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F M Harold

Publications and source records attributed to F M Harold.

At least 19 recordsLinked to original sources
PubMed1991

Biochemical topology: from vectorial metabolism to morphogenesis.

In living cells, many biochemical processes are spatially organized: they have a location, and often a direction, in cellular space. In the hands of Peter Mitchell and Jennifer Moyle, the chemiosmotic formulation of this principle proved to be the key to understanding biological energy transduction and related aspects of cellular physiology. For H. E. Huxley and A. F. Huxley, it provided the basis for unravelling the mechanism of muscle contraction; and vectorial biochemistry continues to reverberate through research on cytoplasmic transport, motility and organization. The spatial deployment of biochemical processes serves here as a point of departure for an inquiry into morphogenesis and self-organization during the apical growth of fungal hyphae.

Biological Transport, Active↗
PubMed1991

Responses of growth cones to changes in osmolality of the surrounding medium.

The possible involvement of osmotically generated hydrostatic pressure in driving actin-rich extensions of the cell surface was examined using cultures of chick neurons. Estimation of the excess internal osmotic pressure of chick neural tissue by vapor pressure deficit osmometry, and of the excess internal hydrostatic pressure in cultured chick neurons using a calibrated pressure pipette, gave upper limits of 10 mosM and 0.1 atmosphere (1 atmosphere = 101325 Pa), respectively. Increases in the osmolality of the medium surrounding cultured neurons by addition of sucrose, mannitol or polyethylene glycol by amounts that should eliminate any internal pressure not only failed to arrest the growth of filopodia but caused them to increase in length up to twofold in 3-5 min. Lamellipodia remained unchanged following hyperosmotic shifts of 20 mosM, but higher levels caused a small decrease in area. Reduction of osmolality by the addition of water to the culture fluid down to 50% of its normal value failed to show any detectable change in either filopodial length or lamellipodia area. These observations argue against an osmotic mechanism for growth cone extension and show that the growth of filopodia, in particular, is unlikely to be driven by osmotically generated hydrostatic pressure. In contrast to the short-term effects on growth cone morphology, the slower elongation of the neuritic cylinder showed a consistent osmotic response. Growth rates were reduced following addition of osmolytes and increased in rate (as much as sixfold) following addition of water to the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

To shape a cell: an inquiry into the causes of morphogenesis of microorganisms.

We recognize organisms first and foremost by their forms, but how they grow and shape themselves still largely passes understanding. The objective of this article is to survey what has been learned of morphogenesis of walled eucaryotic microorganisms as a set of problems in cellular heredity, biochemistry, physiology, and organization. Despite the diversity of microbial forms and habits, some common principles can be discerned. (i) That the form of each organism represents the expression of a genetic program is almost universally taken for granted. However, reflection on the findings with morphologically aberrant mutants suggests that the metaphor of a genetic program is misleading. Cellular form is generated by a web of interacting chemical and physical processes, whose every strand is woven of multiple gene products. The relationship between genes and form is indirect and cumulative; therefore, morphogenesis must be addressed as a problem not of molecular genetics but of cellular physiology. (ii) The shape of walled cells is determined by the manner in which the wall is laid down during growth and development. Turgor pressure commonly, perhaps always, supplies the driving force for surface enlargement. Cells yield to this scalar force by localized, controlled wall synthesis; their forms represent variations on the theme of local compliance with global force. (iii) Growth and division in bacteria display most immediately the interplay of hydrostatic pressure, localized wall synthesis, and structural constraints. Koch's surface stress theory provides a comprehensive and quantitative framework for understanding bacterial shapes. (iv) In the larger and more versatile eucaryotic cells, expansion is mediated by the secretion of vesicles. Secretion and ancillary processes, such as cytoplasmic transport, are spatially organized on the micrometer scale. The diversity of vectorial physiology and of the forms it generates is illustrated by examples: apical growth of fungal hyphae, bud formation in yeasts, germination of fucoid zygotes, and development of cells of Nitella, Closterium, and other unicellular algae. (v) Unicellular organisms, no less than embryos, have a remarkable capacity to impose spatial order upon themselves with or without the help of directional cues. Self-organization is reviewed here from two perspectives: the theoretical exploration of morphogens, gradients, and fields, and experimental study of polarization in Fucus cells, extension of hyphal tips, and pattern formation in ciliates. Here is the heart of the matter, yet self-organization remains nearly as mysterious as it was a century ago, a subject in search of a paradigm.

Animals↗
PubMed1989

Chemotropism and branching as alternative responses of Achlya bisexualis to amino acids.

Hyphae of Achlya bisexualis growing on lean media orient their extension towards a source of amino acids, and also put forth branches. Micropipettes were used to generate gradients of amino acids in the vicinity of individual hyphae. Phenylalanine and methionine were the most powerful attractants: 0.04 mM amino acid in the pipette produced reorientation, and higher concentrations made the hyphae curl around the pipette and grow into its tip. Hyphae detected gradients as low as 5% across their width. Methionine and phenylalanine appeared to bind to different receptors. Local application of these amino acids also elicited the emergence of single branches, next to the pipette and on the high side of the gradient; comparison of diverse amino acids and their analogues suggested that branching and chemotropism share common receptors. By contrast, cytochalasin A and various ionophores induced branches at random sites, without receptor involvement. We propose that binding of amino acids to their receptors determines the site of precursor vesicle exocytosis, and consider possible mechanisms.

Amino Acids↗
PubMed1988

Transcellular ion currents and extension of Neurospora crassa hyphae.

Hyphae of Neurospora crassa, like many other tip-growing 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 that Neurospora 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.

Calcium↗
PubMed1988

Transcellular proton current in Achlya bisexualis hyphae: relationship to polarized growth.

Growing hyphae of Achlya bisexualis drive an electric current through themselves, such that positive charge flows into the apical region (the anterior 300 micron) and exits distally along the hyphal trunk. They also generate a gradient of extracellular pH, such that the medium surrounding the apex is slightly alkaline whereas that along the hyphal trunk is acid. To explore the genesis of these gradients and their relationship to polarized extension, we examined the effects of changes in the composition of the growth medium. The transcellular electric current was most pronounced in medium rich in amino acids. In leaner medium, containing limited amounts of amino acids or none at all, the current was attenuated or absent. We interpret the results to mean that inward current represents H+/amino acid symport, mediated by porters that are preferentially localized in the apical region. Apical alkalinity may be due to ammonia production. Outward current, and perhaps also the generation of metabolic acid, reflects the distribution of the H+-ATPase, which is excluded from the apex but is abundant along the hyphal trunk. Thanks to the spatial segregation of transport functions, protons characteristically flow into the apical region. However, since hyphae grow apically and at the same rate despite wide variations in current pattern, the flow of electric charge through the hyphae cannot be required to polarize extension or to localize the tip.

Amino Acids↗
PubMed1988

Transcellular ion currents during sporangium development in the water mould Achlya bisexualis.

Changes in the pattern of electric currents that accompany the transformation of growing hyphae of Achlya bisexualis into sporangia have been examined. When hyphae were transferred to a non-nutrient buffer, they continued to extend for several hours and then gave rise to sporangia. Throughout this process, current (positive charge) flowed into the apical region that corresponds approximately to the future sporangium. The current ceased after the crosswall appeared. The sporangium then remained electrically quiescent, except for a brief intense burst of outward current at the 'homogeneous' stage of spore cleavage. The inward current during sporangium formation largely represents an influx of protons. Addition of nitrate abolished the flow of electric current with little effect on sporulation. The late burst of outward current is most probably an artefact, generated by the discharge of salts from the sporangial vacuole. The transcellular electric current apparently plays no role in sporangium formation or in spore cleavage. Calcium ions, however, are required and may traverse the plasma membrane.

Calcium↗
PubMed1988

Dual roles for calcium ions in apical growth of Neurospora crassa.

We report initial attempts to define the role of Ca2+ in the polarized extension of Neurospora crassa. Growth of the organism was diminished in media containing less than 1 mM-Ca2+; extension was more severely impaired than biomass synthesis, resulting in the formation of stubby, bulbous hyphae, even of spherical cells. Reduced extension and abnormal morphology were correlated with the loss of surface-bound Ca2+, probably associated with the cell wall. Intracellular Ca2+ may be represented by material that fluoresces brightly in the presence of chlortetracycline. Punctate fluorescent bodies and diffuse fluorescence were both arrayed in a longitudinal gradient, maximum apically. Addition of the calcium ionophore A23187 induced dissipation of the fluorescence; concurrently, the hyphae lost as much as one half of their Ca2+ content. Extension continued almost unabated, but multiple branches quickly emerged from the apex. The observations suggest that a cytoplasmic Ca2+ gradient is not required for polarized extension, but may play a role in ensuring the dominance of the apex.

Calcimycin↗
PubMed1986

Calcium-dependent anion channel in the water mold, Blastocladiella emersonii.

Injection of depolarizing current into vegetative cells of the water mold Blastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about -40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near -100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr2+ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr2+. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.

Action Potentials↗
PubMed1986

Ionophores and cytochalasins modulate branching in Achlya bisexualis.

Hyphae of Achlya bisexualis growing on a medium deficient in amino acids elongated but produced relatively few branches. Branching was enhanced by three classes of compound: cytochalasins A and E, the calcium ionophores A23187 and ionomycin and proton ionophores such as tetrachlorosalicylanilide (TCS), carbonylcyanide m-chlorophenylhydrazone (CCCP), and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the effects of cytochalasins reflect the disruption of a microfilament-based system for vesicle transport. Enhancement of branching by ionophores implicates cytoplasmic ions in the control of branch initiation. There may be links between these phenomena and the earlier discovery that a new point of proton entry precedes the emergence of a branch and predicts its locus.

Calcimycin↗
PubMed1985

ATP-driven exchange of Na+ and K+ ions by Streptococcus faecalis.

We describe the characterization of KtrII, a novel potassium transport system of Streptococcus faecalis, first discovered by H. Kobayashi [1982) J. Bacteriol. 150, 506-511). KtrII requires sodium ions and mediates the stoichiometric exchange of internal Na+ for external K+. Potassium accumulation is not energized by the electrochemical potentials of either H+ or Na+; the energy source is probably ATP. Two lines of evidence indicate that KtrII is a manifestation of the sodium-stimulated ATPase reported earlier (Heefner, D. L., and Harold, F. M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2798-2802). (i) Mutants that lack the ATPase also lack KtrII, and revertants recover both in parallel. (ii) KtrII and the Na+-ATPase are induced in parallel when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential. KtrII is not induced in response to K+ deprivation. We propose that the Na+-ATPase exchanges Na+ for K+ ions.

Adenosine Triphosphatases↗
PubMed1985

Electrobiology of fungal hyphae.

Hyphae of many fungi drive electric currents through themselves. In Achlya bisexualis the current is carried by protons which enter the apical region by symport with amino acids and are expelled distally. The proton current may play a role in the spatial localization of apical growth.

Electric Conductivity↗
PubMed1984

Transcellular ion currents in the water mold Achlya. Amino acid proton symport as a mechanism of current entry.

Achlya, like other tip-growing organisms, generates an endogenous electrical current such that positive charge flows into the hyphal apex and exits from the trunk. The present study is concerned with the mechanism of current generation by hyphae growing in a defined, complete medium. The intensity of the current, measured in the extracellular medium with a vibrating probe, was unaffected by the removal of all the inorganic constituents of the growth medium. However, an increase in the external pH or the deletion of amino acids abolished the current. Removal of methionine alone diminished the current by two thirds. Hyphae also generated a longitudinal pH gradient in the extracellular medium; the region surrounding the tip was more alkaline than the bulk medium, whereas the region around the trunk was relatively acidic. These findings suggest that a flux of protons, dependent upon amino acids in the medium, carries current into the tip and creates the surrounding alkaline zone. The proton current appears to result from the transport of amino acids rather than their metabolism. Conditions that abolished the current also inhibited methionine uptake but had little effect on the respiratory rate. The findings imply a connection between the proton current and chemiosmotic energy transduction. We propose that protons flow into the hyphal tip through amino acid/proton symporters that are preferentially localized there. The proton flux energizes the uptake of amino acids into the growing zone and may also contribute to the polarization of hyphal growth.

Amino Acids↗
PubMed1984

Growing hyphae of Achlya bisexualis generate a longitudinal pH gradient in the surrounding medium.

Growing hyphae of Achlya bisexualis were found to generate a longitudinal pH gradient in the surrounding medium; the medium adjacent to the tip was slightly more alkaline than the bulk phase, while that near distal parts was acidic. The profile of external pH paralleled that of electric current, as measured with a vibrating probe; the apical alkaline zone corresponded to the region of current inflow. In organisms grown in complete medium, both current flow and apical alkalinization were inhibited when amino acid uptake was blocked, either by removing amino acids from the medium or by raising the external pH to 8.5. Achlya could, however, adapt to a medium deficient in organic nutrients; elongating hyphae again generated both the pH profile and the transcellular electric current. It is proposed that both the pH profile and the electric current are manifestations of a transcellular proton current, which arises from the segregation of proton pumps from proton leaks. Symport of protons with amino acids may be one mechanism by which protons enter the hyphal apex.

Adaptation, Physiological↗