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     

UV microirradiation implicates F-actin in reinforcing growing hyphal tips

Summary The cell walls of plants and fungi are thought to provide the strength required to resist turgor and thus maintain the integrity and morphology of these cells. However, during growth, walls must undergo rapid expansion which requires them to be plastic and therefore weak. In most tip-growing cells there is an apical concentration of F-actin associated with the rapidly expanding cell wall. Disruption of F-actin in the growing tips of hyphae ofSaprolegnia ferax by a localized irradiation, beginning 2–6 m behind the apex, with actin-selective 270 nm uv light caused the hyphae to burst, suggesting that actin supports the weak apical wall against turgor pressure. Bursting was pH dependent and Ca²⁺ independent at neutral pH. Hyphae burst in the very tip, where the cell wall is expected to be weakest and actin is most concentrated, as opposed to the lower part of the apical taper where osmotic shock induces bursting when actin is intact. When hyphae were irradiated with a wavelength of light that is less effective at disrupting actin, growth was slowed but they failed to burst, demonstrating that bursting was most likely due to F-actin damage. We conclude that F-actin reinforces the expanding apical wall in growing hyphae and may be the prime stress bearing structure resisting turgor pressure in tip growing cells.Abbreviations RP rhodamine phalloidin - F-actin filamentous actin - EGTA ethylene-glycol-bis-(-amino-ethyl ether) N,N-tetra-acetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - uv ultraviolet     

The involvement of F-actin inUromyces cell differentiation: The effects of cytochalasin E and phalloidin

Summary The role of F-actin in cell differentiation ofUromyces appendiculatus (bean rust fungus) germlings was examined by treating differentiating and nondifferentiating germlings with the actin-binding drugs cytochalasin E (CE) and phalloidin. Prolonged exposure of urediospores to 5×10^–3–5 × 10^–5 M CE induced nuclear division in up to 28–45% of the resulting germlings, whereas the rate of mitosis in established germlings exposed to these concentrations of CE was significantly lower (4–11%). Germlings treated with CE shifted from polarized apical growth to spherical expansion, cytoplasmic microfilaments were depolymerized, and nuclear inclusions became enlarged. Differentiating germlings exposed to a 10 minute pulse of 5×10^–6M CE before the initiation of septum formation prevented the establishment of the F-actin septal ring and growth of the crosswall delimiting the appressorium. Although these CE treatments resulted in morphological and nuclear events similar to those occurring during normal appressorium formation, transient microfilament depolymerization was not sufficient to induce differentiation. Phalloidin stabilized cytoplasmic microfilaments, especially posteriorly-located microfilaments, but did not affect differentiation, nor did it significantly inhibit the effects of CE.Abbrevations CE cytochalasin E - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - F-actin filamentous actin     

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Summary Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.Abbrevations FM fluorescence microscopy - EM electron microscopy - rh rhodamine - CA cytochalasin A - CD cytochalasin D - PBS phosphate-buffered saline - DMSO dimethylsulfoxide - GA glutaraldehyde     

Growth and morphogenesis inSaprolegnia ferax: Is turgor required?

Summary The oomyceteSaprolegnia ferax, unlike most walled organisms, does not regulate turgor. When hyphae were subjected to water stress by the addition of sucrose or other solutes to the growth medium, turgor pressure diminished progressively; yet the hyphae continued to extend with deposition of a more plastic apical wall. Even when turgor was no longer measurable with a micropipet-based pressure probe (0.02 MPa or less, compared with 0.4 MPa in unsupplemented medium) they produced regular hyphal tubes and tips. Such turgorless hyphae extended as rapidly, or more rapidly, than normal ones, but they were wider and their tips blunter. Despite the loss of turgor, hyphae put forth branches and cysts germinated. The organization of actin microfilaments was essentially normal, and the response to cytochalasin A was similar in turgorless and standard hyphae. However, as turgor diminished the hyphae's capacity to penetrate solid media was progressively impaired; aerial hyphae were no longer produced, and zoospore formation was inhibited. The results contradict the common belief that turgor supplies the driving force for hyphal extension, tip morphogenesis, and branching. Evidently, these functions do not intrinsically require hydrostatic pressure. Turgorless hyphae are, however, crippled by their inability to exploit solid media.Abbreviations PEG-300 polyethylene glycol-300 - Rh-Phal rhodamine phalloidin - F-actin filamentous actin - DMSO dimethyl sulfoxide - PYG peptone, yeast extract, glucose - MPa megapascals     

Evidence that actin reinforces the extensible hyphal apex of the oomyceteSaprolegnia ferax

