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1.
Nat Commun ; 13(1): 7110, 2022 11 19.
Article in English | MEDLINE | ID: mdl-36402845

ABSTRACT

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.


Subject(s)
Heparitin Sulfate , N-Acetylglucosaminyltransferases , Humans , N-Acetylglucosaminyltransferases/metabolism , Cryoelectron Microscopy , Heparitin Sulfate/metabolism , Proteins/metabolism , Nucleotidyltransferases , Glycosyltransferases/metabolism
2.
Nat Commun ; 11(1): 1153, 2020 03 02.
Article in English | MEDLINE | ID: mdl-32123169

ABSTRACT

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Endotoxins/chemistry , Endotoxins/metabolism , Hemolysin Proteins/chemistry , Hemolysin Proteins/metabolism , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Cell Membrane/drug effects , Crystallography, X-Ray , Disulfides/chemistry , Endotoxins/genetics , Endotoxins/pharmacology , HEK293 Cells , Hemolysin Proteins/genetics , Hemolysin Proteins/pharmacology , Humans , Hydrogen-Ion Concentration , Insecticides/chemistry , Insecticides/metabolism , Insecticides/pharmacology , Mice , Microscopy, Atomic Force , NIH 3T3 Cells , Protein Conformation , Sf9 Cells
3.
Cell Rep ; 29(8): 2295-2306.e6, 2019 11 19.
Article in English | MEDLINE | ID: mdl-31747602

ABSTRACT

The death receptor CD95 is expressed in every cancer cell, thus providing a promising tool to target cancer. Activation of CD95 can, however, lead to apoptosis or proliferation. Yet the molecular determinants of CD95's mode of action remain unclear. Here, we identify an optimal distance between CD95Ligand molecules that enables specific clustering of receptor-ligand pairs, leading to efficient CD95 activation. Surprisingly, efficient CD95 activation leads to apoptosis in cancer cells in vitro and increased tumor growth in vivo. We show that allowing a 3D aggregation of cancer cells in vitro switches the apoptotic response to proliferation. Indeed, we demonstrate that the absence or presence of cell-cell contacts dictates the cell response to CD95. Cell contacts increase global levels of phosphorylated tyrosines, including CD95's tyrosine. A tyrosine-to-alanine CD95 mutant blocks proliferation in cells in contact. Our study sheds light into the regulatory mechanism of CD95 activation that can be further explored for anti-cancer therapies.


Subject(s)
Protein-Tyrosine Kinases/metabolism , fas Receptor/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Communication/genetics , Cell Communication/physiology , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/physiology , Fas Ligand Protein/genetics , Fas Ligand Protein/metabolism , Humans , Phosphorylation/genetics , Phosphorylation/physiology , Protein-Tyrosine Kinases/genetics , Signal Transduction/genetics , Signal Transduction/physiology , fas Receptor/genetics
4.
Nat Methods ; 16(8): 707-710, 2019 08.
Article in English | MEDLINE | ID: mdl-31285624

ABSTRACT

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.


Subject(s)
B7-2 Antigen/analysis , Cell Tracking/methods , Luminescent Proteins/analysis , Microscopy, Fluorescence/methods , Animals , B7-2 Antigen/genetics , COS Cells , Chlorocebus aethiops , Crystallography, X-Ray , HeLa Cells , Humans , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Mutation , Photochemical Processes , Protein Conformation
5.
J Exp Med ; 216(9): 2113-2127, 2019 09 02.
Article in English | MEDLINE | ID: mdl-31270246

ABSTRACT

NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor-mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor-mediated cytotoxicity are differentially regulated during NK cell serial killing.


Subject(s)
Cytotoxicity, Immunologic , Granzymes/metabolism , Killer Cells, Natural/immunology , Receptors, Death Domain/metabolism , Caspase 8/metabolism , HeLa Cells , Humans , Kinetics , Perforin/metabolism , fas Receptor/metabolism
6.
Front Immunol ; 9: 1840, 2018.
Article in English | MEDLINE | ID: mdl-30135688

ABSTRACT

Natural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes via perforin pores or by activation of caspases via death receptors. In order to understand how NK cells combine different cell death mechanisms, it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here, we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 min. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K, and H. The approach presented here is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.


Subject(s)
Caspases/metabolism , Gene Expression , Genes, Reporter , Granzymes/metabolism , Killer Cells, Natural/metabolism , Apoptosis , Cell Death , Humans , Killer Cells, Natural/immunology , Proteolysis , Single-Cell Analysis
7.
Methods Mol Biol ; 1663: 115-126, 2017.
Article in English | MEDLINE | ID: mdl-28924663

ABSTRACT

Photoswitchable or photoactivatable fluorophores are the key in single-molecule localization microscopy. Next to providing fluorescence images with subdiffraction spatial resolution, additional information is available from observing single fluorophores over time. This includes the characteristic photophysical phenomenon of "blinking" that is exhibited by single fluorescent proteins or fluorophores and follows well-defined kinetic laws. Analyzing the kinetics of "blinking" allows determining the number of fluorophores in a multi-molecular complex. As such, quantitative information at the molecular level can be extracted, representing a tremendously useful extension of single-molecule super-resolution microscopy. This concept is in particular useful to study homo- and heterooligomeric signaling protein complexes in the plasma membrane of an intact cell with molecular resolution. Here, we provide an experimental framework for deciphering the stoichiometry of membrane proteins on the basis of SMLM and photoswitching statistics.


Subject(s)
Membrane Proteins/metabolism , Microscopy, Fluorescence/methods , Single Molecule Imaging/methods , Fluorescent Dyes/metabolism , HeLa Cells , Humans , Kinetics
8.
Biophys J ; 109(11): 2352-62, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26636946

ABSTRACT

The number of fluorophores within a molecule complex can be revealed by single-molecule photobleaching imaging. A widely applied strategy to analyze intensity traces over time is the quantification of photobleaching step counts. However, several factors can limit and bias the detection of photobleaching steps, including noise, high numbers of fluorophores, and the possibility that several photobleaching events occur almost simultaneously. In this study, we propose a new approach, to our knowledge, to determine the fluorophore number that correlates the intensity decay of a population of molecule complexes with the decay of the number of visible complexes. We validated our approach using single and fourfold Atto-labeled DNA strands. As an example we estimated the subunit stoichiometry of soluble CD95L using GFP fusion proteins. To assess the precision of our method we performed in silico experiments showing that the estimates are not biased for experimentally observed intensity fluctuations and that the relative precision remains constant with increasing number of fluorophores. In case of fractional fluorescent labeling, our simulations predicted that the fluorophore number estimate corresponds to the product of the true fluorophore number with the labeling fraction. Our method, denoted by spot number and intensity correlation (SONIC), is fully automated, robust to noise, and does not require the counting of photobleaching events.


Subject(s)
Fluorescent Dyes/chemistry , Models, Statistical , Photobleaching , Automation , Base Sequence , DNA/chemistry , DNA/genetics , Image Processing, Computer-Assisted , Microscopy , Models, Molecular , Nucleic Acid Conformation , Protein Multimerization , Protein Structure, Quaternary , fas Receptor/chemistry
9.
Sci Rep ; 5: 14072, 2015 Sep 11.
Article in English | MEDLINE | ID: mdl-26358640

ABSTRACT

Probing the oligomeric state of abundant molecules, such as membrane proteins in intact cells, is essential, but has not been straightforward. We address this challenge with a simple counting strategy that is capable of reporting the oligomeric state of dense, membrane-bound protein complexes. It is based on single-molecule localization microscopy to super-resolve protein structures in intact cells and basic quantitative evaluation. We validate our method with membrane-bound monomeric CD86 and dimeric cytotoxic T-lymphocyte-associated protein as model proteins and confirm their oligomeric states. We further detect oligomerization of CD80 and vesicular stomatitis virus glycoprotein and propose coexistence of monomers and dimers for CD80 and trimeric assembly of the viral protein at the cell membrane. This approach should prove valuable for researchers striving for reliable molecular counting in cells.


