In polarized motile cells, stress fibers display specific three-dimensional organization. Ventral stress fibers, attached to focal adhesions at both ends, are restricted to the basal side of the cell and nonprotruding cell sides. Dorsal fibers, transverse actin arcs, and perinuclear actin fibers emanate from protruding cell front toward the nucleus and toward apical side of the cell. Perinuclear cap fibers further extend above the nucleus, associate with nuclear envelope through LINC (linker of nucleoskeleton and cytoskeleton) complex and terminate in focal adhesions at cell rear. How are perinuclear actin fibers formed is poorly understood. We show that the formation of perinuclear actin fibers requires dorsal stress fibers that polymerize from focal adhesions at leading edge, and transverse actin arcs that are interconnected with dorsal fibers in spots rich in α-actinin-1. During cell polarization, the interconnected dorsal fibers and transverse arcs move from leading edge toward dorsal side of the cell. As they move, transverse arcs associate with one end of stress fibers present at nonprotruding cell sides, move them above the nucleus thus forming perinuclear actin fibers. Furthermore, the formation of perinuclear actin fibers induces temporal rotational movement of the nucleus resulting in nuclear reorientation to the direction of migration. These results suggest that the network of dorsal fibers, transverse arcs, and perinuclear fibers transfers mechanical signal between the focal adhesions and nuclear envelope that regulates the nuclear reorientation in polarizing cells.
- MeSH
- aktinin fyziologie MeSH
- aktiny fyziologie MeSH
- buněčné jádro fyziologie MeSH
- buněčné linie MeSH
- buněčný převod mechanických signálů fyziologie MeSH
- fibroblasty fyziologie MeSH
- fokální adheze fyziologie MeSH
- kontraktilní svazky fyziologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- pohyb buněk fyziologie MeSH
- pohyb fyziologie MeSH
- polarita buněk fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- úvodníky MeSH
The spreading of adhering cells is a morphogenetic process during which cells break spherical or radial symmetry and adopt migratory polarity with spatially segregated protruding cell front and non-protruding cell rear. The organization and regulation of these symmetry-breaking events, which are both complex and stochastic, are not fully understood. Here we show that in radially spreading cells, symmetry breaking commences with the development of discrete non-protruding regions characterized by large but sparse focal adhesions and long peripheral actin bundles. Establishment of this non-protruding static region specifies the distally oriented protruding cell front and thus determines the polarity axis and the direction of cell migration. The development of non-protruding regions requires ERK2 and the ERK pathway scaffold protein RACK1. RACK1 promotes adhesion-mediated activation of ERK2 that in turn inhibits p190A-RhoGAP signaling by reducing the peripheral localization of p190A-RhoGAP. We propose that sustained ERK signaling at the prospective cell rear induces p190A-RhoGAP depletion from the cell periphery resulting in peripheral actin bundles and cell rear formation. Since cell adhesion activates both ERK and p190A-RhoGAP signaling this constitutes a spatially confined incoherent feed-forward signaling circuit.
- MeSH
- aktiny metabolismus MeSH
- biologické modely MeSH
- buněčná adheze MeSH
- fenotyp MeSH
- fibroblasty cytologie enzymologie metabolismus MeSH
- fokální adhezní tyrosinkinasy metabolismus MeSH
- genový knockdown MeSH
- krysa rodu rattus MeSH
- MAP kinasový signální systém * MeSH
- mitogenem aktivovaná proteinkinasa 1 metabolismus MeSH
- pohyb buněk MeSH
- proteiny vázající GTP nedostatek metabolismus MeSH
- represorové proteiny metabolismus MeSH
- tvar buňky MeSH
- umlčování genů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis.
- MeSH
- alfa-galaktosidasa genetika metabolismus MeSH
- Fabryho nemoc enzymologie genetika metabolismus MeSH
- hmotnostní spektrometrie MeSH
- imunochemie MeSH
- ledviny chemie metabolismus MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši knockoutované MeSH
- myši MeSH
- sfingolipidy chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIMS: Production of minor asukamycin congeners and its new derivatives by combination of targeted genetic manipulations with specific precursor feeding in the producer of asukamycin, Streptomyces nodosus ssp. asukaensis. METHODS AND RESULTS: Structural variations of manumycins lie only in the diverse initiation of the 'upper' polyketide chain. Inactivation of the gene involved in the biosynthesis of cyclohexanecarboxylic acid (CHC) turned off the production of asukamycin in the mutant strain and allowed an increased production of other manumycins with the branched end of the upper chain. The ratio of produced metabolites was further affected by specific precursor feeding. Precursor-directed biosynthesis of a new asukamycin analogue (asukamycin I, 28%) with linear initiation of the upper chain was achieved by feeding norleucine to the mutant strain. Another asukamycin analogue with the unbranched upper chain (asukamycin H, 14%) was formed by the CHC-deficient strain expressing a heterologous gene putatively involved in the formation of the n-butyryl-CoA starter unit of manumycin A. CONCLUSIONS: Combination of the described techniques proved to be an efficient tool for the biosynthesis of minor or novel manumycins. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of two novel asukamycin derivatives, asukamycins H and I, was achieved. Variations appeared in the upper polyketide chain, the major determinant of enzyme-inhibitory features of manumycins, affecting their cancerostatic or anti-inflammatory features.
