ into models of the intracellular and extracellular facing leaflets of neuronal plasma membranes. We show that phosphoinositide mole fractions in the lipid membrane reach physiological levels at equilibrium with reasonable solution concentrations. Finally we explore the consequences of our results for cellular electrophysiology. In particular, we find that TRPV1 is more selective for PI(4,5)P₂ than PI(4)P and activated by extremely low membrane mole fractions of PIPs. We conclude by discussing how the logic of our work extends to other experiments with short-chain phosphoinositides. For delayed rectifier K⁺ channels, consideration of the membrane mole fraction of PI(4,5)P₂ lipids with different acyl chain lengths suggests a different mechanism for PI(4,5)P₂ regulation than previously proposed. Inward rectifier K⁺ channels apparent lack of selectivity for certain short-chain PIPs may require reinterpretation in view of the PIPs different membrane partitioning.     

Adaptation of the Spore Discharge Mechanism in the Basidiomycota

Background

Spore discharge in the majority of the 30,000 described species of Basidiomycota is powered by the rapid motion of a fluid droplet, called Buller''s drop, over the spore surface. In basidiomycete yeasts, and phytopathogenic rusts and smuts, spores are discharged directly into the airflow around the fungal colony. Maximum discharge distances of 1–2 mm have been reported for these fungi. In mushroom-forming species, however, spores are propelled over much shorter ranges. In gilled mushrooms, for example, discharge distances of <0.1 mm ensure that spores do not collide with opposing gill surfaces. The way in which the range of the mechanism is controlled has not been studied previously.

Methodology/Principal Findings

In this study, we report high-speed video analysis of spore discharge in selected basidiomycetes ranging from yeasts to wood-decay fungi with poroid fruiting bodies. Analysis of these video data and mathematical modeling show that discharge distance is determined by both spore size and the size of the Buller''s drop. Furthermore, because the size of Buller''s drop is controlled by spore shape, these experiments suggest that seemingly minor changes in spore morphology exert major effects upon discharge distance.

Conclusions/Significance

This biomechanical analysis of spore discharge mechanisms in mushroom-forming fungi and their relatives is the first of its kind and provides a novel view of the incredible variety of spore morphology that has been catalogued by traditional taxonomists for more than 200 years. Rather than representing non-selected variations in micromorphology, the new experiments show that changes in spore architecture have adaptive significance because they control the distance that the spores are shot through air. For this reason, evolutionary modifications to fruiting body architecture, including changes in gill separation and tube diameter in mushrooms, must be tightly linked to alterations in spore morphology.     

An Analysis of Spore Discharge in Sordaria

Sordaria fimicola can develop mature perithecia from which sporesare discharged when grown in darkness or in light. Under conditionsof alternating dark and light (12 hrs.: 12 hrs.) each day, sporedischarge is periodic with a low rate during the dark period,succeeded by a gradual rise to a relatively high rate in thelight period followed by a decline before the onset of the nextdark period. There is no trace of an endogenous rhythm. Transferfrom darkness to light always leads to an increase in the rateof discharge, and from light to dark to a decrease. The heightof the peak of discharge rate attained in light following adark period seems to be related to the length of the precedingdark period. Experiments with light of different colours but of roughly thesame energy value show that it is the blue rays that are mainlyeffective. From cultures of filter-paper yeast-extract mediuman orange pigment can be extracted with a maximum absorption,in the visible spectrum, at 470 mµ. It is possible thatthis is important in connexion with the sensitivity of the fungusto blue light.     

Iron Reduction and Trans Plasma Membrane Electron Transfer in the Yeast Saccharomyces cerevisiae

The ferri-reductase activity of whole cells of Saccharomyces cerevisiae (washed free from the growth medium) was markedly increased 3 to 6 h after transferring the cells from a complete growth medium (preculture) to an iron-deficient growth medium (culture). This increase was prevented by the presence of iron, copper, excess oxygen, or other oxidative agents in the culture medium. The cells with increased ferri-reductase activity had a higher reduced glutathione content and a higher capacity to expose exofacial sulfhydryl groups. Plasma membranes purified from those cells exhibited a higher reduced nicotinamide adenine phosphate (NADPH)-dependent ferri-reductase specific activity. However, the intracellular levels of NADPH, NADH, and certain organic acids of the tricarboxylic acids cycle were unchanged, and the activity of NADPH-generating enzymes was not increased. Addition of Fe(III)-EDTA to iron-deprived and iron-rich cells in resting suspension resulted in a decrease in intracellular reduced glutathione in the case of iron-deprived cells and in an increase in organic acids and a sudden oxidation of NADH in both types of cells. The depolarizing effect of Fe³⁺ was more pronounced in iron-rich cells. The metabolic pathways that may be involved in regulating the trans-plasma membrane electron transfer in yeast are discussed.     

A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH₂-proximal part of mature IEP110 (110N). The NH₂-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80–90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH₂-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.     

Phylogenetic Study of Organic Anion Transfer from Plasma into the Liver

THE discovery of two intracellular proteins, Y and Z, with the unique property of binding certain organic anions, may help to account for the selective and rapid transfer of bilirubin and various dyes, drugs, steroids and metabolites from plasma into the liver, which is known to occur among mammals^1–3. In the rat, from which they were first isolated³, Y and Z have been shown to possess the following characteristics, (i) They selectively bind organic anions, such as bilirubin, sulphobromophthalein (BSP) and indocyanine green, in vivo and in vitro³, (ii) The principal organic anion binding protein, Y, is present predominantly in the liver^3,4, (iii) The concentration of Y increases after administration of phenobarbital, DDT and other drugs and concomitantly the rate of organic anion transfer into the liver is increased⁵. Furthermore, in the newborn monkey, the concentration of Y and the transfer into the liver of BSP, as measured by plasma disappearance rates, are both low and subsequently increase⁶. We report here the results of a phylogenetic study involving various vertebrates. These results further support the theory that Y and Z are intracellular acceptors which facilitate the transfer of certain organic anions from plasma into the liver.     

An Endogenous Rhythm of Spore Discharge in Sordaria fimicola

The sporulation process in Sordaria fimicola has been studiedunder controlled conditions, with particular reference to discharge.Discharge occurred with a circadian periodicity for the greaterpart of the sporulation life of the culture, with peaks at about18.00 hours in either light or darkness. The consistency ofthis phenomenon was confirmed by an analysis of previous work.Fluctuations in light intensity, temperature, relative humidity,carbon dioxide concentration, and vibration were eliminatedas far as possible, as causes of this periodicity. Minor fluctuationsin discharge rate with a period of less than 24 h were probablyrandom. The general nature of the periodicity was shown to be consistentwith some aspects of endogenous rhythms in other organisms.However, spore discharge in S. fimicola is very sensitive tolight, and it is readily entrained to discharge out of phasewith the natural period. The rate of development of two stages of spore formation (delimitationand maturation) were determined, and these were related to therate and periodicity of discharge. From the data it was impossibleto establish at which stage of sporulation the periodicity arose.     

Stimulation of Spore Discharge by Reduced Humidity in Sordaria

An account is given of experiments on the effect of the humidityof an ambient air-stream on spore discharge in Sordaria fimicola.Change from saturated to dry (35 per cent. R. H.) conditionsinvariably led to a striking increase in the rate of spore discharge,and conversely change from dry to damp conditions produced amarked decrease.     

Effect of Light on Spore Discharge of Selected Basidiomycetes

Following entrainment of nine selected species of basidiomyceteswith alternating 12 h light and 12 h dark periods, circadianrhythms with nocturnal peaks of spore discharge were found topersist in continuous light or dark for periods of 4 –9d. However, when light and dark periods were reversed followingentrainment, Piptoporus belulinus. Panellus stipticus, Awiculariaauricula and Dacrymyces deliquescens made immediate readjustmentof their rhythms; Coriolus Dersicolor, Stereum hirsutum andClitocybe nebularis took 24 h to readjust with peaks of sporedischarge synchronized with the new dark time periods, whileGanoderma applanatum and Trametes betulina took 48 h to readjust.These reactions indicate exogenous rhythms Light effects, spore discharge, basidiomycetes     

Stimulation of Spore Discharge in Sordaria by Carbon Dioxide

The effect on the rate of spore discharge in Sordaria fimicolaof small concentrations of carbon dioxide has been studied.Substitution of an air-stream containing 0.2-2.5 per cent carbondioxide for air freed from this gas circulating over the peritheciaalways led to a marked increase in the rate of spore discharge.     

Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM''s cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM''s whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRγ found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.     

Further Observations on Light and Spore Discharge in Certain Pyrenomycetes

A ‘spore-clock’ for studying the hourly rate ofspore discharge over a 24-hour period is described. A numberof the experiments reported in this paper have involved theuse of this apparatus. In Sordaria fimicola there is a distinct positive light-dischargereaction in a dark-conditioned culture, the rate of spore dischargeincreasing steeply to a peak 2–3 hours after brief stimulationby bright light. Although darkening a light-conditioned cultureleads to an immediate decrease in the rate of discharge, thereis no evidence of a delayed negative dark-discharge reaction. In S. verruculosa with a 12-hours light: 12-hours dark dailyreëgime, more spores are discharged in the dark than inthe light periods if the intensity of illumination is low. Withhigher light intensity there is no significant difference betweenthe number of spores discharged in light and dark periods. Asin S. fimicola there is a positive light-discharge reaction,the interval between stimulus and maximum response being muchlonger (8–12 hours). When a dark-conditioned culture istransferred to light for 48 hours and then returned to darknessfor a further 48 hours it is apparent that not only is therea positive light-discharge reaction but also a negative dark-dischargeresponse. The ‘plateau’ level of discharge is essentiallythe same in light and darkness. It is confirmed that in Hypoxylon fuscum light inhibits discharge.