Summary Filamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.Abbrevations CE cytochalasin E - DMSO dimethyl sulphoxide - RP rhodamine phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

Growth inhibition and excessive branching in <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis> induced by 2,4-diacetylphloroglucinol is linked to disruption of filamentous actin cytoskeleton in the hyphae

We observed that 2,4-diacetylphloroglucinol (DAPG), a major antimicrobial metabolite produced by a rhizoplane bacterium Pseudomonas fluorescens ECO-001 inhibited mycelial growth of a damping-off phytopathogen Aphanomyces cochlioides AC-5 through inducing excessive branching and curling in the hyphae. This study aimed to unravel the mode of action of DAPG caused excessive branching, curling and growth inhibition of AC-5 hyphae by detecting localized changes in the cortical filamentous actin (F-actin) organization by rhodamine-conjugated phalloidin. Confocal laser scanning microscopic observations revealed that both living bacteria and DAPG severely disrupted the organization of F-actin in the A. cochlioides hyphae in a similar manner. Furthermore, an inhibitor of F-actin polymerization, latrunculin B also induced similar growth inhibition, excessive branching and caused disruption of F-actin in the AC-5 hyphae. Our results suggested that growth inhibition and excessive branching induced in A. cochlioides by DAPG is likely to be linked to the disruption of F-actin cytoskeleton in the affected hyphae. This is the first report on disruption of cytoskeleton of a eukaryotic A. cochlioides by a well-known biocontrol metabolite DAPG secreted from a prokaryotic bacterium ECO-001.     

The cytoskeleton of Cobaea seed hairs:

The cell wall of Cobaea scandens seed hairs developed in a characteristic sequence, with the deposition of a cellulose thread onto a pectic swelling layer was the final event. The cellulose thread was intracellularly accompanied by a band of 10–18 microtubules. During the formation of the swelling layer the microtubules were homogeneously distributed; they ran circumferentially normal to the cell axis. When cellulose-thread formation started, the microtubules became arranged in a helical band. The density of the microtubules varied during the different phases of development. The highest density was observed before cellulosethread formation and ranged from 6–15 m·m^-2. The length of the microtubules, 20–30 m, was determined by direct measurements, as well as estimated from the total microtubular length in a given area and the counted free ends. With the indirect immunofluorescence technique the microtubules of the band stained inhomogeneously. Those which were located at the edges of the band fluoresced more intensely than those of the central part. Attempts to visualize actin filaments in the hair cells with rhodaminyl-conjugated phalloidin resulted in a homogeneous staining of the area of the microtubular band, indicating that actin filaments may be present in this region. Though, in thin sections and dry-cleaved cells, filamentous structures were observed between the microtubules, caution is expressed that the observed fluorescence was, indeed, due to actin filaments. The role of the filamentous structures is discussed with respect to formation and maintenance of the microtubular band. Microtubules apparently did not cross coated pits which were visualized in the plasma membrane through the dry-cleaving technique.Abbreviations IFT indirect immunofluorescence technique - RP rhodaminyl-conjugated phalloidin - SEM scanning electron microscopy     

Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans

The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.     

The effect of diffusion,depolymerization and nucleation promoting factors on actin gel growth

In eukaryotic cells, localized actin polymerization is able to deform the plasma membrane and push the cell forward. Depolymerization of actin filaments and diffusion of actin monomers ensure the availability of monomers at sites of polymerization, and therefore these processes must play an active role in cellular actin dynamics. Here we reveal experimental evidence that actin gel growth can be limited by monomer diffusion, consistent with theoretical predictions. We study actin gels formed on beads coated with ActA (and ActA fragments), the bacterial factor responsible for actin-based movement of Listeria monocytogenes. We observe a saturation of gel thickness with increasing bead radius, the signature of diffusion control. Data analysis using an elastic model of actin gel growth gives an estimate of 2×10^–8 cm^–2 s^–1 for the diffusion coefficient of actin monomers through the gel, ten times less than in buffer, and in agreement with literature values in bulk cytoskeleton, providing corroboration of our model. The depolymerization rate of actin filaments and the elastic modulus of the gel are also evaluated. Furthermore, we qualitatively examine the different actin gels produced when ActA fragments interact with either VASP or the Arp2/3 complex.     

Configuration of actin microfilaments during sporangium development inAchlya bisexualis: comparison of two staining protocols

Summary The spatial organization of actin microfilaments during the asexual life cycle ofAchlya bisexualis has been examined by two methods. One is the standard procedure described by Heath [Eur J Cell Biol (1987) 44: 10–16], in which specimens are fixed with formaldehyde and then stained with rhodamine-phalloidin. In the other, introduced by Sonobe and Shibaoka [Protoplasma (1989) 148: 80–86], specimens are treated with the protein crosslinking agent MBS (m-maleimidobenzoyl-N-hydroxysuccinimide) before fixation and staining. Both methods display the actin-rich cleavage zones that outline the developing zoospores. However, in extending hyphae and young sporangia the images are significantly different. Specimens pretreated with MBS display more prominent axial microfilament cables than do standard specimens, while peripheral actin plaques are sparse or absent. The results suggest that actin microfilaments occur in several configurations, some of which may be obscured by the standard fixation procedure. Pretreatment with MBS, though probably subject to artefacts of its own, may help preserve some features that would otherwise be missed.Abbreviations Rh-Phal rhodamine phalloidin - MBS m-maleimidobenzoyl-N-hydroxysuccinimide - PIPES piperazine-N,N-bis [2-ethanesulfonic acid] - EGTA ethylene glycol-bis (-aminoethyl ether) N,N-tetraacetic acid - DMSO dimethyl sulfoxide     

Effects of exogenous calcium ions on tip growth,intracellular Ca2+ concentration,and actin arrays in hyphae of the fungus Saprolegnia ferax

The maintenance of growth of hyphae of Saprolegnia ferax was dependent on the presence of external Ca²⁺ and the growth rate increased with increased external Ca²⁺ up to 5 × 10⁻² m Ca²⁺. When Ca²⁺ was greater than 5 × 10⁻² m, growth rates decreased. Internal membrane-associated Ca²⁺ was localized with chlortetracycline. Internal Ca²⁺ became depleted in hyphae grown in the absence of Ca²⁺ and was increased in hyphae grown in high concentrations of Ca²⁺, showing that internal Ca²⁺ can be modulated by external Ca²⁺. However, the range of the internal change was not as great as the range of external concentration used, indicating that the hyphae are capable of regulating Ca²⁺ in the presence of a large concentration gradient. In the absence of external Ca²⁺, growth can occur for a limited time through use of internal Ca²⁺. The actin cytoskeleton was altered in hyphae grown in both high and low Ca²⁺. Hyphae grown in 10⁻³ m Ca²⁺ had more actin in their apical network and peripheral plaques of actin were further from the apex than in more slowly growing hyphae in 10⁻¹ m and 0 Ca²⁺. The tips of hyphae growing in low Ca²⁺ also had a tendency to swell, giving these hyphae irregular shapes. Ca²⁺ is known to affect cell wall rigidity and the consistency of actin gels, two factors that can be expected to affect hyphal growth. External Ca²⁺ does play a role in hyphal growth possibly directly by acting on the cell wall and indirectly by altering internal Ca²⁺, thus affecting the actin cytoskeleton and possibly other growth processes.     

Microinjection of pollen‐specific actin‐depolymerizing factor, ZmADF1, reorientates F‐actin strands in Tradescantia stamen hair cells

Maize actin-depolymerizing factor (ADF) binds both monomeric and filamentous actin and increases actin dynamics in vitro. To test its effects in vivo, recombinant pollen ADF1 was expressed in bacteria and microinjected into Tradescantia stamen hair cells. Initially, all cytoplasmic streaming ceased and the central, longitudinal transvacuolar strands were disrupted. After 20–45 min, streaming resumed but in the form of conspicuous transverse pathways of movement in the cortex. Staining the actin filaments by a second injection of fluorescein-conjugated phalloidin showed that the longitudinal actin cables seen in controls had been replaced by a thickening of the transverse cortical arrays, whose orientation matched the new pattern of streaming. Microinjection of rhodamine–tubulin confirmed that the microtubules also formed a transverse cortical array and it is suggested that the spatial cues for re-modelling the actin after ADF1 injection may be provided by the microtubular system.     

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g⁻¹ with productivities of 3.2–5.0 g l⁻¹ h⁻¹. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l⁻¹ h⁻¹ by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l⁻¹ furfural and 10 g l⁻¹ acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.     

Rac1-dependent actin filament organization in growth cones is necessary for β1-integrin–mediated advance but not for growth on poly-D-lysine

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine–phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine–phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D -lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine–phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for β1 integrin–mediated growth cone advance, but not for growth on poly-D lysine. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 524–540, 1998     

Differential cytoplasm-plasma membrane-cell wall adhesion patterns and their relationships to hyphal tip growth and organelle motility

Summary Plasmolysis of hyphae of the oomycetesSaprolegnia ferax andAchlya ambisexualis and the ascomyceteNeurospora crassa produced abundant cytoplasmic strands between the retracted cytoplasm and punctate adhesions of the plasma membrane to the cell wall. These strands formed throughout the length of mature hyphae and are the first demonstration of Hechtian strands in hyphae. In contrast to similar strands in various plant cells, the strands inSaprolegnia lacked endoplasmic reticulum but contained F-actin, suggesting similarity between their adhesion sites and focal contacts in animal cells. However, strand adhesion to the wall was insensitive to RGD-containing peptides, suggesting that the trans-membrane adhesion molecules differ from animal integrins. The pattern of plasma membrane-cell wall adhesion varied in different zones along hyphae, with broad, irregular connections in the extreme apex, uniform and continuous connection in a transition zone, and small, punctate adhesions in the mature subapical zone, suggesting differential functions in these different regions. The apical adhesions are important in tip growth, as diverse inhibitors induced concomitant changes in hyphal growth and the adhesions in the apical and transition zones. Plasmolysis also induced cytoplasmic migrations throughout hyphae. Such migrations were dominated by the central cytoplasm, and produced distorted organelles which spanned central and peripheral cytoplasm, thus supporting the idea that the adhesions in mature zones of hyphae anchor the peripheral cytoplasm and facilitate cytoplasmic and organelle migrations.Abbreviations OM organic medium - RP rhodamine phalloidin - DIC differential interference contrast - PIPES piperazine-N,N-bis-2-ethanosulphonic acid     

The dominant attached filamentous algae of Georgian Bay,the North Channel and Eastern Lake Huron: Field ecology and biomonitoring potential during 1980

Field investigations during the ice-free period of 1980 confirm that the dominant attached filamentous algae in the Canadian waters of Lake Huron are the green algae Ulothrix zonata and Cladophora glomerata, and the red alga Bangia atropurpurea. It is believed that nutrient availability limits the distribution of these algae, while temperature controls their seasonal periodicity. Because of favourable physical characteristics, the study area represents a vast potential habitat for attached filamentous algae. It is expected that eastern Georgian Bay, in particular, will suffer significant environmental degradation from the growth of Cladophora unless existing phosphorus levels are maintained indefinitely (i.e., < 0.005 mg total P 1^–1). Attached filamentous algae accumulate (10³ to 10⁵ x) a variety of elements primarily in proportion to availability in the surrounding water. The occurrence of maximum algal metal concentrations at municipal waste water outfalls, river mouths and harbour areas (e.g., in µg g^–1, Cr 29.0, Cu 46.4, Ni 34.0, Pb 55.0) is indicative of discrete source loadings, while elevated levels at remote sites in eastern Georgian Bay (e.g., in µg g^–1, Cr 12.0–15.5, Cu 18.0, Ni 15.0–16.0, Pb 8.5–8.8) are suggestive of generalized loadings from the Canadian Shield, possibly due to the effects of acidic precipitation.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Rapid wound responses ofSaprolegnia ferax hyphae depend upon actin and Ca2+-involving deposition of callose plugs

Summary Growing hyphae of the oomyceteSaprolegnia ferax wounded by impalement with a ca. 0.2 m diameter glass microelectrode normally respond within seconds with an apically directed cytoplasmic contraction followed by production of a plug which encases the electrode and occludes its recording of transmembrane potentials. This plug contains callose and Ca²⁺-associated membranes. To characterize the rapid wounding response, we disrupted specific filamentous (F) actin populations and Ca²⁺ regulation. Plug formation is inhibited by disruption of F-actin populations and low exogenous Ca²⁺ but not by inhibition of stretch-activated Ca²⁺ channels with Gd³⁺. Therefore, stretch-activated channels are not the immediate sensor. Instead, sensing may involve strain on the actin cytoskeleton which triggers the occlusion response. This wound response is qualitatively similar to the production of septa which isolate developing sporangia and seal severed hyphae, indicating the use of a normal basic cellular developmental system as a protective mechanism against environmental damage. The wound response is essential, since an inability to seal sites of mechanical damage is potentially catastrophic in acellular coenocytic organisms.Abbreviations APW artificial pond water - BAPTA 1,2-bis(orthzo-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - CTC chlortetracycline - DIC Nomarski differential interference contrast microscopy - F-actin filamentous actin - LatB latrunculin B - PM plasma membrane - RP rhodamine-labeled phalloidin - SA channels stretch-activated channels