Subject(s)
Membrane Proteins/metabolism , Microscopy/methods , Models, Theoretical , Algorithms , B7-1 Antigen/chemistry , B7-1 Antigen/metabolism , Flow Cytometry , Humans , Membrane Proteins/chemistry , Protein Multimerization , Reproducibility of Results , Viral Proteins/chemistry , Viral Proteins/metabolism
10.
Sci Signal ; 7(316): ra23, 2014 Mar 11.
Article in English | MEDLINE | ID: mdl-24619646

ABSTRACT

Apoptosis in response to the ligand CD95L (also known as Fas ligand) is initiated by caspase-8, which is activated by dimerization and self-cleavage at death-inducing signaling complexes (DISCs). Previous work indicated that the degree of substrate cleavage by caspase-8 determines whether a cell dies or survives in response to a death stimulus. To determine how a death ligand stimulus is effectively translated into caspase-8 activity, we assessed this activity over time in single cells with compartmentalized probes that are cleaved by caspase-8 and used multiscale modeling to simultaneously describe single-cell and population data with an ensemble of single-cell models. We derived and experimentally validated a minimal model in which cleavage of caspase-8 in the enzymatic domain occurs in an interdimeric manner through interaction between DISCs, whereas prodomain cleavage sites are cleaved in an intradimeric manner within DISCs. Modeling indicated that sustained membrane-bound caspase-8 activity is followed by transient cytosolic activity, which can be interpreted as a molecular timer mechanism reflected by a limited lifetime of active caspase-8. The activation of caspase-8 by combined intra- and interdimeric cleavage ensures weak signaling at low concentrations of CD95L and strongly accelerated activation at higher ligand concentrations, thereby contributing to precise control of apoptosis.


Subject(s)
Apoptosis/physiology , Caspase 8/metabolism , Fas Ligand Protein/metabolism , Models, Biological , Signal Transduction/physiology , Blotting, Western , Caspase 8/chemistry , Computer Simulation , Cytosol/metabolism , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Dimerization , Flow Cytometry , HeLa Cells , Humans , Image Processing, Computer-Assisted , Single-Cell Analysis
11.
FEBS Lett ; 586(8): 1179-89, 2012 Apr 24.
Article in English | MEDLINE | ID: mdl-22575653

ABSTRACT

Endosomes constitute a central layer in the regulation of growth factor signaling. We applied flow cytometry, confocal microscopy and automated image quantification to define the role of Caveolin1 (Cav1) in epidermal growth factor (EGF) receptor (i) internalization and (ii) endosomal trafficking. Antisense-downregulation of Cav1 did not affect internalization of EGF:EGFR-complexes from the plasma membrane. Instead, Cav1-knockdown had a profound effect on endosomal trafficking and caused a shift in EGF vesicle distribution towards Rab7-negative compartments at late timepoints. Moreover, image quantification with single-endosome resolution revealed that EGF:Cav1-complexes undergo a maturation pattern reminiscent of late endosomes. Our data suggest a model in which Caveolin1 acts upon EGF endosomes internalized via the Clathrin-pathway and functions at the transition from early to late endosomes.


Subject(s)
Caveolin 1/metabolism , Endosomes/metabolism , ErbB Receptors/metabolism , Caveolin 1/chemistry , Caveolin 1/genetics , Down-Regulation , Endocytosis , Epidermal Growth Factor/metabolism , HeLa Cells , Humans , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding Proteins
12.
J Cell Biol ; 190(3): 377-89, 2010 Aug 09.
Article in English | MEDLINE | ID: mdl-20696707

ABSTRACT

Cellular FADD-like interleukin-1beta-converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP(L)], c-FLIP short [c-FLIP(S)], and c-FLIP Raji [c-FLIP(R)]) regulate caspase-8 activation and death receptor (DR)-induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP(L) has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP(S) or c-FLIP(R). Our findings resolve the present controversial discussion on the function of c-FLIP(L) as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.


Subject(s)
Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Models, Biological , Signal Transduction , fas Receptor/metabolism , Algorithms , Blotting, Western , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Caspase 8/metabolism , Computer Simulation , HeLa Cells , Humans , Protein Isoforms/metabolism , fas Receptor/genetics
13.
Mol Syst Biol ; 6: 352, 2010.
Article in English | MEDLINE | ID: mdl-20212524

ABSTRACT

This study explores the dilemma in cellular signaling that triggering of CD95 (Fas/APO-1) in some situations results in cell death and in others leads to the activation of NF-kappaB. We established an integrated kinetic mathematical model for CD95-mediated apoptotic and NF-kappaB signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c-FLIP(L) cleavage in the death-inducing signaling complex (DISC) and the NF-kappaB pathway. We validated experimentally that CD95 stimulation resulted in an interaction of p43-FLIP with the IKK complex followed by its activation. Furthermore, we showed that the apoptotic and NF-kappaB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c-FLIP(L) and procaspase-8 determines life/death decisions in a nonlinear manner. We present an integrated model describing the complex dynamics of CD95-mediated apoptosis and NF-kappaB signaling.


Subject(s)
Death Domain Receptor Signaling Adaptor Proteins/metabolism , Signal Transduction , fas Receptor/metabolism , Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspases/metabolism , Cell Death , Cell Lineage , Cell Survival , Enzyme Activation , HeLa Cells , Humans , I-kappa B Kinase/metabolism , Kinetics , Models, Biological , NF-kappa B/metabolism , Protein Binding , fas Receptor/genetics
14.
Biophys J ; 97(11): 2876-85, 2009 Dec 02.
Article in English | MEDLINE | ID: mdl-19948116

ABSTRACT

Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the repressive heterochromatin state. To elucidate its mobility and interactions, we conducted a comprehensive analysis on different time and length scales by fluorescence fluctuation microscopy in mouse cell lines. The local mobility of HP1alpha and HP1beta was investigated in densely packed pericentric heterochromatin foci and compared with other bona fide euchromatin regions of the nucleus by fluorescence bleaching and correlation methods. A quantitative description of HP1alpha/beta in terms of its concentration, diffusion coefficient, kinetic binding, and dissociation rate constants was derived. Three distinct classes of chromatin-binding sites with average residence times t(res)

Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Animals , Cell Line , Cell Survival , Chromobox Protein Homolog 5 , Diffusion , Epigenesis, Genetic , Fluorescence Recovery After Photobleaching , Heterochromatin/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Kinetics , Mice , Microscopy, Fluorescence , Movement , Protein Transport , Spectrometry, Fluorescence
15.
EMBO J ; 28(24): 3785-98, 2009 Dec 16.
Article in English | MEDLINE | ID: mdl-19927119

ABSTRACT

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.


Subject(s)
Cell Nucleus/metabolism , Chromatin/chemistry , Heterochromatin/chemistry , Animals , Cell Nucleolus/metabolism , DNA/metabolism , Fractals , Kidney/cytology , Kinetics , Mice , Microscopy, Fluorescence/methods , Models, Biological , RNA/metabolism , Rats , Spectrometry, Fluorescence/methods
16.
Mol Biol Cell ; 19(7): 3147-62, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18480407

ABSTRACT

Promyelocytic leukemia nuclear bodies (PML NBs) have been proposed to be involved in tumor suppression, viral defense, DNA repair, and/or transcriptional regulation. To study the dynamics of PML NBs during mitosis, we developed several U2OS cell lines stably coexpressing PML-enhanced cyan fluorescent protein with other individual marker proteins. Using three-dimensional time-lapse live cell imaging and four-dimensional particle tracking, we quantitatively demonstrated that PML NBs exhibit a high percentage of directed movement when cells progressed from prophase to prometaphase. The timing of this increased dynamic movement occurred just before or upon nuclear entry of cyclin B1, but before nuclear envelope breakdown. Our data suggest that entry into prophase leads to a loss of tethering between regions of chromatin and PML NBs, resulting in their increased dynamics. On exit from mitosis, Sp100 and Fas death domain-associated protein (Daxx) entered the daughter nuclei after a functional nuclear membrane was reformed. However, the recruitment of these proteins to PML NBs was delayed and correlated with the timing of de novo PML NB formation. Together, these results provide insight into the dynamic changes associated with PML NBs during mitosis.


Subject(s)
Intranuclear Inclusion Bodies/metabolism , Leukemia, Promyelocytic, Acute/metabolism , Mitosis , Antigens, Nuclear/metabolism , Autoantigens/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Chromatin/chemistry , Cyclin B/metabolism , Cyclin B1 , Green Fluorescent Proteins/metabolism , Humans , Metaphase , Microscopy, Fluorescence/methods , Prophase , Time Factors , Transcription, Genetic
17.
Bioinformatics ; 22(21): 2709-10, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-16940327

ABSTRACT

UNLABELLED: Tropical is a software for simulation and parameter estimation of reaction-diffusion models. Based on spatio-temporal microscopy images, Tropical estimates reaction and diffusion coefficients for user-defined models. Tropical allows the investigation of systems with an inhomogeneous distribution of molecules, making it well suited for quantitative analyses of microscopy experiments such as fluorescence recovery after photobleaching (FRAP). AVAILABILITY: Tropical is available free of charge for academic use at http://www.dkfz.de/tbi/projects/modellingAndSimulationOfCelluarSystems/tropical.jsp after signing a material transfer agreement.


Subject(s)
Cell Physiological Phenomena , Image Interpretation, Computer-Assisted/methods , Microscopy, Fluorescence/methods , Models, Biological , Protein Interaction Mapping/methods , Signal Transduction/physiology , Software , Algorithms , Computer Simulation , Diffusion
18.
Biophys J ; 90(6): 1878-94, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16387760

ABSTRACT

Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from such data are lacking. Here, we implemented a reaction-diffusion model to determine the contributions of protein interaction and diffusion on fluorescence redistribution. The model was validated and applied to five chromatin-interacting proteins probed by photoactivation in living cells. We found that very transient interactions are common for chromatin proteins. Their observed mobility was limited by the amount of free protein available for diffusion but not by the short residence time of the bound proteins. Individual proteins thus locally scan chromatin for binding sites, rather than diffusing globally before rebinding at random nuclear positions. By taking the real cellular geometry and the inhomogeneous distribution of binding sites into account, our model provides a general framework to analyze the mobility of fluorescently tagged factors. Furthermore, it defines the experimental limitations of fluorescence perturbation experiments and highlights the need for complementary methods to measure transient biochemical interactions in living cells.


Subject(s)
Active Transport, Cell Nucleus/physiology , Kidney/chemistry , Kidney/metabolism , Models, Biological , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Interaction Mapping/methods , Animals , Cells, Cultured , Computer Simulation , Diffusion , Microscopy, Fluorescence/methods , Models, Chemical , Motion , Rats
19.
J Cell Biol ; 167(1): 51-63, 2004 Oct 11.
Article in English | MEDLINE | ID: mdl-15479736

ABSTRACT

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15-20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.


Subject(s)
Cell Nucleolus/ultrastructure , Sarcoplasmic Reticulum/metabolism , Telophase , Cell Nucleus/enzymology , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , DNA, Complementary/metabolism , HeLa Cells , Humans , Microscopy, Electron , Microscopy, Fluorescence , Nuclear Proteins/metabolism , Nucleolus Organizer Region , Phosphorylation , RNA Polymerase II/metabolism , RNA, Messenger/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Time Factors , Transcription, Genetic , Transfection
20.
J Biol Chem ; 279(27): 28715-23, 2004 Jul 02.
Article in English | MEDLINE | ID: mdl-15102844

ABSTRACT

Focal adhesion kinase (FAK) activity and Ca(2+) signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca(2+) sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca(2+) variations at FA sites and FA dynamics. Discrete subcellular Ca(2+) oscillators initiate both propagating and abortive Ca(2+) waves in migrating U87 astrocytoma cells. Ca(2+)-dependent FA disassembly occurs when the Ca(2+) wave reaches individual FAs, indicating that local but not global Ca(2+) increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca(2+) elevation. FAK-related non-kinase domain-Ycam had a faster, Ca(2+)-insensitive recovery half-time (11 s), which is consistent with the effect of Ca(2+) on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca(2+) and FAK autophosphorylation was correlated to intracellular Ca(2+) levels, we propose that local Ca(2+) elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.


Subject(s)
Calcium/metabolism , Focal Adhesions/metabolism , Protein-Tyrosine Kinases/metabolism , Blotting, Western , Cell Line, Tumor , Cell Movement , Chelating Agents/pharmacology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Green Fluorescent Proteins , Humans , Ionomycin/pharmacology , Light , Luminescent Proteins/metabolism , Lymphocytes/metabolism , Microscopy, Fluorescence , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Signal Transduction , Time Factors , Transfection , Tyrosine/metabolism
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