The physicist Ernest Rutherford said, "If your experiment needs statistics, you ought to have done a better experiment." Although this aphorism remains true for much of today's research in cell biology, a basic understanding of statistics can be useful to cell biologists to help in monitoring the conduct of their experiments, in interpreting the results, in presenting them in publications, and when critically evaluating research by others. However, training in statistics is often focused on the sophisticated needs of clinical researchers, psychologists, and epidemiologists, whose conclusions depend wholly on statistics, rather than the practical needs of cell biologists, whose experiments often provide evidence that is not statistical in nature. This review describes some of the basic statistical principles that may be of use to experimental biologists, but it does not cover the sophisticated statistics needed for papers that contain evidence of no other kind.
A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.
It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.
Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2-associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak.
Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.
Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness.
Understanding mechanisms of information processing in cellular signaling networks requires quantitative measurements of protein activities in living cells. Biosensors are molecular probes that have been developed to directly track the activity of specific signaling proteins and their use is revolutionizing our understanding of signal transduction. The use of biosensors relies on the assumption that their activity is linearly proportional to the activity of the signaling protein they have been engineered to track. We use mechanistic mathematical models of common biosensor architectures (single-chain FRET-based biosensors), which include both intramolecular and intermolecular reactions, to study the validity of the linearity assumption. As a result of the classic mechanism of zero-order ultrasensitivity, we find that biosensor activity can be highly nonlinear so that small changes in signaling protein activity can give rise to large changes in biosensor activity and vice versa. This nonlinearity is abolished in architectures that favor the formation of biosensor oligomers, but oligomeric biosensors produce complicated FRET states. Based on this finding, we show that high-fidelity reporting is possible when a single-chain intermolecular biosensor is used that cannot undergo intramolecular reactions and is restricted to forming dimers. We provide phase diagrams that compare various trade-offs, including observer effects, which further highlight the utility of biosensor architectures that favor intermolecular over intramolecular binding. We discuss challenges in calibrating and constructing biosensors and highlight the utility of mathematical models in designing novel probes for cellular signaling.
The nuclear envelope (NE) surrounds the nucleus and separates it from the cytoplasm. The NE is not a passive structural component, but rather contributes to various cellular processes such as genome organization, transcription, signaling, and stress responses. Although the NE is mostly a smooth surface, it also forms invaginations that can reach deep into the nucleoplasm and may even traverse the nucleus completely. Cancer cells are generally characterized by irregularities and invaginations of the NE that are of diagnostic and prognostic significance. In the current chapter, we describe the link between nuclear invaginations and irregularities with cancer and explore possible mechanistic roles they might have in tumorigenesis.
Loss of inhibitor of apoptosis proteins (IAPs), particularly cIAP1, can promote production of tumor necrosis factor (TNF) and sensitize cancer cell lines to TNF-induced necroptosis by promoting formation of a death-inducing signaling complex containing receptor-interacting serine/threonine-protein kinase (RIPK) 1 and 3. To define the role of IAPs in myelopoiesis, we generated a mouse with cIAP1, cIAP2, and XIAP deleted in the myeloid lineage. Loss of cIAPs and XIAP in the myeloid lineage caused overproduction of many proinflammatory cytokines, resulting in granulocytosis and severe sterile inflammation. In vitro differentiation of macrophages from bone marrow in the absence of cIAPs and XIAP led to detectable levels of TNF and resulted in reduced numbers of mature macrophages. The cytokine production and consequent cell death caused by IAP depletion was attenuated by loss or inhibition of TNF or TNF receptor 1. The loss of RIPK1 or RIPK3, but not the RIPK3 substrate mixed lineage kinase domain-like protein, attenuated TNF secretion and thereby prevented apoptotic cell death and not necrosis. Our results demonstrate that cIAPs and XIAP together restrain RIPK1- and RIPK3-dependent cytokine production in myeloid cells to critically regulate myeloid homeostasis.
Amyloid formation is a hallmark of protein misfolding diseases (e.g. Type II diabetes mellitus). The energetically unfavourable nucleation step of amyloidogenesis can be accelerated by seeding, during which pre-formed aggregates act as templates for monomer recruitment. Hydrophobic-hydrophilic interfaces [e.g. AWI (air-water interface)] can also catalyse amyloidogenesis due to the surfactant properties of amyloidogenic polypeptides. Using thioflavin T fluorescence and electron microscopy, we demonstrate that the outcome of seeding on human islet amyloid polypeptide amyloidogenesis is dependent upon whether the AWI is present or absent and is dictated by seed type. Seeding significantly inhibits (with AWI) or promotes (without AWI) plateau height compared with seedless controls; with short fibrils being more efficient seeds than their longer counterparts. Moreover, promotion of nucleation by increasing monomer concentrations can only be observed in the absence of an AWI. Using biophysical modelling, we suggest that a possible explanation for our results may reside in lateral interactions between seeds and monomers determining the fibril mass formed in seeded reactions at steady-state. Our results suggest that in vivo hydrophobic-hydrophilic interfaces (e.g. the presence of membranes and their turnover rate) may dictate the outcome of seeding during amyloidogenesis and that factors affecting the size of the pre-aggregate may be important.
Lamins are intermediate filament proteins that form a major component of the nuclear lamina, a protein complex at the surface of the inner nuclear membrane. Numerous clinically diverse conditions, termed laminopathies, have been found to result from mutation of LMNA. In contrast, coding or loss of function mutations of LMNB1, encoding lamin B1, have not been identified in human disease. In mice, polymorphism in Lmnb1 has been shown to modify risk of neural tube defects (NTDs), malformations of the central nervous system that result from incomplete closure of the neural folds.
We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz-sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked ?-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein ?-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER-Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.
Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.
The nuclear envelope is not only important for the structural integrity of the nucleus, but also involved in a number of cellular functions. It has been shown to be important for maintaining and controlling chromatin organization, sequestering transcription factors, replication, transcription and signalling. The nuclear envelope is thus important for development and differentiation, and some of its components are essential for cell viability. Among the many functions which are emerging for the nuclear envelope is its involvement in protecting the cell against different types of cellular stress. In the present paper, we review key findings which describe the roles of nuclear envelope components in responses to common types of stress conditions.
The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.
Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-? (CCT-?, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.
According to the somatic mutation theory (SMT), cancer begins with a genetic change in a single cell that passes it on to its progeny, thereby generating a clone of malignant cells. It is strongly supported by observations of leukemias that bear specific chromosome translocations, such as Burkitts lymphoma, in which a translocation activates the c-myc gene, and chronic myeloid leukemia (CML), in which the Philadelphia chromosome causes production of the BCR-ABL oncoprotein. Although the SMT has been modified and extended to encompass tumor suppressor genes, epigenetic inheritance, and tumor progression through accumulation of further mutations, perhaps the strongest validation comes from the successful treatment of certain malignancies with drugs that directly target the product of the mutant gene.
The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.
RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-?B, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-?B2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.
B cells require signals delivered through B-cell activating factor of the TNF family receptor (BAFF-R) and CD40 to survive and produce antibody responses in vivo. In vitro data indicate that these signals are controlled by the homologous RING finger proteins cIAP1 and cIAP2, in collaboration with TRAF2 and TRAF3. There is also mounting evidence that all 4 of these signaling molecules can act as tumor suppressors in human B-lineage malignancies. However, it has not been possible to identify the roles of cIAP1 and cIAP2 in controlling B-cell physiology because of the absence of an appropriate in vivo model. Here we describe a unique genetically modified mouse in which the linked cIap1 and cIap2 genes can be independently inactivated. Deletion of cIAP1 plus cIAP2 (but not either protein alone) rendered primary B cells independent of BAFF-R for their survival and led to their uncontrolled accumulation in vivo. B cells deficient in cIAP1 and cIAP2 were also incapable of forming germinal centers, a key step in antibody-mediated immunity. These data define a fundamental role for cIAP1/cIAP2 in regulating B-cell survival and responsiveness, show this requires direct binding to TRAF2, and suggest how mutations of TRAF2, TRAF3, and cIAP1/cIAP2 contribute to B-lineage malignancies, such as multiple myeloma.
BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain-interacting proteins. Ezrin-radixin-moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA1(1634-1863) acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA1(1634-1863) in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.
The nuclear envelope (NE) physically separates nucleoplasm and cytoplasm, contributes to nuclear structural integrity, controls selective bidirectional transport of ions and macromolecular cargo, regulates gene expression, and acts as a mechanotransducer and a platform for signalling. It is noteworthy however that the NE is not simply a smooth-surfaced outer boundary but is interrupted by invaginations that reach deep within the nucleoplasm and could even traverse the nucleus completely. The existence of such a complex branched network of invaginations forming a nucleoplasmic reticulum (NR) provides sites that are capable of carrying out the conventional NE functions deep within the nucleus in regions that would otherwise be remote from the nuclear periphery. In this review, we describe the structural features of NR in normal and pathological states and discuss the current understanding of their functional and possible pathological roles.
Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.
When cells kill themselves, they usually do so by activating mechanisms that have evolved specifically for that purpose. These mechanisms, which are broadly conserved throughout the metazoa, involve two processes: activation in the cytosol of latent cysteine proteases (termed caspases), and disruption of mitochondrial functions. These processes are linked in a number of different ways. While active caspases can cleave proteins in the mitochondrial outer membrane, and cleave and thereby activate certain pro-apoptotic members of the Bcl-2 family, proteins released from the mitochondria can trigger caspase activation and antagonise IAP family proteins. This review will focus on the pro-apoptotic molecules that are released from the mitochondria of cells endeavouring to kill themselves. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.
Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.
Cellular inhibitor of apoptosis protein (cIAP) 1 and cIAP2 set the balance between transcription factor and apoptosis signaling downstream of tumor necrosis factor (TNF) receptor superfamily members by acting as ubiquitin E3 ligases for substrates that are part of the TNF receptor complex. To fulfill this role, cIAPs must be recruited to the receptor complex by TNF-receptor-associated factor (TRAF) 2. In this study, we reconstituted the complex between baculoviral IAP repeat (BIR) 1 of cIAP1 and the coiled-coil region of TRAF2, solved the structure of BIR1 from cIAP1, and mapped key binding residues on each molecule using mutagenesis. Biophysical analysis indicates that a single BIR1 domain binds the trimeric TRAF2 coiled-coil domain. This suggests that only one IAP molecule binds to each TRAF trimer and makes it likely that the dimeric cIAPs crosslink two TRAF trimers.
Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4(+) T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.
The nuclear envelope can regulate gene expression through its interaction with chromatin and by the sequestration of specific transcription factors. In this study, we show that such regulation can be achieved via microRNA regulation. We identify a set of miRNAs that are dysregulated in the absence of a fully functional nuclear lamina. We then focus on miRNA-31 and experimentally confirm its targets. The target set identified is significantly enriched in genes involved in controlling progress through the cell cycle such as Cdkn2a. Normalizing miRNA-31 levels, either using a specific inhibitor or by restoration of the nuclear lamina, also normalizes cell cycle distribution and cell proliferation rates. We show that the 3UtR of p16(Ink4a)/p19(Arf) has a functional miRNA-31 binding site which contributes to the observed regulation of cell cycle progression. Our findings are the first demonstration that the nuclear envelope can control gene expression by regulating specific miRNA levels, and that miRNA-31 is involved in the regulation of cell proliferation and progress through the cell cycle at least in part by regulating the levels of p16(Ink4a)/p19(Arf).
HIV-PIs (HIV protease inhibitors) have proved to be of great benefit for the millions of people suffering from AIDS. However, one of the side effects of this component of combined highly active antiretroviral therapy is lipodystrophy, which affects a large number of the patients taking this class of drug. It has been shown that many of these protease inhibitors inhibit the ZMPSTE24 enzyme responsible for removing the farnesylated tail of prelamin A, which is a nuclear lamina component that has been implicated in some of the nuclear laminopathies. Build up of this protein somehow leads to acquired lipodystrophy, possibly through its interaction with a transcription factor called SREBP-1 (sterol-regulatory-element-binding protein-1). The downstream effect of this is altered fatty acid metabolism and sterol synthesis, which may cause lipodystrophy in patients. The build-up of this protein also appears to have morphological consequences on the nucleus and we reveal, by dual-axis electron tomography, a complex nucleoplasmic reticulum that forms after HIV-PI treatment as a result of acute farnesylated prelamin A accumulation. A greater understanding of the molecular mechanisms leading to lipodystrophy will hopefully facilitate the design of improved HIV-PIs that do not cause this debilitating side effect.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.
