Protein labeling Dotaz Zobrazit nápovědu
Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes.
- MeSH
- barvení a značení metody MeSH
- luminescentní proteiny analýza genetika MeSH
- mikrobiální genetika metody MeSH
- molekulární biologie metody MeSH
- optické zobrazování metody MeSH
- plazmidy MeSH
- rekombinace genetická MeSH
- rekombinantní fúzní proteiny analýza genetika MeSH
- reportérové geny * MeSH
- Saccharomyces cerevisiae cytologie genetika MeSH
- selekce (genetika) MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.
Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.
- MeSH
- bakteriální geny MeSH
- bakteriální proteiny genetika metabolismus MeSH
- barvení a značení * MeSH
- fluorescenční barviva * MeSH
- fluorescenční mikroskopie * MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- myši MeSH
- průtoková cytometrie MeSH
- Spirochaetales cytologie genetika metabolismus MeSH
- spirochetové infekce mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.
- MeSH
- barvení a značení MeSH
- biotinylace MeSH
- endocytóza MeSH
- endozomy metabolismus MeSH
- klathrin metabolismus MeSH
- lidé MeSH
- mikroskopie metody MeSH
- nádorové buněčné linie MeSH
- receptor fibroblastových růstových faktorů, typ 4 metabolismus MeSH
- signální transdukce MeSH
- trans-Golgiho síť metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pelizaeus Merzbacherova choroba (PMD) je dědičnou poruchou centrální myelinizace způsobenou hiutacemi proteolipid protein (PLP) genu lokalizovaném na X-chromozomu. Klinicky je PMD charakterizovaná typickými příznaky s časným nystagmem, opožděným psychomotorickjón vývojem různého stupně - podle typu PMD, spasticitou s hyperreflexií a pyramidovými jevy, cerebelární symptomatologií, výrazně prodlouženými latencemi sluchových i zrakových evokovaných potenciálů á typickými hyperintenzivními nálezy v bílé hmotě mozku na MRI. Podle tíže postižení se většinou odlišuje častější a mírnější tzv. klasická forma od vzácnější a těžší tzv. konnatální formy. PLP gen může být poškozen dvěma typy mutací - častější (60-70 %) je duplikace celého genu, která vede ke klasické formě PMD a vzácnější (20-30 %) různé bodové mutace, které vedou ke spektru klinických fenotypu od konnatálního až k velmi mírným formám spastické paraplegic typ 2 (SPG2). Jsou popsány klinické nálezy a výsledky vyšetření u prvního souboru čtyř českých pacientů s klasickou formou PMD, u kterých po stanovení klinické diagnózy byla její správnost následně potvrzena průkazem duplikace PLP genu. U dvou chlapců byla původně nesprávně stanovena diagnóza hypotonické formy dětské mozkové obniy a u dvou jsme stanovením diagnózy PMD vysvětlili příčinu jejich psychomotorické retardace. Signálním příznakem u všech 4 chlapců byl časný nystagmus, hypotonie a opožďování motorického vývoje. Duplikace PLP genu byla prokázána pomocí kvantitativní komparativní PCR s fluorescenčním značením (QCFPCR) a pomocí intragenového mikrosatelitového markem. Ve všech případech byla nosičkou duplikace PLP genu - přenašečkou PMD i matka pacienta, i když ve třech případech šlo klinicky a anamnestický o sporadický případ postižení a pouze u jedné rodiny šlo o familiární, mnohočetný výskyt PMD v rodině. Poučení z typického fenotypu těchto pacientů s molekulárně geneticky ověřenou diagnózou by mělo vést ke snadnějšímu, přímějšímu a časnému stanovení správné diagnózy i U dalších pacientů s PMD v naší zemi včetně dospělých pacientů.
Pelizaeus Merzbacher's disease (PMD) is a hereditary disorder of central myelinization caused by mutations of the proteolipid protein (PLP) gene situated on the X chromosome. Clinically PMD is characterized by typical symptoms with early nystagmus, late psychomotor development of varying grades - depending on the type of PMD, spasticity with hyperreflexia and pyramidal phenomena, cerebellar symptomatology, markedly prolonged latencies of auditory and optically evoked potentials and typical hyperintense findings in the white matter of the brain on MRI. According to the severity of the affection usually the more fi'equent and milder so-called classical form is differentiated firom the rarer and more severe, so-called connatal form. The PLP gene can be damaged by two types of mutations - more fi:equently (60-70%) duplication of the whole gene which leads to the classical form of PMD and the less frequent (20-30%) various point mutations which lead to a spectrum of clinical phenot5T>es from connatal to very mild forms of spastic paraplegia type 2 (SPG2). The authors describe the clinical findings and results of examination in the first group od four Czech patients with the classical form of PMD where after assessment of the clinical diagnosis its correctness was subsequently confirmed by evidence of duplication of the PLP gene. In two boys originally incorrectly the diagnosis of the hypotonie form of infantile cerebral palsy was made and in two the authors established the diagnosis of PMD and explained the psychomotor retardation of the patients. The signalling symptoms in all four boys were early nystagmus, hypotonia and retarded motor development. Duplication of the PLP gene was proved by quantitative comparative PCR with fluorescent labelling (QCFPCR) and by means of an intragenic microsatellite marker. In all instances carrier of duplication of the PLP gene - was also the patient's mother although in three instances a clinically and anamnestically sporadic affection was iiivoled and only in one family a familial, multiple incidence of PMD in the family was recorded. Findings from a typical phenotype of these patients with molecular genetically confirmed diagnosis should lead to an easier, more direct and earlier assessment of the correct diagnosis also in other patients with PMD in our country, incl. adult patients.
- MeSH
- chromozom X MeSH
- dítě MeSH
- finanční podpora výzkumu jako téma MeSH
- kongenitální nystagmus MeSH
- lidé MeSH
- mutace MeSH
- myelinový proteolipidový protein MeSH
- Pelizaeusova-Merzbacherova nemoc diagnóza genetika klasifikace MeSH
- psychomotorické poruchy MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Geografické názvy
- Česká republika 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
