The nuclear pore complex (NPC) has emerged as a hub for the transcriptional regulation of a subset of genes, and this type of regulation plays an important role during differentiation. Nucleoporin TPR forms the nuclear basket of the NPC and is crucial for the enrichment of open chromatin around NPCs. TPR has been implicated in the regulation of transcription; however, the role of TPR in gene expression and cell differentiation has not been described. Here we show that depletion of TPR results in an aberrant morphology of murine proliferating C2C12 myoblasts (MBs) and differentiated C2C12 myotubes (MTs). The ChIP-Seq data revealed that TPR binds to genes linked to muscle formation and function, such as myosin heavy chain (Myh4), myocyte enhancer factor 2C (Mef2C) and a majority of olfactory receptor (Olfr) genes. We further show that TPR, possibly via lysine-specific demethylase 1 (LSD1), promotes the expression of Myh4 and Olfr376, but not Mef2C. This provides a novel insight into the mechanism of myogenesis; however, more evidence is needed to fully elucidate the mechanism by which TPR affects specific myogenic genes.
- MeSH
- buněčná diferenciace MeSH
- buněčné linie MeSH
- exprese genu MeSH
- komplex proteinů jaderného póru metabolismus MeSH
- kosterní svalová vlákna * cytologie metabolismus MeSH
- myoblasty kosterní * cytologie metabolismus MeSH
- myši MeSH
- protoonkogenní proteiny metabolismus MeSH
- regulace genové exprese MeSH
- těžké řetězce myosinu metabolismus MeSH
- vývoj svalů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1.
- MeSH
- aktiny metabolismus MeSH
- Arabidopsis metabolismus ultrastruktura MeSH
- autofagie MeSH
- autofagozomy metabolismus ultrastruktura MeSH
- biologické modely MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- endocytóza * MeSH
- endoplazmatické retikulum metabolismus ultrastruktura MeSH
- fylogeneze MeSH
- komplex proteinů 2-3 souvisejících s aktinem metabolismus MeSH
- mikrofilamenta metabolismus MeSH
- mikrofilamentové proteiny metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.
- MeSH
- HeLa buňky MeSH
- jaderná lamina metabolismus ultrastruktura MeSH
- jaderný obal metabolismus ultrastruktura MeSH
- komplex proteinů jaderného póru antagonisté a inhibitory genetika metabolismus MeSH
- lamin typ A antagonisté a inhibitory genetika metabolismus MeSH
- lamin typ B genetika metabolismus MeSH
- lidé MeSH
- malá interferující RNA genetika metabolismus MeSH
- molekulární zobrazování MeSH
- protoonkogenní proteiny antagonisté a inhibitory genetika metabolismus MeSH
- regulace genové exprese MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and non-active chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatin-modifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP.
- MeSH
- buněčné jádro metabolismus MeSH
- chromatin chemie metabolismus MeSH
- epigeneze genetická MeSH
- fluorescenční mikroskopie MeSH
- HeLa buňky MeSH
- heterochromatin chemie MeSH
- histondemethylasy metabolismus MeSH
- histony chemie MeSH
- jaderný obal metabolismus MeSH
- jaderný pór metabolismus MeSH
- lidé MeSH
- mikroskopie metody MeSH
- software MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components, and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo, or real 3D at magnifications ranging from about 10× to 1,000,000×. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualized. In order to identify that protein at high resolution, or to see and quantify its distribution at lower magnifications, samples can be labeled with antibodies. Any surface that can be exposed can potentially be studied in this way. Presented here is a generic method for immunogold labeling for scanning electron microscopy, using two examples of specimens: isolated nuclear envelopes and the cytoskeleton of mammalian culture cells. Various parameters for sample preparation, fixation, immunogold labeling, drying, metal coating, and imaging are discussed so that the best immunogold scanning electron microscopy results can be obtained from different types of specimens.
