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.

• MeSH
• aktiny * metabolismus   MeSH
• endozomy metabolismus   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• transport proteinů MeSH
• vezikulární transportní proteiny * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• aktiny metabolismus   MeSH
• buněčné linie MeSH
• draslíkové kanály aktivované sodíkem metabolismus   MeSH
• HEK293 buňky MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• metoda terčíkového zámku metody   MeSH
• mutace genetika   MeSH
• myši MeSH
• neurony metabolismus   MeSH
• proteinfosfatasa 1 metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Complex visual signaling through various combinations of colors and patterns has been well documented in a number of diurnal reptiles. However, there are many nocturnal species with highly sensitive vision, being able to discriminate colors in night conditions, as was shown in geckos. Because of their sensitivity to chromatic signals, including UV (ultraviolet), they may have potential hidden features in their coloration, which may play role in intraspecific communication (e.g. mate choice) or interspecific signals (e.g. antipredatory function). We explored this hypothesis in nocturnal Leopard geckos (Eublepharis macularius), a species using visual signals in both antipredation defense and courtship, having ontogenetic color change accompanied by a shift in behavior. We used UV photography and visual modeling in order to compare various aspects of their coloration (luminance, contrast, color proportions) between sexes, age groups and populations. We found that Leopard geckos have considerable UV reflectance in white patches on their tails (and on the head in juveniles). Though, no prominent differences were detected in their coloration between various groups. We hypothesize that the limitation of UV reflectance to the head and tail, which are both actively displayed during defense, especially in juveniles, might potentially boost the effect of antipredation signaling.

• MeSH
• ještěři fyziologie   MeSH
• noční vidění fyziologie   MeSH
• rozpoznávání obrazu fyziologie   MeSH
• sexuální faktory MeSH
• světelná stimulace MeSH
• ultrafialové záření MeSH
• věkové faktory MeSH
• vidění barevné fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The subject of our investigation was the visual features of wing color with special focus on the UV reflectance in the green-veined white butterfly (Pieris napi). Previous studies had concluded that UV reflectance on dorsal wing surfaces is found only in the female P. napi. Based on UV sensitive photography, we analyzed a correlation between 12 geographic and environmental factors and UV reflectance patterns on 3 patches on the forewings of 407 P. napi specimens from the Palaearctic region. Results had shown that females significantly differ from males: they exhibit a 25% higher UV reflectance. To investigate whether and how UV reflectance levels on the forewings and hindwings of both sexes are influenced by the environment, we performed a principal component analysis (PCA) with several environmental variables. For several variables (in particular, latitude and longitude, mean annual temperature and precipitation, and temperature annual range and altitude), the generalized linear model (GLM) model revealed a significant correlation in both sexes. This suggests a link between UV reflectance levels and the environment and distribution of P. napi. We found that stronger UV reflectance is associated with generally more hostile environments and concluded that large-scale environmental factors influence the UV reflectance on the forewings of both male and female green-veined white butterflies.

• MeSH
• motýli * MeSH
• pigmentace * MeSH
• pohlavní dimorfismus MeSH
• ultrafialové záření MeSH
• životní prostředí * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

• MeSH
• draslíkové kanály řízené napětím chemie   metabolismus   MeSH
• endozomální třídící komplexy pro transport metabolismus   MeSH
• lidé MeSH
• mikrofilamentové proteiny chemie   metabolismus   MeSH
• multimerizace proteinu * MeSH
• proteasomový endopeptidasový komplex chemie   metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 1 chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• ubikvitinace * MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

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
• aktiny antagonisté a inhibitory   MeSH
• antifungální látky farmakologie   MeSH
• bicyklické sloučeniny heterocyklické farmakologie   MeSH
• buněčné dělení účinky léků   MeSH
• Cryptococcus neoformans účinky léků   MeSH
• fluorescenční mikroskopie MeSH
• lidé MeSH
• mikrofilamenta účinky léků   MeSH
• mikrotubuly účinky léků   MeSH
• protinádorové látky farmakologie   MeSH
• racionální návrh léčiv MeSH
• thiazolidiny farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• biologické hodiny fyziologie   MeSH
• buněčný převod mechanických signálů fyziologie   MeSH
• gastrointestinální motilita fyziologie   MeSH
• kultivované buňky MeSH
• mikrofilamenta metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• osmotický tlak fyziologie   MeSH
• telocyty fyziologie   MeSH
• vápníková signalizace fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• buňky * MeSH
• molekulární biologie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• NLK Publikační typ
• učebnice vysokých škol

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
• aktiny antagonisté a inhibitory   MeSH
• antifungální látky farmakologie   MeSH
• bicyklické sloučeniny heterocyklické farmakologie   MeSH
• mikrobiální viabilita účinky léků   MeSH
• mikroskopie MeSH
• počet mikrobiálních kolonií MeSH
• Saccharomyces cerevisiae cytologie   účinky léků   fyziologie   MeSH
• Saccharomycetales cytologie   účinky léků   fyziologie   MeSH
• thiazolidiny farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH

Úvod: Do diagnostiky kardiomyopatií ve forenzní medicíně patří metody světelné i elektronové mikroskopie a v současné době i metody molekulární biologie. Případ: Vyšetření myokardu u 27-letého muž, který zkolaboval při cestě do práce, přivolané RZS se nepodařilo obnovit vitální funkce. Výsledky: Světelnou a elektronovou mikroskopií byly prokázány změny svědčící pro kardiomyopatii (nepravidelně probíhající hypertrofické kardiomyocyty, ultrastrukturálně neuspořádanost Z proužků, zmnožení mitochondrií, myofibrily probíhající longitudinálně i transverzálně). Genetické vyšetření prokázalo mutaci beta-aktinu. Závěr: Kardiomyopatie může být příčinou náhlého úmrtí u mladých jedinců a její diagnostika vyžaduje interdisciplinární spolupráci.

Introduction: Light microscopy and electron microscopy rank among methods to diagnose of cardiomyopathy in forensic medicine, and, recently, the methods of molecular biology. Methods: Investigation of 27 year old man who collapsed on his way to work. The Rescuers did not succeed resuscitation of vital function. Samples were H-E stained and processed for the electron microscopy. RNA was isolated from the tissue for the alpha, beta, gama actine primer investigation. Results: By H-E staining we proved irregular hypertrophic cardiomyocytes (disarray) with the links and loci patches of thin fibrosis. Ultrastructurally we diagnosed a disarray of Z-bands, accumulation of mitochondria, rectangular nuclei of cardiomyocytes. We have detected rare plasmocytes and leucocytes with specific granules in cytoplasma. In the electronogrames we can see myofibriles oriented longitudinally and transversally. A genetic examination demonstrated beta actin mutation. Conclusion: Cardiomyopathy can be a cause of sudden and unexpected death in young individuals and its diagnostics requires an interdisciplinary collaboration.

• Klíčová slova
• ultrastruktura kardiomyocytu, genové mutace,
• MeSH
• aktiny genetika   MeSH
• bodová mutace MeSH
• dospělí MeSH
• elektronová mikroskopie MeSH
• genetické testování MeSH
• hypertrofická kardiomyopatie * patologie   MeSH
• kardiomyocyty patologie   ultrastruktura   MeSH
• lidé MeSH
• myokard patologie   ultrastruktura   MeSH
• náhlá srdeční smrt * etiologie   MeSH
• pitva MeSH
• polymerázová řetězová reakce MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH