Saccharomyces cerevisiae forms actin ring structures in sporulation, similarly to Zygosaccharomyces rouxii

Yeast filamentous actin (F-actin) exists mainly as patches and cables. Previously, we investigated the behavior of F-actin during sporulation of Zygosaccharomyces rouxii and found a novel actin ring localized around the spore periphery in zygotic asci at a late stage of sporulation. To clarify whether the actin rings are also formed in sporulation in the model yeast Saccharomyces cerevisiae, we observed the distribution of F-actin in sporulating S. cerevisiae by rhodamine-phalloidin staining and confocal laser scanning microscopy. Ringlike actin structures were detected at the peripheral regions of S. cerevisiae spores in globose asci. When asci of S. cerevisiae were induced to become zygotic, actin rings were more obvious than those in globose asci. These results indicate that S. cerevisiae forms characteristic actin ring structures at a late stage of sporulation, similarly to Z. rouxii.     

Cortical actin filaments fragment and aggregate to form chloroplast-associated and free F-actin rings in mechanically isolatedZinnia mesophyll cells

Summary Changes in F-actin organization following mechanical isolation ofZinnia mesophyll cells were documented by rhodamine-phalloidin staining. Immediately after isolation, most cells contained irregular cortical actin fragments of varying lengths, and less than 5% of cells contained intact cortical filaments. During the first 8 h of culture, filament fragments were replaced by actin rings, stellate actin aggregates, and bundled filament fragments. Some of these aggregates had no association with organelles (free actin aggregates). Other aggregates were associated with chloroplasts, which changed in shape and location at the same time actin aggregates appeared. F-actin was concentrated within or around the nucleus in a small percentage of cells. After 12 h in culture, the percentage of cells with free actin rings and chloroplast-associated actin aggregates began to decline and the percentage of cells having intact cortical actin filaments increased greatly. Intermediate images were recorded that strongly indicate that free actin rings, chloroplast-associated actin rings, and other actin aggregates self-assemble by successive bundling of actin filament fragments. The fragmentation and bundling of F-actin observed in mechanically isolatedZinnia cells resembles changes in F-actin distribution reported after diverse forms of cell disturbance and appears to be an example of a generalized response of the actin cytoskeleton to cell stress.Abbreviations FITC fluorescein isothiocyanate - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - RhPh tetramethylrhodamine isothiocyanate-phalloidin     

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     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole     

Different extent of F-actin bundling in walled cells and in protoplasts ofMougeotia scalaris

Summary F-actin was localized inMougeotia interphase cells by rhodamine phalloidin (RLP) using an extended, formaldehyde-based fixation protocol, which included a minimal concentration of 0.05% (v/v) glutardialdehyde and stabilization of the calcium-binding vesicles by presaturation with neutral red. Staining revealed a low level of RLP-fluorescence throughout the cytoplasm. An enhanced level of RLP-fluorescence was found around the nucleus and in mostly two parallel fringes along each longitudinal chloroplast edge; also close to the chloroplast edge, quite regularly spaced patches of RLP-fluorescence were seen possibly associated with dictyosomes. The diffuse staining indicates lack of F-actin bundles inMougeotia filamentous cells, in contrast toSpirogyra interphase cells orMougeotia protoplasts. The observations upon staining with RLP confirm previous findings by electron microscopy and indicate seemingly single actin filaments throughout the entireMougeotia filamentous cell. Thus, a special F-actin organization is evident here which for the chloroplast movement is in support of the hypothesis of pigment regulated plasmalemma anchorage sites to actin filaments.Abbreviations CaBV calcium-binding vesicle - DIC differential interference contrast - EGTA ethyleneglycol-bis-(-aminoethyl ether) N, N, N, N tetraacetic acid - FA formaldehyde - GA glutardialdehyde - MFSB microfilament stabilizing buffer - PIPES piperazine-N, N-bis(2-ethanesulfonic acid) - RLP rhodamine (labeled) phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

ThePenicillium chrysogenum andAspergillus nidulans wetA developmental regulatory genes are functionally equivalent

Aspergillus nidulans andPenicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. InA. nidulans, spore maturation is controlled by thewetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) fromP. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. ThePwetA andAwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5 flanking regions, including conserved binding sites for the product of the regulatory geneabaA.PwetA fully complemented anA. nidulans wetA deletion mutation, demonstrating thatPwetA and its 5 regulatory sequences function normally inA. nidulans. These results indicate that the mechanisms controlling sporulation inA. nidulans andP. chrysogenum are evolutionarily conserved.     