- MeSH
- acylkoenzym A metabolismus MeSH
- aminokyseliny metabolismus MeSH
- antibakteriální látky biosyntéza MeSH
- genetické inženýrství MeSH
- inzerční mutageneze MeSH
- kultivační média MeSH
- kyseliny cyklohexankarboxylové metabolismus MeSH
- mutace MeSH
- polyeny metabolismus MeSH
- polynenasycené alkamidy metabolismus MeSH
- Streptomyces genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Corynebacterium glutamicum genome codes for 7 sigma subunits (factors) of RNA polymerase (RNAP): primary sigma factor SigA (σ(A)), primary-like SigB and 5 other alternative sigma factors (SigC, SigD, SigE, SigH and SigM). Each sigma factor is responsible for recognizing promoters of genes belonging to a regulon (sigmulon) involved in specific functions of the cell. Most promoters of C. glutamicum housekeeping genes are recognized by RNAP+σ(A), whereas σ(B) is involved in transcription of a large group of genes active during the transition phase between the exponential and stationary growth phases when various stress factors threaten to damage the cell. The σ(H) regulon consists of the genes involved in heat shock response including those coding for regulators and other sigma factors. It seems therefore that σ(H) occupies a central position in the cross-regulated network of sigma factors and controls their concerted response to various stress conditions in C. glutamicum. The σ(M) factor was found to regulate genes responding to oxidative stress. The main role of σ(E) is to activate genes involved in response to a cell surface stress. Promoters of individual classes recognized by different sigma factors are compiled and the respective consensus sequences of their key recognition motifs (-35 and -10 regions) are derived. In a number of genes, two or more promoters controlled by the same or different sigma factors were discovered. These multiple, overlapping or dual promoters contribute to a complex gene transcription control mechanisms that integrate internal and external signals and tune gene expression in cells as required by environmental and physiological conditions.
- MeSH
- Corynebacterium glutamicum genetika metabolismus MeSH
- DNA řízené RNA-polymerasy genetika metabolismus MeSH
- fyziologický stres genetika MeSH
- genové regulační sítě MeSH
- molekulární sekvence - údaje MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u bakterií MeSH
- regulon MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sigma faktor genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Microorganisms in nature form organized multicellular structures (colonies, biofilms) possessing properties absent in individual cells. These are often related to the better ability of communities to survive long-lasting starvation and stress and include mechanisms of adaptation and cell specialization. Thus, yeast colonies pass through distinct developmental phases characterized by changes in pH and the production of ammonia-signalling molecules. Here, we show that Saccharomyces cerevisiae colony transition between major developmental phases (first acidic, alkali, second acidic) is accompanied by striking transcription changes, while the development within each particular phase is guided mostly at the post-transcriptional level. First- and second-acidic-phase colonies markedly differ. Second-acidic-phase colonies maintain the adaptive metabolism activated in the ammonia-producing period, supplemented by additional changes, which begin after colonies enter the second acidic phase. Cells with particular properties are not homogenously dispersed throughout the colony population, but localize to specific colony regions. Thus, cells located at the colony margin are able to export higher amounts of ammonium than central cells and to activate an adaptive metabolism. In contrast, central chronologically aged cells are unable to undergo these changes but they maintain higher levels of various stress-defence enzymes. These divergent properties of both cell types determine their consequent dissimilar fate.
he white-rot fungus Irpex lacteus has been reported to be an efficient degrader of polycyclic aromatic hydrocarbons, polychlorinated biphenyls and pentachlorophenol. The fungus produces ligninolytic enzymes laccase, lignin peroxidase and manganese peroxidase (MnP), the latter being the major one produced. MnP was purified using anion exchange and size exclusion chromatography. SDS-PAGE showed the purified MnP to be a monomeric protein of 37 kDa (37.5 kDa using MALDI-TOF) with an isoelectric point at 3.55. The pH optimum was relatively broad, from 4.0 to 7.0 with a peak at pH 5.5. Kinetic constants K(m) were 8 microM for H(2)O(2) and 12 or 31 microM for Mn(2+) depending on the substrate. The enzyme did not perform oxidation in the absence of H(2)O(2) or Mn(2+). MnP was active at 5-70 degrees C with an optimum between 50-60 degrees C. At temperatures above 65 degrees C the enzyme rapidly lost activity. Degradation of four representatives of PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) was tested and the enzyme showed the ability to degrade them in vitro. Major degradation products of anthracene were identified. The results confirm the role of MnP in PAH degradation by I. lacteus, including cleavage of the aromatic ring.
- MeSH
- Basidiomycota enzymologie MeSH
- biodegradace MeSH
- chromatografie iontoměničová MeSH
- financování organizované MeSH
- látky znečišťující životní prostředí metabolismus MeSH
- pentachlorfenol metabolismus MeSH
- peroxidasy izolace a purifikace metabolismus MeSH
- polychlorované bifenyly metabolismus MeSH
- polycyklické aromatické uhlovodíky metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
