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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. ispartof: NATURE METHODS vol:16 issue:8 pages:707-+ ispartof: location:United States status: published

Summary: 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. : Gülcüler Balta et al. show that CD95 receptor activation is determined through the presentation of its ligand at a certain intermolecular distance. The type of signaling triggered by CD95 is, however, decided by the cellular environment. CD95 triggers survival in cancer cells in contact with other cells and death in isolated ones. Keywords: CD95, CD95 ligand, death receptors, apoptosis, survival, cell-cell contact, cancer, tyrosine kinase, supported membrane

Natural killer cells can kill infected and transformed cells via two different cell death mechanisms. Prager et al. show that NK cells quickly kill their first targets by releasing cytotoxic granules and only use the slower death receptor–mediated cytotoxicity for their final kill., 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., Graphical Abstract

1.AbstractNatural 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 minutes. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K and H. The here presented approach is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.

Cancer cells react to CD95 activation with either apoptotic or tumorigenic responses. Yet, the determinants of these two antithetic reactions are fundamentally not understood. Here, we show that pre-confined CD95L molecules activate apoptosis of cancer cells in-vitro. For particular CD95L pre-confinement, apoptosis activation is most efficient. Surprisingly, in tumor models, the same pre-confinement yields enhanced proliferation of cancer cells. This shift is rooted in cell-cell interactions, as proliferation was also observed in tumorspheres in-vitro. Indeed, proliferation required death-domain tyrosine phosphorylation of CD95 that was facilitated by cell-cell contacts, whereas decreasing the levels of global tyrosine kinase activity favored apoptosis. Altogether, the response to CD95 activation is cell context-dependent and tunable by CD95L pre-confinement, thereby opening therapeutic opportunities in cancer.One Sentence SummaryCell-cell contact tunes tyrosine-kinase activity thereby dictating life or death upon CD95 activation by pre-confined CD95L.

CD95 (Fas, APO-1, TNFRSF6) is a widely expressed single-pass transmembrane protein that is implicated in cell death, inflammatory response, proliferation and cell migration. CD95 ligand (CD95L, FasL, TNFSF6), is a potent apoptotic inducer in the membrane form but not when cleaved into soluble CD95L (sCD95L). Here, we aimed at understanding the relation between ligand-receptor multimerization and receptor activation by correlating the kinetics of ligand binding, receptor oligomerization, FADD (FAS-Associated via Death Domain) recruitment and caspase-8 activation inside living cells. Using single molecule localization microscopy and Förster resonance energy transfer imaging we show that the majority of CD95 receptors on the plasma membrane are monomeric at rest. This was confirmed functionally as the wild-type receptor is not blocked by a receptor mutant that cannot bind ligand. Moreover, using time-resolved fluorescence imaging approaches we demonstrated that receptor multimerization follows instantaneously ligand binding, whereas FADD recruitment is delayed. This process can explain the typical delay time seen with caspase-8 activity reporters. Finally, the low activity of sCD95L, which was caused by inefficient FADD recruitment, was not explained by the low avidity for the receptor but by a receptor clustering mechanism that was different from the one induced by the strong apoptosis inducer IZ-sCD95L. Our results reveal that receptor activation is modulated by the capacity of its ligand to trimerize it.HighlightsAt a density of less than 10 receptors per µm2CD95 exists as monomer (58%) and dimer (42%)Pre-formed dimers do not contribute to ligand-induced CD95 apoptotic signalingThe PLAD of CD95 attenuates overexpression-induced, ligand-independent cell deathsoluble CD95L can rapidly multimerize CD95 after binding but it is still a poor inducer of apoptosis through inefficient FADD recruitmentFADD recruitment kinetics but not ligand binding kinetics correlates with caspase-8 onset of activity

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.

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.

Apoptosis occurs through a tightly regulated cascade of caspase activation. In the context of extrinsic apoptosis, caspase-8 is activated by dimerization inside a death receptor complex, cleaved by auto-proteolysis and subsequently released into the cytosol. This fully processed form of caspase-8 is thought to cleave its substrates BID and caspase-3. To test if the release is required for substrate cleavage, we developed a novel approach based on localization probes to quantitatively characterize the spatial-temporal activity of caspases in living single cells. Our study reveals that caspase-8 is significantly more active at the plasma membrane than within the cytosol upon CD95 activation. This differential activity is controlled by the cleavage of caspase-8 prodomain. As a consequence, targeting of caspase-8 substrates to the plasma membrane can significantly accelerate cell death. Subcellular compartmentalization of caspase-8 activity may serve to restrict enzymatic activity before mitochondrial pathway activation and offers new possibilities to interfere with apoptotic sensitivity of the cells.

Background The CRISPR/Cas9 genome editing system has greatly facilitated and expanded our capacity to engineer mammalian genomes, including targeted gene knock-outs. However, the phenotyping of the knock-out effect requires a high DNA editing efficiency. Results Here, we report a user-friendly strategy based on the extrinsic apoptosis pathway that allows enrichment of a polyclonal gene-edited cell population, by selecting Cas9-transfected cells that co-express dominant-negative mutants of death receptors. The extrinsic apoptosis pathway can be triggered in many mammalian cell types, and ligands are easy to produce, do not require purification and kill much faster than the state-of-the-art selection drug puromycin. Stringent assessment of our advanced selection strategy via Sanger sequencing, T7 endonuclease I (T7E1) assay and direct phenotyping confirmed a strong and rapid enrichment of Cas9-expressing cell populations, in some cases reaching up to 100 % within one hour. Notably, the efficiency of target DNA cleavage in these enriched cells reached high levels that exceeded the reliable range of the T7E1 assay, a conclusion that can be generalized for editing efficiencies above 30 %. Moreover, our data emphasize that the insertion and deletion pattern induced by a specific gRNA is reproducible across different cell lines. Conclusions The workflow and the findings reported here should streamline a wide array of future low- or high-throughput gene knock-out screens, and should largely improve data interpretation from CRISPR experiments. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0250-4) contains supplementary material, which is available to authorized users.

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)or= 0.2 s (class I, dominant in euchromatin), 7 s (class II, dominant in heterochromatin), and approximately 2 min (class III, only in heterochromatin) were identified. HP1 was present at low micromolar concentrations at heterochromatin foci, and required histone H3 lysine 9 methylases Suv39h1/2 for two- to fourfold enrichment at these sites. These findings impose a number of constraints for the mechanism by which HP1 is able to maintain a heterochromatin state.

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.

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.

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.

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.

Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex.

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.

International audience; The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.

Live-cell imaging technology using fluorescent proteins (green fluorescent protein and its homologues) has revolutionized the study of cellular dynamics. But tools that can quantitatively analyse complex spatiotemporal processes in live cells remain lacking. Here we describe a new technique--fast multi-colour four-dimensional imaging combined with automated and quantitative time-space reconstruction--to fill this gap. As a proof of principle, we apply this method to study the re-formation of the nuclear envelope in live cells. Four-dimensional imaging of three spectrally distinct fluorescent proteins is used to simultaneously visualize three different cellular compartments at high speed and with high spatial resolution. The highly complex data, comprising several thousand images from a single cell, were quantitatively reconstructed in time-space by software developed in-house. This analysis reveals quantitative and qualitative insights into the highly ordered topology of nuclear envelope formation, in correlation with chromatin expansion - results that would have been impossible to achieve by manual inspection alone. Our new technique will greatly facilitate study of the highly ordered dynamic architecture of eukaryotic cells.

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121–green fluorescent protein (GFP) and GFP-Nup153, and GFP–lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

The CRISPR/Cas9 system currently revolutionizes the gene therapy field due to the unique ease and efficiency with which it can be exploited for targeted DNA editing in mammalian cells. It only requires a small g(uide)RNA that directs the Cas9 protein to a specific DNA sequence which is then cleaved by Cas9. In the absence of a homologous exogenous template, the ensuing double-stranded DNA break is repaired by non-homologous end joining, an error-prone cellular mechanism frequently resulting in insertions or deletions that disrupt the original sequence. While useful in principle to perturb the expression of disease-associated genes, rapidly evolving targets such as human viruses will require concurrent expression of multiple gRNAs to thwart adverse mutational escape. To fill in this gap, we created novel highly customizable AAV vectors permitting multiplexing of up to three gRNAs in a single self-complementary vector backbone. Notably, our constructs readily accommodate and deliver minimal expression cassettes only consisting of gRNA(s) and promoter(s) with total sizes as little as 250 bp, which reduces the amount of ectopic DNA and improves vector safety. We demonstrated the power of our vectors by their use for simultaneous knock-out of three key players in apoptosis signaling, exemplifying their usefulness for dissection of cellular pathways. In addition, we engineered corresponding AAV backbones for shRNA expression, to be able to eventually juxtapose targeting on the DNA (CRISPR) and the RNA (RNAi) level in a single vector. Therefore, we made all constructs compatible with Golden Gate Assembly which minimizes work while maximizing flexibility, in turn paving the way for high-throughput cloning and screening of combinatorial RNAi/CRISPR libraries. Notably, the vectors offer three different RNA polymerase III promoters – U6, H1 or 7SK – for expression of gRNA or shRNA, thus allowing users to fine-tune g/shRNA levels and to alleviate toxic effects from over-expression. Finally, we also assembled a library of promoters for Cas9 expression comprising viral, synthetic and mammalian variants, to further expand the options for customization of our AAV/TRISPR system according to specific targets and needs. Indeed, as described in more detail in a separate abstract (Borner et al.), the selection of optimal gRNA/Cas9 vector designs even permits potent disruption of very challenging targets, such as integrated proviral HIV-1 DNA in T cells. Likewise, the use of proper regulatory elements is crucial for potent in vivo CRISPR-mediated targeting of endogenous genes in the mouse liver, as noted for the miR-122 microRNA. Considering the high degree of versatility of our vector designs, as well as the additional options for in vivo re-targeting through AAV capsid engineering, we expect our novel AAV-TRISPR kit for combinatorial gRNA/shRNA expression to substantially foster future efforts to co-exploit RNAi and CRISPR, and to benefit a multitude of human gene therapy applications.

Fluorescence Recovery After Photobleaching (FRAP) is a popular and versatile family of methods used to estimate mobility and reaction parameters in cellular systems. Part of an area containing a fluorescently labeled species is bleached using a laser, and the effect of the perturbation of the spatial concentration profile of the fluorescent species is monitored. Subsequently, the collected data is reconciled with a model of the dynamics, thus yielding estimates for the parameters of interest. While originally devised to elucidate transport parameters, it was soon extended to the estimation of reaction rates, and is also used nowadays to answer a variety of questions on the organization of cellular systems.In this chapter, we review a variety of different approaches, classifying them according to the sources of uncertainty that are addressed or ignored, and the type of parameter that they attempt to estimate. We would like to highlight the importance of the general methodology as a tool that can be widely applied to a large number of situations.

CD95-mediated apoptotic and NF-κB signaling were described by a simple kinetic model. We used a model reduction technique to reduce the number of reactions from 92 to 23 while maintaining a good model fit. p43-FLIP, which is generated at the CD95 DISC by procaspase-8 cleavage, was found to be the link between the CD95 DISC and the NF-κB pathway. P43-FLIP interacts with the IKK complex and leads to its activation. The CD95 DISC complex acts as a signal processor that diverges signals into the apoptotic and NF-κB pathways depending on the amounts of specific DISC proteins. Life/death decisions in CD95 signaling are determined by c-FLIPL and procaspase-8 in a non-linear way., The CD95 protein (APO-1/Fas; Krammer et al, 2007) is a member of the death receptor family. Signal transduction of CD95 starts with the formation of the death-inducing signaling complex (DISC) detectable within seconds after receptor stimulation (Kischkel et al, 1995). The DISC consists of CD95, the adaptor molecule FADD, procaspase-8/10 and c-FLIPL/S/R (Muzio et al, 1996; Scaffidi et al, 1999; Sprick et al, 2002; Golks et al, 2005; Krammer et al, 2007). Procaspase-8 is converted at the DISC, in a series of autoproteolytic cleavage steps, to p43/p41 and p18, which leads to the activation of effector caspase-3 and demolition of the cell. Recently, experiments have demonstrated that CD95L also activates the induction of transcription factor NF-κB (Barnhart et al, 2004; Kreuz et al, 2004; Peter et al, 2007). It was shown that DED-containing proteins at the DISC, such as procaspase-8 and c-FLIP have a complex role in NF-κB activation (Chaudhary et al, 2000; Hu et al, 2000; Kreuz et al, 2004; Dohrman et al, 2005; Su et al, 2005). These findings motivated our systems biology approach and prompted us to determine whether CD95-mediated signaling should be considered a dynamic system, resulting in life/death decisions. We observed simultaneous apoptosis and NF-κB induction on CD95 stimulation in HeLa cells stably overexpressing CD95–GFP (HeLa-CD95) using biochemical approaches and live-cell imaging. To understand the crosstalk between CD95-mediated apoptosis and NF-κB activation, we created a mathematical model of CD95 signaling. Our model assumes a trimerized ligand (L) that binds to a trimerized CD95 receptor (R) that can recruit three copies of FADD (F) leading to the DISC formation. Subsequently, DED-containing proteins, such as procaspase-8 (C8), c-FLIPL (FL) and c-FLIPS (FS) can bind to FADD. The order of protein binding gives rise to a combinatorial variety of intermediates, resulting either in the formation of the cleavage product of procaspase-8: p43/p41, or in the formation of the cleavage product of c-FLIPL: p43-FLIP. p43/p41 gives rise to signaling in the apoptotic branch of the model, whereas the cleavage product p43-FLIP triggers the activation of NF-κB. The model postulates that p43-FLIP interacts with the IKK complex leading to the phosphorylation of IκB (NF-κB·IκB·P), which entails its degradation and the translocation of p65 to the nucleus (NF-κB*). As a validation of the model topology, we confirmed experimentally that p43-FLIP interacts with the IKK complex and subsequently leads to its activation. The complete model could be fitted well to a data set derived from quantitative western blots of a number of key proteins of the apoptotic and NF-κB pathways. However, we tested whether all the 92 reactions were required to reproduce the observed dynamics, as a small model would yield more reliable parameter estimates, which in turn would increase its usefulness as a predictive tool. To determine the most important interactions, we simplified the complete model in a step-wise manner obtaining a model of considerably lower complexity (Figure 5A, simplification steps are listed in Figure 5B). The final reduced model still approximated well the experimental data set (Figure 5D), whereas the number of reactions decreased from 92 to 23 (Figure 5C). To better understand the interplay of DISC proteins in the determination of cell fate, we analyzed the activity of caspase-3 and NF-κB as a function of procaspase-8 and c-FLIPL levels (Figure 8A). We observed in our simulations that the decision over apoptosis and NF-κB is controlled by both proteins. Different scenarios occur that show combination or absence of either caspase-3 or NF-κB activity. The phase diagram shown in Figure 8A predicts that either increasing or decreasing the amount of c-FLIPL leads to a different signaling mode. We sought to validate this prediction by downregulating or overexpressing procaspase-8 and c-FLIPL, respectively, in HeLa-CD95 cells and measuring CD95-mediated signaling. In agreement with the phase diagram (Figure 8A), we observed that c-FLIPL overexpression resulted in a strong reduction of apoptosis (Figure 8D). Furthermore, we could further confirm by western blot analysis that the stable knockdown of c-FLIPL and procaspase-8 led to a reduction of the levels of p43-FLIP and phosphorylated IκBα after receptor stimulation (Figure 8C and D). In addition, to control the specificity of c-FLIP downregulation and further confirm the requirement of cleavage of c-FLIPL to p43-FLIP, we performed a reconstitution experiment in HeLa-CD95–c-FLIP-deficient cells (Figure 8E). Cells reconstituted with WT c-FLIPL were able to generate p43-FLIP and increased IκBα phosphorylation on CD95 stimulation. In contrast, cells reconstituted with the noncleavable mutant of c-FLIPL (D376E) did not show processing to p43-FLIP (Figure 8E; Supplementary Figure S9). Noticeably, as postulated by the model, this resulted in a strong reduction of the levels of IκBα phosphorylation on CD95 stimulation. Hence, by perturbing the ratio of procaspase-8 to c-FLIPL at the DISC, we directed the induction of apoptosis and NF-κB activation as predicted by our model. Taken together, we found that the DISC protein levels determine cell fate in a nonlinear manner, highlighting the role of signal processing within the DISC. In this study, we propose, to the best of our knowledge, the first integrated kinetic model of CD95-mediated apoptosis and NF-κB signaling. This was achieved by integrating mechanistic knowledge of DISC assembly and caspase activation with a simple scheme of NF-κB activation. We observed that c-FLIPL levels crucially determine the balance between apoptotic and NF-κB signaling by shaping the dynamics of DISC assembly. Although this finding is based on experiments performed in cell lines, we expect that the nonlinear dynamics of DISC assembly is a generic systems property of life/death decision making in CD95 signaling pathways. This is especially important for understanding the regulation of cell death in physiologically relevant cells, such as cancer cells often showing resistance against death receptor-induced apoptosis., 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-κB. We established an integrated kinetic mathematical model for CD95-mediated apoptotic and NF-κB signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c-FLIPL cleavage in the death-inducing signaling complex (DISC) and the NF-κB 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-κB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c-FLIPL 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-κB signaling.

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.

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.

Here, we report on the identification of nucleolar spindle–associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

We investigated positioning of chromosomes during the cell cycle in live mammalian cells with a combined experimental and computational approach. By non-invasive labeling of chromosome subsets and tracking by 4D imaging, we could show that no global rearrangements occurred in interphase. Using the same assay, we also observed a striking order of chromosomes throughout mitosis. By contrast, our computer simulation based on stochastic movements of individual chromosomes predicted randomization of chromosome order in mitosis. In vivo, a quantitative assay for single chromosome positioning during mitosis revealed strong similarities between daughter and mother cells. These results demonstrate that global chromosome positions are heritable through the cell cycle in mammalian cells. Based on tracking of labeled chromosomes and centromeres during chromosome segregation and experimental perturbations of chromosomal order, we propose that chromosome specific timing of sister chromatid separation transmits chromosomal positions from one cell generation to the next.

The mechanism of nuclear envelope breakdown (NEBD) was investigated in live cells. Early spindle microtubules caused folds and invaginations in the NE up to one hour prior to NEBD, creating mechanical tension in the nuclear lamina. The first gap in the NE appeared before lamin B depolymerization, at the site of maximal tension, by a tearing mechanism. Gap formation relaxed this tension and dramatically accelerated the rate of chromosome condensation. The hole produced in the NE then rapidly expanded over the nuclear surface. NE fragments remaining on chromosomes were removed toward the centrosomes in a microtubule-dependent manner, suggesting a mechanism mediated by a minus-end-directed motor.

CD95 is a plasma membrane receptor that can mediate apoptosis after activation by its specific ligand CD95L. Upon stimulation the death receptor recruits various proteins that form the so-called death inducing complex (DISC). The DISC contains the protease procaspase-8 that becomes active by dimerization and self-processing. This process is thought to require high local concentration, that can be achieved by CD95 and DISC clustering. Several mechanisms have been proposed to achieve this clustering, including CD95 self-association, lipid raft localization and oligomerization of the DISC protein FADD.Our goal is to understand how DISC clusters correlate with CD95 spatial organization on the membrane and more generally how this organization can regulate cell sensitivity to CD95-induced apoptosis.We visualize the distribution of CD95 by immunofluorescence and GFP fusion, using confocal microscopy, stimulated emission depletion microscopy and total internal reflection microscopy. As this distribution appears as a dotty pattern (fig.), we determine the dot density in different cell types. We investigate the influence of system perturbations such as cholesterol depletion and actin depolymerization on the CD95 distribution and on apoptosis sensitivity. We also investigate the dependence of clustering on the receptor amount.View Large Image | View Hi-Res Image | Download PowerPoint Slide

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.