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1.
Genes Chromosomes Cancer ; 62(12): 732-739, 2023 12.
Article in English | MEDLINE | ID: mdl-37530573

ABSTRACT

Rhabdomyosarcomas (RMS) constitute a heterogeneous spectrum of tumors with respect to clinical behavior and tumor morphology. The paternal uniparental disomy (pUPD) of 11p15.5 is a molecular change described mainly in embryonal RMS. In addition to LOH, UPD, the MLPA technique (ME030kit) also determines copy number variants and methylation of H19 and KCNQ1OT1 genes, which have not been systematically investigated in RMS. All 127 RMS tumors were divided by histology and PAX status into four groups, pleomorphic histology (n = 2); alveolar RMS PAX fusion-positive (PAX+; n = 39); embryonal RMS (n = 70) and fusion-negative RMS with alveolar pattern (PAX-RMS-AP; n = 16). The following changes were detected; negative (n = 21), pUPD (n = 75), gain of paternal allele (n = 9), loss of maternal allele (n = 9), hypermethylation of H19 (n = 6), hypomethylation of KCNQ1OT1 (n = 6), and deletion of CDKN1C (n = 1). We have shown no difference in the frequency of pUPD 11p15.5 in all groups. Thus, we have proven that changes in the 11p15.5 are not only specific to the embryonal RMS (ERMS), but are often also present in alveolar RMS (ARMS). We have found changes that have not yet been described in RMS. We also demonstrated new potential diagnostic markers for ERMS (paternal duplication and UPD of whole chromosome 11) and for ARMS PAX+ (hypomethylation KCNQ1OT1).


Subject(s)
Rhabdomyosarcoma, Alveolar , Rhabdomyosarcoma, Embryonal , Rhabdomyosarcoma , Humans , Rhabdomyosarcoma, Embryonal/genetics , Rhabdomyosarcoma, Embryonal/pathology , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma, Alveolar/genetics , DNA Methylation , Uniparental Disomy , Chromosomes
2.
Target Oncol ; 15(5): 673-679, 2020 10.
Article in English | MEDLINE | ID: mdl-32748047

ABSTRACT

BACKGROUND: Sarcopenia is common in advanced cancer and correlates with poor performance status, increased risk of treatment-related toxicity, and shortened survival. Inhibitors of the vascular endothelial growth factor pathway have been associated with development or deterioration of sarcopenia. OBJECTIVE: To assess the prevalence and impact of sarcopenia on survival in patients with metastatic renal cell carcinoma (mRCC) treated with cabozantinib, a novel, highly potent multikinase inhibitor. PATIENTS AND METHODS: Patients treated with cabozantinib for mRCC progressing on other targeted therapies with available computed tomography (CT) scans acquired at the time of initiation of cabozantinib and on the first restaging were evaluated retrospectively. Muscle mass was assessed based on striated muscle area at the level of the third lumbar vertebra. RESULTS: The median muscle mass index at CT1 and CT2 was 52.2 cm2/m2 (range 33.0-69.2 cm2/m2) and 49.1 cm2/m2 (range 33.1-68.2 cm2/m2), respectively. Sarcopenia was initially present in 13 (44.8%) patients. The mean muscle mass change between CT1 and CT2 was - 2.2 cm2/m2 (range - 10.1 to + 4.8cm2/m2). Six-month progression-free survival (PFS) was significantly shorter in patients with at least 10% muscle loss, reaching 50% (95% CI 9.9-90) versus 79.8% (95% CI 62.1-90.6) in others (p = 0.022). The presence of initial sarcopenia was not associated with grade 3-4 toxicity, which was reported in six (46.2%) and seven (46.7%) patients with and without sarcopenia, respectively. CONCLUSIONS: Significant and early skeletal muscle loss occurs during treatment with cabozantinib in a high proportion of patients and is associated with poor PFS.


Subject(s)
Anilides/therapeutic use , Carcinoma, Renal Cell/complications , Pyridines/therapeutic use , Sarcopenia/drug therapy , Sarcopenia/etiology , Adult , Aged , Anilides/pharmacology , Female , Humans , Male , Middle Aged , Neoplasm Metastasis , Pyridines/pharmacology , Sarcopenia/pathology
3.
Chemotherapy ; 61(3): 117-21, 2016.
Article in English | MEDLINE | ID: mdl-26650399

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/drug effects , Antifungal Agents/pharmacology , Antineoplastic Agents/pharmacology , Cryptococcus neoformans/drug effects , Actins/antagonists & inhibitors , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Division/drug effects , Drug Design , Humans , Microscopy, Fluorescence , Microtubules/drug effects , Thiazolidines/pharmacology
4.
Microbiology (Reading) ; 161(7): 1348-55, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25858300

ABSTRACT

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 ß-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 ß-1,3-glucan microfibrils in the bud cell wall. LA has fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae.


Subject(s)
Actins/antagonists & inhibitors , Antifungal Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomycetales/drug effects , Thiazolidines/pharmacology , Colony Count, Microbial , Microbial Viability/drug effects , Microscopy , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/physiology , Saccharomycetales/cytology , Saccharomycetales/physiology
5.
Chemotherapy ; 60(2): 99-106, 2014.
Article in English | MEDLINE | ID: mdl-25720843

ABSTRACT

BACKGROUND: Cryptococcus neoformans is one of the most important human fungal pathogens. Its cells contain rich microtubules required for nuclear division and rich F-actin cytoskeletons for cell division. Disruption of microtubules by a microtubule inhibitor should block nuclear division, and disruption of F-actin by an actin inhibitor should block cell division. We investigated the effects of microtubule and actin inhibitors to find out whether the cytoskeletons of C. neoformans can become a new anti-fungal target for the inhibition of cell division, when examined at the ultrastructural level. METHODS: Cells treated with the microtubule inhibitors vincristine (VIN) and methyl benzimidazole-2-ylcarbamate (BCM) and the actin inhibitor latrunculin A (LA), in yeast extract peptone dextrose medium, were examined by scanning (SEM) and transmission electron microscopy (TEM), and the cell number was counted using a Bürker chamber. RESULTS: After 2 days of inhibition with VIN, BCM or LA, the cells did not divide, but later, resistant, proliferating cells appeared in all samples. With combined microtubule and actin inhibitors (VIN + LA or BCM + LA), cells did not divide during 6 or even 14 days, and no resistant cells originated. TEM showed that the inhibited cells were without cytoplasm and were dead; only empty cell walls persisted with reduced capsules, shown on SEM. CONCLUSION: Combined microtubule and actin inhibitors (VIN + LA or BCM + LA), have lethal effects on C. neoformans cells and no resistant cells originate.


Subject(s)
Actins/antagonists & inhibitors , Benzimidazoles/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carbamates/pharmacology , Cryptococcus neoformans/drug effects , Microscopy, Electron, Transmission/methods , Microtubules/drug effects , Thiazolidines/pharmacology , Vincristine/pharmacology , Actins/ultrastructure , Cryptococcus neoformans/ultrastructure , Humans , Microscopy, Electron, Scanning/methods , Microtubules/ultrastructure , Treatment Outcome
6.
Chemotherapy ; 60(3): 185-90, 2014.
Article in English | MEDLINE | ID: mdl-25823986

ABSTRACT

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.


Subject(s)
Actins/antagonists & inhibitors , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cryptococcus neoformans/drug effects , Thiazolidines/pharmacology , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Antifungal Agents/pharmacology , Cell Division/drug effects , Cell Proliferation/drug effects , Cell Wall/drug effects , Cell Wall/metabolism , Cryptococcosis/drug therapy , Cryptococcosis/metabolism , Cryptococcosis/microbiology , Cryptococcus neoformans/metabolism , Humans , Microscopy, Electron/methods , Microscopy, Fluorescence/methods
7.
Microscopy (Oxf) ; 62(2): 295-301, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23155113

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/ultrastructure , Actins/analysis , Cell Nucleus/ultrastructure , Cryptococcus neoformans/ultrastructure , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/physiology , Dimethyl Sulfoxide/pharmacology , Free Radical Scavengers/pharmacology , Marine Toxins/pharmacology , Microscopy, Electron , Microscopy, Fluorescence , Microtubules/drug effects , Nocodazole/pharmacology , Phalloidine/analogs & derivatives , Rhodamines , Thiazolidines/pharmacology , Tubulin Modulators/pharmacology
8.
Microscopy (Oxf) ; 62(3): 327-39, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23160360

ABSTRACT

Polysaccharides account for more than 90% of the content of fungal cell walls, but the mechanism underlying the formation of the architecture of the cell walls, which consist of microfibrils embedded in an amorphous wall matrix, remains unknown. We used electron microscopy to investigate ten different fungal cell-wall polysaccharides to determine whether they could self-assemble into the fibrillar or amorphous component of fungal cell walls in a test tube without enzymes. The ultrastructures formed by precipitating ß-1,3-glucan and ß-1,6-glucan are different depending on the existence of branching in the molecule. Linear ß-1,3-glucan and linear ß-1,6-glucan precipitate into a fibrillar ultrastructure. Branched ß-1,6-glucan, mannan and glycogen precipitates are amorphous. Branched ß-1,3-glucan forms a fibrillar plus amorphous ultrastructure. Self-assembly among combinations of different linear and branched cell-wall polysaccharides results in an ultrastructure that resembles that of a yeast cell wall, which suggests that self-assembly of polysaccharides may participate in the development of the three-dimensional architecture of the yeast cell wall.


Subject(s)
Cell Wall/ultrastructure , Fungal Polysaccharides/metabolism , Saccharomyces cerevisiae/metabolism , beta-Glucans/metabolism , Fungal Polysaccharides/biosynthesis , Mannans/chemistry , Mannans/metabolism , Microfibrils/metabolism , Microscopy, Electron , beta-Glucans/chemistry
9.
J Electron Microsc (Tokyo) ; 61(4): 249-55, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22717791

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/ultrastructure , Actins/metabolism , Basidiomycota/metabolism , Cell Nucleus/metabolism , Actin Cytoskeleton/metabolism , Basidiomycota/drug effects , Basidiomycota/growth & development , Basidiomycota/ultrastructure , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Microscopy, Fluorescence , Thiazolidines/pharmacology
10.
J Basic Microbiol ; 52(5): 531-8, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22144013

ABSTRACT

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.


Subject(s)
Basidiomycota/growth & development , Basidiomycota/ultrastructure , Actins/ultrastructure , Antifungal Agents/pharmacology , Basidiomycota/classification , Basidiomycota/drug effects , Cell Nucleus/ultrastructure , Cytoplasm/ultrastructure , Microscopy , Microtubules/ultrastructure , Spores, Fungal/drug effects , Spores, Fungal/growth & development , Spores, Fungal/ultrastructure
11.
J Electron Microsc (Tokyo) ; 60(6): 379-91, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22003229

ABSTRACT

Temperature-sensitive actin mutant of Saccharomyces cerevisiae act1-1 was studied at a permissive temperature of 23°C by light, fluorescent and electron microscopy to elucidate the roles of actin cytoskeleton in the cycling eukaryotic cells. Mutant cells that grew slowly at the permissive temperature showed aberrations in the cytoskeleton and cell cycle. Mutant cells contained aberrant 'faint actin cables,' that failed in directing of mitochondria, vacuoles and secretory vesicles to the bud and the stray vesicles delivered their content to the mother wall instead of the bud. Bud growth was delayed. Spindle pole bodies and cytoplasmic microtubules did not direct to the bud, and nucleus failed to migrate to the bud. Repeated nuclear divisions produced multinucleated cells, indicating continued cycling of actin mutant cells that failed in the morphogenetic checkpoint, the spindle position checkpoint and cytokinesis. Thus, a single actin mutation appears to indicate uncoupling in space and time of the 'actin cytoskeleton-dependent cytoplasmic pathway of bud development and organelle positioning and inheritance' from the 'microtubule-dependent nuclear division pathway' in a budding yeast cell cycle.


Subject(s)
Actin Cytoskeleton/ultrastructure , Actins/genetics , Actins/ultrastructure , Microtubules/genetics , Microtubules/ultrastructure , Saccharomyces cerevisiae/genetics , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actins/metabolism , Cell Division , Cytoplasm/metabolism , Microscopy, Electron, Scanning/methods , Microscopy, Electron, Scanning Transmission/methods , Microscopy, Fluorescence/methods , Microtubules/metabolism , Mutation , Saccharomyces cerevisiae/growth & development , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
12.
Chemotherapy ; 56(3): 197-202, 2010.
Article in English | MEDLINE | ID: mdl-20551635

ABSTRACT

BACKGROUND: The aim of this basic study was to investigate by scanning electron microscopy the effects of cytoskeleton inhibitors on conidiogenesis and capsule in the yeast Fellomyces fuzhouensis CBS 8243, related to Cryptococcus neoformans. METHODS: Cells were treated by methyl benzimidazole-2-ylcarbamate (BCM) and latrunculin A (LAT) in yeast extract peptone dextrose medium and examined by scanning electron microscopy. RESULTS: During conidiogenesis, mother cells covered by capsule formed hypha-like stalks and at the hyphal tip yeast-like conidium developed. LAT blocked both stages of conidiogenesis. Inhibited mother cells and conidia became spherical and their capsule disappeared. BCM did not block formation of conidia that were neckless, or affect capsule. Combined application of LAT and BCM blocked both stages of conidiogenesis, cells became spherical and their capsule disappeared. CONCLUSION: Yeast cells with disrupted actin cytoskeleton do not reproduce by conidiogenesis and do not retain inherited cell shape and capsule.


Subject(s)
Basidiomycota/drug effects , Basidiomycota/ultrastructure , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Spores, Fungal/drug effects , Spores, Fungal/ultrastructure , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Shape/drug effects , Microscopy, Electron, Scanning/methods , Thiazolidines/pharmacology
13.
J Electron Microsc (Tokyo) ; 59(2): 141-52, 2010.
Article in English | MEDLINE | ID: mdl-19815613

ABSTRACT

Phenotypes of the two temperature-sensitive actin mutants of Saccharomyces cerevisiae act1-1 and act1-2 at permissive, restrictive and semi-restrictive temperatures were studied by freeze fracture and thin section electron microscopy, and fluorescent microscopy. In contrast to secretory mutants where accumulations of either secretory vesicles, Golgi apparatus, or endoplasmic reticulum were reported, act1-1 and act1-2 mutants revealed accumulation of all the three components, even at permissive temperature. However, more distinct accumulation of secretory organelles was evident during cultivation at the sub-restrictive temperature of 30 degrees C. At the restrictive temperature of 37 degrees C, many cells died, and their empty cell walls remained. Some of the few living cells showed features of apoptosis. From the present study, actin cables are concluded to be necessary for (i) correct spatial positioning and orientation of secretary pathway to the bud and septum, and (ii) vectorial movement of vesicles of the secretory pathway along the actin cables to the bud and septum.


Subject(s)
Actins/genetics , Mutation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Secretory Pathway , Freeze Fracturing , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Microscopy, Electron/methods , Microscopy, Fluorescence/methods , Phenotype , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Secretory Pathway/genetics , Secretory Pathway/physiology , Temperature
14.
Chemotherapy ; 55(4): 278-86, 2009.
Article in English | MEDLINE | ID: mdl-19521073

ABSTRACT

BACKGROUND: The cytoskeleton was investigated as a potential target for the inhibition of cell division in Fellomyces fuzhouensis CBS 8243 related to Cryptococcus neoformans. METHODS: Vincristine, vinblastine, paclitaxel, methyl benzimidazole-2-yl carbamate (BCM), thiabendazole, cytochalasins A, B and D and latrunculin A were added to yeast extract peptone dextrose medium containing cells, investigated by phase contrast and fluorescence microscopy, counted in a Burker chamber and absorbance was measured. RESULTS: Vincristine, vinblastine, paclitaxel, cytochalasins A, B and D transiently blocked proliferation. BCM disrupted microtubules and inhibited mitosis, but F-actin patches and cables persisted and neck-less conidia appeared without stalks. Latrunculin disrupted F-actin, cells became spherical, and stalks and necks degenerated; microtubules persisted, but mitosis, cytokinesis and conidiogenesis were blocked. The combined application of latrunculin and BCM disrupted F-actin and microtubules, and inhibited cells became spherical and did not divide. CONCLUSIONS: Microtubules and F-actin are effective targets for permanent inhibition of nuclear and cell division and conidiogenesis by BCM and latrunculin A.


