The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.
- MeSH
- Actins analysis MeSH
- Bridged Bicyclo Compounds, Heterocyclic pharmacology MeSH
- Cell Nucleus ultrastructure MeSH
- Cryptococcus neoformans drug effects physiology ultrastructure MeSH
- Dimethyl Sulfoxide pharmacology MeSH
- Microscopy, Electron MeSH
- Phalloidine analogs & derivatives MeSH
- Microscopy, Fluorescence MeSH
- Actin Cytoskeleton ultrastructure MeSH
- Microtubules drug effects MeSH
- Tubulin Modulators pharmacology MeSH
- Marine Toxins pharmacology MeSH
- Nocodazole pharmacology MeSH
- Rhodamines MeSH
- Free Radical Scavengers pharmacology MeSH
- Thiazolidines pharmacology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The unique long-neck yeast Fellomyces fuzhouensis has F-actin cables and cortical patches. Here, we describe a new F-actin structure present in fungi, a perinuclear F-actin collar ring around the cell nucleus. This F-actin structure can be visualized by fluorescent microscopic imaging of rhodamine-phalloidin-stained F-actin in cells treated with the mitotic drug isopropyl N-(3-chlorophenyl) carbamate or the microtubule inhibitor thiabendazol or when cells were grown in cut dried radish medium or yeast extract pepton dextrose (YEPD) medium. In contrast, these structures were absent in cells treated with Latrunculin A. The hypothetical functions of the F-actin ring are discussed.
- MeSH
- Actins metabolism MeSH
- Basidiomycota drug effects growth & development metabolism ultrastructure MeSH
- Bridged Bicyclo Compounds, Heterocyclic pharmacology MeSH
- Cell Nucleus metabolism MeSH
- Microscopy, Fluorescence MeSH
- Actin Cytoskeleton metabolism ultrastructure MeSH
- Thiazolidines pharmacology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: This basic research aimed to investigate the effects of the actin inhibitor latrunculin A (LA) on the human pathogen Cryptococcus neoformans, by freeze-substitution (FS) and electron microscopy (EM), to determine whether the actin cytoskeleton can become a new antifungal target for inhibition of cell division. METHODS: Cells treated with LA for 20 h in yeast-extract peptone dextrose medium were investigated by phase-contrast and fluorescent microscopy, FS and transmission EM, counted in a Bürker chamber and the absorbance was then measured. RESULTS: The disappearance of actin patches, actin cables and actin rings demonstrated the response of the cells of C. neoformans to the presence of the actin inhibitor LA. The removal of actin cables and patches arrested proliferation and led to the production of cells that had ultrastructural disorder, irregular morphology of the mitochondria and thick aberrant cell walls. Budding cells lysed in the buds and septa. CONCLUSION: LA exerts fungistatic, fungicidal and fungilytic effects on the human pathogenic yeast C. neoformans.
- MeSH
- Actins antagonists & inhibitors MeSH
- Antifungal Agents pharmacology MeSH
- Bridged Bicyclo Compounds, Heterocyclic pharmacology MeSH
- Cell Wall drug effects metabolism MeSH
- Cell Division drug effects MeSH
- Cryptococcus neoformans drug effects metabolism MeSH
- Microscopy, Electron methods MeSH
- Microscopy, Fluorescence methods MeSH
- Cryptococcosis drug therapy metabolism microbiology MeSH
- Humans MeSH
- Actin Cytoskeleton drug effects metabolism MeSH
- Cell Proliferation drug effects MeSH
- Thiazolidines pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Our basic cell biology research was aimed at investigating the effect on eukaryotic cells of the sudden loss of the F-actin cytoskeleton. Cells treated with latrunculin A (LA) in yeast extract peptone dextrose (YEPD) medium were examined using phase-contrast and fluorescent microscopy, freeze-substitution, transmission and scanning electron microscopy, counted using a Bürker chamber and their absorbance measured. The cells responded to the presence of LA, an F-actin inhibitor, with the disappearance of actin patches, actin cables and actin rings. This resulted in the formation of larger spherical cells with irregular morphology in the cell walls and ultrastructural disorder of the cell organelles and secretory vesicles. Instead of buds, LA-inhibited cells formed only 'table-mountain-like' wide flattened swellings without apical growth with a thinner glucan cell-wall layer containing β-1,3-glucan microfibrils. The LA-inhibited cells lysed. Actin cables and patches were required for bud formation and bud growth. In addition, actin patches were required for the formation of β-1,3-glucan microfibrils in the bud cell wall. LA has fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae.
- MeSH
- Actins antagonists & inhibitors MeSH
- Antifungal Agents pharmacology MeSH
- Bridged Bicyclo Compounds, Heterocyclic pharmacology MeSH
- Microbial Viability drug effects MeSH
- Microscopy MeSH
- Colony Count, Microbial MeSH
- Saccharomyces cerevisiae cytology drug effects physiology MeSH
- Saccharomycetales cytology drug effects physiology MeSH
- Thiazolidines pharmacology MeSH
- Publication type
- Journal Article MeSH
Microtubular and actin cytoskeletons were investigated in the lipophilic yeast Malassezia pachydermatis by fluorescence and electron microscopy. To detect microtubules by indirect immunofluorescence using monoclonal anti-tubulin antibody, a prolonged incubation with lysing enzymes was necessary due to its very thick cell wall. Cytoplasmic microtubules were detected in interphase and a spindle with astral microtubules was seen in M-phase. The disintegration of cytoplasmic microtubules and migration of the nucleus to the bud before mitosis were characteristic features of the basidiomycetous yeast Malassezia pachydermatis. The visualisation of F-actin structures (patches, cables and cytokinetic rings) by fluorescence microscopy using both monoclonal anti-actin antibody and rhodamine-phalloidin failed, but actin was detected by electron microscopy with immunogold labelling. Clusters of gold particles indicating actin structures were detected at the plasma membrane of cells with unique cortical ultrastructural features characteristic of the genus Malassezia. A possible association of these with the actin cytoskeleton is suggested.
BACKGROUND: We investigated the targeting of microtubules (MT) and F-actin cytoskeleton (AC) of the human pathogenic yeast Cryptococcus neoformans with agents for cancer therapy, in order to examine whether this yeast cytoskeleton could become a new antifungal target for the inhibition of cell division. METHODS: Cells treated with 10 cytoskeleton inhibitors in yeast extract peptone dextrose medium were investigated by phase-contrast and fluorescence microscopy, and growth inhibition was estimated by cell counts using a Bürker chamber and measuring absorbance for 6 days. RESULTS: Docetaxel, paclitaxel, vinblastine sulfate salt, cytochalasin D and chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] did not inhibit proliferation. The MT inhibitors methyl benzimidazole-2-ylcarbamate (BCM), nocodazole, thiabendazole (TBZ) and vincristine (VINC) disrupted MT and inhibited mitoses, but anucleated buds emerged on cells that increased in size, vacuolated and seemed to die after 2 days. The response of the cells to the presence of the actin inhibitor latrunculin A (LA) included the disappearance of actin patches, actin cables and actin rings; this arrested budding and cell division. However, in 3-4 days, resistant budding cells appeared in all 5 inhibitors. Disruption of the MT and AC and inhibition of cell division and budding persisted only when the MT and AC inhibitors were combined, i.e. VINC + LA, BCM + LA or TBZ + LA. CONCLUSION: The MT and AC of C. neoformans are new antifungal targets for the persistent inhibition of cell division by combined F-actin and MT inhibitors.
- MeSH
- Actins antagonists & inhibitors MeSH
- Antifungal Agents pharmacology MeSH
- Antineoplastic Agents pharmacology MeSH
- Bridged Bicyclo Compounds, Heterocyclic pharmacology MeSH
- Cell Division drug effects MeSH
- Cryptococcus neoformans drug effects MeSH
- Microscopy, Fluorescence MeSH
- Humans MeSH
- Actin Cytoskeleton drug effects MeSH
- Microtubules drug effects MeSH
- Drug Design MeSH
- Thiazolidines pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Distension is a regular mechanical stimulus in gastrointestinal (GI) tract. This study was designed to investigate the effect of hypotonic stress on pacemaking activity and determine whether actin microfilament is involved in its mechanism in cultured murine intestinal interstitial cells of Cajal (ICCs) by using whole-cell patch-clamp and calcium imaging techniques. Hypotonic stress induced sustained inward holding current from the baseline to -650+/-110 pA and significantly decreased amplitudes of pacemaker current. Hypotonic stress increased the intensity of basal fluorescence ratio (F/F0) from baseline to 1.09+/-0.03 and significantly increased Ca(2+) oscillation amplitude. Cytochalasin-B (20 microM), a disruptor of actin microfilaments, significantly suppressed the amplitudes of pacemaker currents and calcium oscillations, respectively. Cytochalasin-B also blocked hypotonic stress-induced sustained inward holding current and hypotonic stress-induced increase of calcium oscillations. Phalloidin (20 microM), a stabilizer of actin microfilaments, significantly enhanced the amplitudes of pacemaker currents and calcium oscillations, respectively. Despite the presence of phalloidin, hypotonic stress was still able to induce an inward holding current and increased the basal fluorescence intensity. These results suggest that hypotonic stress induces sustained inward holding current via actin microfilaments and the process is mediated by alteration of intracellular basal calcium concentration and calcium oscillation in cultured intestinal ICCs.
- MeSH
- Biological Clocks physiology MeSH
- Mechanotransduction, Cellular physiology MeSH
- Gastrointestinal Motility physiology MeSH
- Cells, Cultured MeSH
- Actin Cytoskeleton metabolism MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Osmotic Pressure physiology MeSH
- Telocytes physiology MeSH
- Calcium Signaling physiology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2 ) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.
The yeast strains VKM Y-2977 and VKM Y-2978, derived from the isolate Pa-202, were examined for their physiological properties and mycocin sensitivities and studied by light, phase-contrast, fluorescence, transmission and scanning electron microscopy. The cells of the first strain produced long stalk-like conidiophores, whereas the cells of the second one had the appearance of a typical budding yeast under the light microscope. Transmission and scanning electron microscopy showed the formation of stalk-like conidiophores and long necks in VKM Y-2977, similar in appearance to Fellomyces fuzhouensis. The actin cytoskeleton, microtubules and nuclei were similar as well, but due to presence of a capsule, they were not clearly visible. The second isolate, VKM Y-2978, had very short stalk-like conidiophores, and the neck, microtubules and actin cables were shorter as well. The actin patches, actin cables, and microtubules were similar in VKM Y-2977 and VKM Y-2978 and not clearly visible. The physiological characteristics and mycocin sensitivity patterns, together with the microscopic structures and ultrastructures, led us to conclude that both strains belong to Fellomyces penicillatus, even though they differ in the lengths of their stalk-like conidiophores and necks.
- MeSH
- Actins ultrastructure MeSH
- Antifungal Agents pharmacology MeSH
- Basidiomycota classification drug effects growth & development ultrastructure MeSH
- Cell Nucleus ultrastructure MeSH
- Cytoplasm ultrastructure MeSH
- Microscopy MeSH
- Microtubules ultrastructure MeSH
- Spores, Fungal drug effects growth & development ultrastructure MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The Slack (KCNT1) gene encodes sodium-activated potassium channels that are abundantly expressed in the central nervous system. Human mutations alter the function of Slack channels, resulting in epilepsy and intellectual disability. Most of the disease-causing mutations are located in the extended cytoplasmic C-terminus of Slack channels and result in increased Slack current. Previous experiments have shown that the C-terminus of Slack channels binds a number of cytoplasmic signaling proteins. One of these is Phactr1, an actin-binding protein that recruits protein phosphatase 1 (PP1) to certain phosphoprotein substrates. Using co-immunoprecipitation, we found that Phactr1 is required to link the channels to actin. Using patch clamp recordings, we found that co-expression of Phactr1 with wild-type Slack channels reduces the current amplitude but has no effect on Slack channels in which a conserved PKC phosphorylation site (S407) that regulates the current amplitude has been mutated. Furthermore, a Phactr1 mutant that disrupts the binding of PP1 but not that of actin fails to alter Slack currents. Our data suggest that Phactr1 regulates the Slack by linking PP1 to the channel. Targeting Slack-Phactr1 interactions may therefore be helpful in developing the novel therapies for brain disorders associated with the malfunction of Slack channels.
- MeSH
- Actins metabolism MeSH
- Cell Line MeSH
- Potassium Channels, Sodium-Activated metabolism MeSH
- HEK293 Cells MeSH
- Rats MeSH
- Humans MeSH
- Membrane Potentials physiology MeSH
- Patch-Clamp Techniques methods MeSH
- Mutation genetics MeSH
- Mice MeSH
- Neurons metabolism MeSH
- Protein Phosphatase 1 metabolism MeSH
- Signal Transduction physiology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
