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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., Bacillus thuringiensis israelensis (Bti) produces the naturally-crystalline proteinaceous toxin Cyt1Aa that is toxic to mosquito larvae. Here the authors grow recombinant nanocrystals of the Cyt1Aa protoxin in vivo and use serial femtosecond crystallography to determine its structure at different redox and pH conditions and by combining their structural data with further biochemical, toxicological and biophysical analyses provide mechanistic insights into the Cyt1Aa bioactivation cascade.

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

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.

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.

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.

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.

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.