Subject(s)
Actin Cytoskeleton/drug effects , Antifungal Agents/pharmacology , Basidiomycota/drug effects , Microtubules/drug effects , Tubulin Modulators/pharmacology , Yeasts/drug effects , Basidiomycota/cytology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cytochalasins/pharmacology , Cytokinesis/drug effects , Paclitaxel/pharmacology , Spores, Fungal/drug effects , Thiabendazole/pharmacology , Thiazolidines/pharmacology , Vinblastine/pharmacology , Vincristine/pharmacology , Yeasts/cytology
15.
Cell Biol Int ; 31(1): 16-23, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17055753

ABSTRACT

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.


Subject(s)
Actins/ultrastructure , Cytoskeleton/ultrastructure , Malassezia/ultrastructure , Microtubules/ultrastructure , Cell Cycle , Cytoskeleton/metabolism , Freeze Fracturing , Immunohistochemistry , Microscopy, Fluorescence , Microtomy , Models, Biological
16.
Antonie Van Leeuwenhoek ; 92(1): 29-36, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17136567

ABSTRACT

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.


Subject(s)
Cryptococcus/metabolism , Cryptococcus/ultrastructure , Cytoskeleton/metabolism , Actins/metabolism , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cell Wall/metabolism , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Microtubules/metabolism , Mitosis , Models, Biological , Phalloidine/analogs & derivatives , Phalloidine/chemistry , Rhodamines/chemistry
17.
Microbiology (Reading) ; 149(Pt 4): 865-876, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12686629

ABSTRACT

Organization of the cytoskeleton was studied in the ascomycetous black yeast Aureobasidium pullulans, an opportunistic human pathogen, in an effort to present it as a potential target of antifungal therapy. Long cytoplasmic microtubules, extending along the hyphae from the base to the growing apex, were the dominant structures in multinucleate interphase cells. Before mitosis these microtubules disappeared and were replaced by intranuclear spindles. This reorganization of microtubules occurred along the whole length of hypha before synchronous division of the nuclei. Actin cytokinetic rings were rarely seen. Cortical actin in the form of patches accumulated in areas of cell wall growth, i.e. in the hyphal apex and near the occasionally formed septum. Actin cables were not seen. During synchronous conidiogenesis, the cytoplasmic microtubules extended along developing conidia, and actin patches lined their subcortical areas. Actin rings were formed regularly at the base of uninuclear conidia. Microtubule inhibitor methyl benzimidazol-2-ylcarbamate disintegrated the microtubules, and inhibited nuclear division, development of hyphae and conidiogenesis. Actin inhibitor Cytochalasin D induced swelling of hyphal apexes and developing conidia. This inhibitory activity ceased after 5 to 12 h when the occasional septa appeared and conidiogenesis was completed. The lack of unicellular organization in multinucleate hyphae of A. pullulans seems be related to a rarity of F-actin structures: i.e. absence of actin cables, the lack of actin cytokinetic rings in particular, resulting in the uncoupling of the nuclear division from cytokinesis; the association of both processes is, however, retained during conidiogenesis.


Subject(s)
Actins/metabolism , Ascomycota/growth & development , Ascomycota/ultrastructure , Microtubules/ultrastructure , Opportunistic Infections/microbiology , Ascomycota/pathogenicity , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Fluorescent Antibody Technique, Indirect , Microscopy, Electron , Mycoses/microbiology
18.
Microbiology (Reading) ; 144 ( Pt 7): 1783-1797, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9695911

ABSTRACT

An excentric position of the nuclei, random orientation of mitoses, and multinuclear budding cells were identified in part of a population of temperature-sensitive (ts) Saccharomyces cerevisiae actin mutants at the permissive temperature of 23 degrees C by fluorescence and electron microscopy. The phenotype resembled that of mutants in beta-tubulin, dynein, JNM1, NUM1, ACT3, ACT5, myosins, profilin, tropomyosin 1, SLA2 and other genes. The question was addressed whether the cause was (i) defects in cell polarity in some ts actin mutants, manifested by lack of asymmetry of actin cortical patches, or (ii) lack of cytoplasmic or astral microtubules. The results indicated that in the cells with the nuclear defects, actin cortical patches showed the normal asymmetric distribution typical of undisturbed polarity. Cytoplasmic astral and spindle microtubules were also preserved. The principal difference found between the wild-type and actin mutant cells was in actin cables, which in the actin mutants were developed insufficiently. It is suggested that actin cables serve as a 'suspensory apparatus' and/or 'intracellular corridor', predetermining: the location of the nucleus in the central position in interphase; the axis of nuclear movement to the bud neck before mitosis; the direction of the elongating nucleus during mitosis; and the motion of each nucleus from an excentric to a central position during cytokinesis, in cooperation with the above-mentioned and other gene products.


Subject(s)
Actins/ultrastructure , Microtubules/ultrastructure , Saccharomyces cerevisiae/ultrastructure , Actins/chemistry , Cell Division , Cell Nucleus/ultrastructure , Genes, Fungal , Microscopy, Electron , Microscopy, Fluorescence , Microtubules/chemistry , Mutation , Phenotype , Saccharomyces cerevisiae/genetics , Temperature
19.
Microbiology (Reading) ; 144 ( Pt 8): 2331-2344, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9720056

ABSTRACT

The cells of Schizosaccharomyces japonicus var. versatilis responded to the presence of cytochalasin D (CD), an inhibitor of actin polymerization, by the disappearance of contractile actin rings (ARs) that had already formed and by inhibition of new ring formation. Actin cables disappeared. Actin patches remained preserved and became co-localized with regions of actual cell wall formation (at cell poles and at the site of septum development). Removal of the AR arrested formation of the primary septum and led to the production of aberrant septum protrusions in that region. Nuclear division was accomplished in the presence of CD but new ARs were not produced. The wall (septum) material was deposited in the form of a wide band at the inner surface of the lateral cell wall in the cell centre. This layer showed a thin fibrillar structure. The removal of CD resulted in rapid formation of new ARs in the equatorial region of the cells. This implies that the signal for AR localization was not abolished either by CD effects or by removal of an AR already formed. Some of the newly developed ARs showed atypical localization and orientation. In addition, redundant, subcortically situated actin bundles were produced. The removal of CD was quickly followed by the development of primary septa co-localized with ARs. Wall protrusions occurred co-localized with the redundant actin bundles. If these were completed in a circle, redundant septa developed. The AR is a mechanism which, in time and space, triggers cytokinesis by building a septum sequentially dependent on the AR. Aberrant septa were not capable of separating daughter cells. However, non-separated daughter cells subsequently gave rise to normal cells.


Subject(s)
Actins/physiology , Cytochalasin D/pharmacology , Schizosaccharomyces/drug effects , Schizosaccharomyces/physiology , Cell Wall/drug effects , Cell Wall/physiology , Cell Wall/ultrastructure , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Protoplasts/drug effects , Protoplasts/physiology , Protoplasts/ultrastructure , Schizosaccharomyces/ultrastructure
20.
Microbiology (Reading) ; 141 ( Pt 6): 1289-1299, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7670632

ABSTRACT

The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts of Saccharomyces cerevisiae DBY 1693 containing the act1-1 allele. Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons. In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized. After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth. This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature. Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts. These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope. A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of beta-(1-->3)-D-glucan wall microfibrils in yeast cells.


Subject(s)
Actins/genetics , Cell Wall/ultrastructure , Cytoskeleton/ultrastructure , Fungal Proteins/genetics , Protoplasts/ultrastructure , Saccharomyces cerevisiae/genetics , Actins/metabolism , Cell Wall/metabolism , Cytoskeleton/metabolism , Freeze Etching , Fungal Proteins/metabolism , Models, Molecular , Protoplasts/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/ultrastructure , Temperature
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