- MeSH
- antigeny genetika metabolismus MeSH
- barvení a značení metody MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- epoxidové pryskyřice chemie MeSH
- exprese genu MeSH
- fixace tkání metody MeSH
- fixativa chemie MeSH
- formaldehyd chemie MeSH
- imunohistochemie metody MeSH
- jaderný obal metabolismus ultrastruktura MeSH
- koloidní zlato chemie MeSH
- komplex proteinů jaderného póru genetika metabolismus MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mikrotomie MeSH
- oocyty metabolismus ultrastruktura MeSH
- polymery chemie MeSH
- protilátky chemie MeSH
- Xenopus laevis MeSH
- zalévání tkání metody MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events.
- MeSH
- barvení a značení metody MeSH
- epoxidové pryskyřice chemie MeSH
- exprese genu MeSH
- fixace tkání metody MeSH
- fixativa chemie MeSH
- glutaraldehyd chemie MeSH
- imunoelektronová mikroskopie metody MeSH
- imunohistochemie metody MeSH
- komplex proteinů jaderného póru genetika metabolismus MeSH
- kryoprezervace metody MeSH
- mikrotomie MeSH
- mrazová substituce metody MeSH
- protilátky chemie MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae metabolismus ultrastruktura MeSH
- zalévání tkání metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Paxillin (PXN) is a focal adhesion protein that has been implicated in signal transduction from the extracellular matrix. Recently, it has been shown to shuttle between the cytoplasm and the nucleus. When inside the nucleus, paxillin promotes cell proliferation. Here, we introduce paxillin as a transcriptional regulator of IGF2 and H19 genes. It does not affect the allelic expression of the two genes; rather, it regulates long-range chromosomal interactions between the IGF2 or H19 promoter and a shared distal enhancer on an active allele. Specifically, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, thus activating IGF2 gene transcription, whereas it restrains the interaction between the enhancer and the H19 promoter, downregulating the H19 gene. We found that paxillin interacts with cohesin and the mediator complex, which have been shown to mediate long-range chromosomal looping. We propose that these interactions occur at the IGF2 and H19 gene cluster and are involved in the formation of loops between the IGF2 and H19 promoters and the enhancer, and thus the expression of the corresponding genes. These observations contribute to a mechanistic explanation of the role of paxillin in proliferation and fetal development.
- MeSH
- buňky Hep G2 MeSH
- chromozomální proteiny, nehistonové genetika MeSH
- extracelulární matrix genetika MeSH
- fokální adheze genetika MeSH
- genomový imprinting genetika MeSH
- insulinu podobný růstový faktor II biosyntéza genetika MeSH
- lidé MeSH
- metylace DNA genetika MeSH
- paxilin aplikace a dávkování MeSH
- proliferace buněk účinky léků genetika MeSH
- promotorové oblasti (genetika) MeSH
- proteiny buněčného cyklu genetika MeSH
- RNA dlouhá nekódující biosyntéza genetika MeSH
- signální transdukce účinky léků MeSH
- vývoj plodu genetika MeSH
- vývojová regulace genové exprese MeSH
- zesilovače transkripce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sample preparation methods that robustly but accurately preserve biological structures. In plants, exposing the object of interest may be difficult due to the existence of a cell wall. This protocol shows how to isolate plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncovering membrane-associated cytoskeletal structures.
- MeSH
- buněčná membrána ultrastruktura MeSH
- buněčné jádro ultrastruktura MeSH
- cytoskelet ultrastruktura MeSH
- mikroskopie elektronová rastrovací metody MeSH
- rostlinné buňky ultrastruktura MeSH
- rostliny anatomie a histologie ultrastruktura MeSH
- tabák anatomie a histologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs. RESULTS: Application of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B. CONCLUSIONS: We show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.
- MeSH
- aktiny antagonisté a inhibitory metabolismus MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- cytoskelet účinky léků MeSH
- embryonální vývoj MeSH
- fylogeneze MeSH
- protein - isoformy metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- RNA rostlin genetika MeSH
- rostlinné geny MeSH
- sekvenční seřazení MeSH
- smrk embryologie růst a vývoj MeSH
- substituce aminokyselin MeSH
- techniky tkáňových kultur MeSH
- thiazolidiny farmakologie MeSH
- vazebná místa MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH