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
Boron-doped nanocrystalline diamond (BNCD) films exhibit outstanding electrochemical properties that make them very attractive for the fabrication of electrodes for novel neural interfaces and prosthetics. In these devices, the physicochemical properties of the electrode materials are critical to ensure an efficient long-term performance. The aim of this study was to investigate the relative contribution of topography and doping to the biological performance of BNCD films. For this purpose, undoped and boron-doped NCD films were deposited on low roughness (LR) and high roughness (HR) substrates, which were studied in vitro by means of protein adsorption and fibroblast growth assays. Our results show that BNCD films significantly reduce the adsorption of serum proteins, mostly on the LR substrates. As compared to fibroblasts cultured on LR BNCD films, cells grown on the HR BNCD films showed significantly reduced adhesion and lower growth rates. The mean length of fibronectin fibrils deposited by the cells was significantly increased in the BNCD coated substrates, mainly in the LR surfaces. Overall, the largest influence on protein adsorption, cell adhesion, proliferation, and fibronectin deposition was due to the underlying sub-micron topography, with little or no influence of boron doping. In perspective, BNCD films displaying surface roughness in the submicron range may be used as a strategy to reduce the fibroblast growth on the surface of neural electrodes.
- MeSH
- 4-aminopyridin analogy a deriváty metabolismus MeSH
- aktiny fyziologie MeSH
- bor chemie MeSH
- buněčná adheze fyziologie MeSH
- diamant chemie MeSH
- fibroblasty fyziologie MeSH
- krevní proteiny chemie MeSH
- kultivované buňky MeSH
- lidé MeSH
- membrány umělé MeSH
- nanočástice * MeSH
- povrchové vlastnosti MeSH
- proliferace buněk MeSH
- testování materiálů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis.
- MeSH
- adenosintrifosfatasy metabolismus MeSH
- aktiny metabolismus fyziologie MeSH
- buněčné jadérko metabolismus fyziologie MeSH
- chromozomální proteiny, nehistonové metabolismus fyziologie MeSH
- genetická transkripce fyziologie MeSH
- glukosa metabolismus MeSH
- HeLa buňky MeSH
- lidé MeSH
- ribozomální DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 oC or 42.5 oC) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 oC) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 oC or 42.5 oC) or standard temperature (control 37.5 oC), post-incubated overnight (16-20 h) at 37.5 oC and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 oC did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 oC. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 oC) and embryos (exposed to 41.5 oC). Following the elevation of temperature to 42.5 oC embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.
- MeSH
- aktiny fyziologie izolace a purifikace MeSH
- apoptóza genetika MeSH
- financování organizované využití MeSH
- indukovaná hypertermie metody využití MeSH
- králíci růst a vývoj MeSH
- proteiny tepelného šoku HSP70 izolace a purifikace škodlivé účinky MeSH
- tvorba embrya pro výzkumné účely metody MeSH
- western blotting metody využití MeSH
- Check Tag
- králíci růst a vývoj MeSH
This work is focused on the function of the microtubule and actin networks in plasmid DNA transport during liposomal transfection. We observed strong binding of plasmid DNA-lipid complexes (lipoplexes) to both networks and directional long-range motion of these lipoplexes along the microtubules. Disruption of either of these networks led to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and accumulation of plasmid DNA in large aggregates at the cell periphery. Our findings show an indispensable but different role of both types of cytoskeleton, actin and microtubular, in the processes of gene delivery.
- MeSH
- aktiny fyziologie metabolismus MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- biologický transport účinky léků MeSH
- cytochalasin D farmakologie MeSH
- DNA genetika metabolismus MeSH
- fibroblasty cytologie metabolismus účinky léků MeSH
- financování organizované MeSH
- imunoblotting MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikrotubuly fyziologie metabolismus MeSH
- plazmidy genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- thiazolidiny farmakologie MeSH
- transfekce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- MeSH
- aktiny diagnostické užití fyziologie MeSH
- apoptóza genetika MeSH
- buněčná smrt fyziologie účinky léků MeSH
- finanční podpora výzkumu jako téma MeSH
- fosfatidylseriny diagnostické užití MeSH
- kaspasy diagnostické užití klasifikace MeSH
- nekróza MeSH
- zinek terapeutické užití MeSH
- Publikační typ
- techniky in vitro MeSH
The P19 mouse embryonal carcinoma cell line was used as a model for a study of apoptosis accompanying differentiation induced by all-trans retinoic acid (ATRA). Apoptosis was detected both on the basis of morphological features (nuclear fragmentation, blebbing of plasma membrane, and formation of apoptotic bodies), and by using DNA electrophoresis and flow-cytometric measurement of DNA content. Actin cytoskeleton was studied both on morphological and submicroscopic levels. ATRA-treated cells manifested apoptosis-specific changes in the distribution of actin foremost in association with their entry into executive phase of apoptosis, when F-actin cables participated in cell disintegration into apoptotic bodies. Using immunogold labeling, actin was also identified in centers of fragmenting apoptotic nuclei, in the disintegration of which it is likely involved as well. At the same time, a cleavage of actin by active caspase-3 was proved, resulting in the emergence of 32 kDa fragment, termed fractin. Measurement of F-actin and fractin content using flow cytometry showed an unequivocal decrease of F-actin and synchronous increase of fractin in the apoptotic population as compared to non-treated cells. Therefore, our results proved both actin proteolysis and active involvement of specific actin structures in the final cell disintegration during apoptosis in the P19 cells.
- MeSH
- aktiny fyziologie chemie metabolismus MeSH
- apoptóza MeSH
- buněčná diferenciace MeSH
- buněčné jádro metabolismus MeSH
- cytoskelet metabolismus MeSH
- DNA metabolismus MeSH
- elektroforéza MeSH
- embryonální karcinom metabolismus MeSH
- financování organizované MeSH
- fluorescenční mikroskopie MeSH
- fluorescenční protilátková technika nepřímá MeSH
- fragmentace DNA MeSH
- HL-60 buňky MeSH
- imunohistochemie MeSH
- kaspasy metabolismus MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- peptidové fragmenty fyziologie chemie MeSH
- průtoková cytometrie MeSH
- radiační rozptyl MeSH
- světlo MeSH
- transmisní elektronová mikroskopie MeSH
- tretinoin metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- MeSH
- aktiny fyziologie MeSH
- finanční podpora výzkumu jako téma MeSH
- mikrofilamentové proteiny aplikace a dávkování MeSH
- myši MeSH
- vestibulární vláskové buňky fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- kongresy MeSH
- MeSH
- aktiny fyziologie genetika MeSH
- cytoskelet fyziologie MeSH
- cytoskeletální proteiny * fyziologie genetika MeSH
- kontrakce myokardu * fyziologie MeSH
- kontraktilní proteiny fyziologie MeSH
- lidé MeSH
- myosiny fyziologie genetika MeSH
- protein - isoformy MeSH
- svalové proteiny * fyziologie genetika metabolismus MeSH
- tropomodulin fyziologie genetika MeSH
- tropomyosin fyziologie genetika MeSH
- troponin fyziologie genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