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.     

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.Download video file.^{(136M, mov)}     

The arrangement of F-actin and microtubules during germination of Mucor rouxii sporangiospores

The distribution of F-actin microfilaments and microtubules was analyzed in germinating sporangiospores of Mucor rouxii by labeling with rhodamine-tagged phalloidin and by immunofluorescence microscopy. The transition from isodiametrical to apical growth was accompanied by a switch from uniform distribution of F-actin patches to a polarized accumulation of F-actin material at the germ tube tips. Immunoblotting of cell-free extracts of M. rouxii with a monoclonal anti-porcine -tubulin antibody (TU-01) disclosed two discrete bands of -tubulin suggesting the existence of two -tubulin genes in this fungus. Immunofluorescence microscopy of germinating cells stained with the same antibody revealed an elaborate network of cytoplasmic microtubules that persisted during the entire germination process and extended into the apex of the germ tube. Although their precise roles remain undetermined, the observed arrangement of cytoskeletal elements during germination is consistent with their presumed involvement in cell wall morphogenesis: the long axial microtubules serving as long-distance conveyors of wall-building vesicles to the apical region while the concentrated F-actin patches mark the participation of microfilaments in the zone of intense vesicle exocytosis at the hyphal apex.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DTT dithiothreitol - EGTA Ethylene glycol-bis (beta-aminoethyl ether) - N,N,N,N tetraacetic acid - F-actin Filamentous actin - MES 2-(N0morpholino)-ethanesulfonic acid - PIPES Piperazine-N,N-bis-2-ethanesulfonic acid - PMSF Phenyl-methylsulphonyl fluoride - TBS Tris-buffered saline     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.     

Identification of aryl-phospho-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidases in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

Four aryl-phospho--d-glucosidases were identified in Bacillus subtilis by using 4-methylumbelliferyl-phospho--d-glucopyranoside as a substrate. Two of these enzymes are the products of the bglA and bglH genes, previously suggested to encode aryl-phospho--d-glucosidases, while the other enzymes are encoded by the yckE and ydhP genes. Together, these four genes account for >99.9% of the glucosidase activity in B. subtilis on aryl-phospho--d-glucosides. yckE was expressed at a low and constant level during growth, sporulation, and spore germination, and was not induced by aryl--d-glucosides. ydhP was also not induced by aryl--d-glucosides. However, while ydhP was expressed at only a very low level in exponential-phase cells and germinating spores, this gene was expressed at a higher levels upon entry into the stationary phase of growth. Strains lacking yckE or ydhP exhibited no defects in growth, sporulation, or spore germination or in growth on aryl--d-glucosides. However, a strain lacking bglA, bglH and yckE grew poorly if at all on aryl--d-glucosides as the sole carbon source.Abbreviations MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--d-glucopyranoside - MUGal 4-Methylumbelliferyl--d-galactopyranoside - MUG-P 4-Methylumbelliferyl--d-glucopyranoside-6-phosphate     

Organization of the cytoskeleton in the coenocytic algaVaucheria longicaulis var.macounii: an experimental study

Summary The organization of the cytoskeleton of vegetative filaments ofVaucheria longicaulis Hoppaugh var.macounii Blum is investigated by fluorescence microscopy using monoclonal anti -tubulin antibodies and fluorescein (FITC)-labelled phalloidin. Confocal laser scanning microscopy observations give further information on the distribution of the cytoskeletal elements. Phalloidin labelling reveals F-actin bundles in the cortical cytoplasm of both fixed and unfixed vegetative filaments of this alga. In addition a more diffuse fluorescent component, seen at higher magnification to be made up of thinner F-actin bundles, can also be detected in unfixed cells. The distribution of the F-actin bundles resemble that of filamentous structures observed with differential interference contrast (DIC) microscopy in living cells. These structures seem to correspond to the microtubule associated reticulum (MAR) described in literature and overall the evidence suggests that actin and MAR elements are co-distributed. F-actin bundles are always found in association with focal masses (foci) of phalloidin-positive material. Foci are also observed by DIC microscopy associated with the cytoplasmic filamentous structures in living cells.Depolymerization of F-actin with cytochalasin D and the subsequent repolymerization that occurs on transfer ofVaucheria vegetative filaments to cytochalasin-free medium suggest that these foci are involved in the organization of the F-actin array. Immunofluorescence for -tubulin reveals microtubule bundles that are shorter in length and straighter in configuration than microfilament bundles. Microtubule bundles are associated with spot-like focal structures that, in many instances, show a close relationship with respect to nuclei. Oryzalin and cold temperature cause the depolymerization of the microtubule bundles and suggest, in conjunction with repolymerization studies, that these fluorescent spots associated with the ends of the microtubule bundles are involved in their organization; hence, they represent microtubule organizing centres or MTOCs. The importance of both microfilament and microtubule bundle focal regions is discussed with respect to the apical growth exhibited by the vegetative filaments of this alga.     

Cytoskeletal structures,ultrastructural characteristics and the capsule of the basidiomycetous yeast <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis>

The cytoskeleton, capsule and cell ultrastructure were studied during the cell cycle of Cryptococcus laurentii. In an encapsulated strain, cytoplasmic microtubules and a mitotic spindle were detected. Mitosis was preceded by migration of the nucleus into the bud. F-actin failed to be visualised by rhodamine-phalloidin (RhPh) in encapsulated cells and therefore an acapsular strain was used. The following actin structures were found: actin dots, actin cables and cytokinetic ring. Ultrastructural studies showed the presence of a nucleus in the bud before mitosis. A collar-shaped structure was seen at the base of bud emergence. A lamellar cell wall and a rough outer surface of the cells were detected. Cytoskeletal structures found in C. laurentii are similar to those in Cryptococcus neoformans, which is a serious human pathogen.     

Effect of cytochalasin A on actin distribution in the fission yeastSchizosaccharomyces pombe studied by fluorescent and electron microscopy

Summary Actin distribution and ultrastructure of the fission yeastSchizosaccharomyces pombe treated with cytochalasin A (CA) were investigated by fluorescence microscopy using rhodamine-conjugated phalloidin (rh-ph) and freeze substitution electron microscopy. Among the cytochalasins tested, CA was most effective and at 5 g/ ml inhibited the appearance of the actin ring at the cell equator at the stage prior to septum formation and the accumulation of actin dots at the septum-forming site both in wild-type cells and the mutantcdc 11, which is defective in septum formation at restrictive temperature. Freeze substitution electron microscopy of CA-treated cells revealed the displacement and morphological alteration of cytoplasmic vesicles and dictyosomes within 30 min and the appearance of dense bodies in the cytoplasm. A sub-population of cytoplasmic vesicles and dictyosomes were insensitive to CA and maintained their original structure. An electron less dense layer containing filamentous material was noted beneath the plasma membrane and thought to be the area of heavy actin patches stained with rh-ph at the cells ends. These results suggest that CA disrupted an actin network that normally maintains the organization of the secretory pathway involving dictyosomes and vesicles.     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

Structure of acetylcholinesterase complexed with (-)-galanthamine at 2.3 A resolution

In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of intense scrutiny. However, to date, only a single actin mutation has been identified. Described here is the isolation and characterization of four new cold-sensitive actin mutations. Sequence analysis of the mutant actin genes indicated that each of these mutations caused alterations in single amino acids that are conserved in all actin sequences. These mutants differ in their phenotypes. One of these mutations (act1-48) was identified as an extragenic suppressor of a mutation in the cdc4 gene, which is required for actin ring formation and cytokinesis. Interestingly, when act1-48 mutant cells were shifted to the restrictive temperature, actin patches were not detected but the actin ring formation and stability was unaffected. The three other mutations, act1-16, act1-32 and act1-67, primarily affected the actin ring formation or stability while F-actin patches did not seem to be substantially different in appearance. Given that the ultrastructural architectures of F-actin patches and the F-actin ring are presently unclear, these mutations, which affect one structure or the other, should be useful for future studies on the role of actin itself in the function of these F-actin-containing structures in S. pombe.     

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     

Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to 98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.