Three companies, Genentech, Aegera Therapeutics/Human Genome Sciences, and Novartis, have commenced phase 1 clinical trials of inhibitor of apoptosis (IAP) antagonist Smac mimetic compounds for the treatment of cancer. These trials represent the culmination of a line of research that commenced with analysis of how insect viruses stop host cells from killing themselves and led to the discovery of a family of proteins that regulate development in insects and signalling by tumour necrosis factor superfamily members in mammals, which prompted development of drugs that mimic natural IAP-binding proteins to promote cell death.
The most thoroughly characterized mammalian IAP is XIAP/BIRC4, which can inhibit caspases 9, 3 and 7, but may also regulate apoptosis through interactions with other proteins such as Smac/DIABLO, HtrA2/Omi, XAF1, TAK1, cIAP1, and cIAP2.High throughput sequencing of the mouse genome revealed the existence of a gene resembling Xiap/Birc4 on mouse chromosome 7. To confirm the existence of this gene, and to determine its functional significance, we performed Southern and Northern blot analysis. This showed the presence of the Xiap-like gene in both wild-type and Xiap gene knock-out mice, but the corresponding mRNA was not detected in any tissues examined by Northern blot. Analysis of the gene sequence in all three possible reading frames predicts that expression of this gene would not give rise to a full-length protein, but only non-functional truncated polypeptides. Because its nucleotide sequence is 92% identical to Xiap, but it has no introns corresponding to those of Xiap, we conclude that Xiap-ps1 is a pseudogene generated by retro-transposition of a spliced Xiap message to chromosome 7.
Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-kappaB-inducing kinase stability and suppress constitutive noncanonical NF-kappaB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-kappaB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-kappaB signaling and that efficient activation of NF-kappaB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.
Protein amyloid fibrils are a form of linear protein aggregates that are implicated in many neurodegenerative diseases. Here, we study the dynamics of amyloid fibril elongation by performing Langevin dynamic simulations on a coarse-grained model of peptides. Our simulation results suggest that the elongation process is dominated by a series of local minimum due to frustration in monomer-fibril interactions. This rugged energy landscape picture indicates that the amount of recycling of monomers at the fibrils ends before being fibrilized is substantially reduced in comparison to the conventional two-step elongation model. This picture, along with other predictions discussed, can be tested with current experimental techniques.
Amyloid accumulation is associated with pathological conditions, including type II diabetes and Alzheimers disease. Lipids influence amyloidogenesis and are themselves targets for amyloid-mediated cell membrane disruption. Amyloid precursors are surface-active, accumulating at hydrophobic-hydrophilic interfaces (e.g., air-water), where their biophysical and kinetic behaviors differ from those in the bulk solution with significant and underappreciated consequences. Biophysical modeling predicted the probability and rate of beta-sheet amyloid dimer formation to be higher and faster at the air-water interface (AWI) than in the bulk (by 14 and approximately 1500 times, respectively). Time-course staining experiments with a typical amyloid dye verified our predictions by demonstrating that without AWI, islet amyloid polypeptide (IAPP) fibrilization was abolished or slowed, depending on the conditions. Our controls included undisturbed IAPP reactions, and we ascertained that the AWI removal process (technical or material) did not itself affect the reaction. Furthermore, we showed that the role of membranes in amyloidogenesis has been previously underestimated; in an in vivo-like situation (with no AWI), anionic liposomes (containing dioleoylphosphatidylglycerol) enhanced IAPP fibrilogenesis far more than described previously in conventional assay conditions (in the presence of an AWI). These findings have implications for the protein misfolding field and in assay design to target toxic protein aggregation.
Interaction of lamins with chromatin and transcription factors regulate transcription. Oct-1 has previously been shown to colocalize partly with B-type lamins and is essential for transcriptional regulation of oxidative stress response genes. Using sequential extraction, co-immunoprecipitation (IP), fluorescence loss in photobleaching, and fluorescence resonance energy transfer, we confirm Oct-1-lamin B1 association at the nuclear periphery and show that this association is lost in Lmnb1(Delta/Delta) cells. We show that several Oct-1-dependent genes, including a subset involved in oxidative stress response, are dysregulated in Lmnb1(Delta/Delta) cells. Electrophoretic mobility shift assay and chromatin IP reveal that Oct-1 binds to the putative octamer-binding sequences of the dysregulated genes and that this activity is increased in cells lacking functional lamin B1. Like Oct1(-/-) cells, Lmnb1(Delta/Delta) cells have elevated levels of reactive oxygen species and are more susceptible to oxidative stress. Sequestration of Oct-1 at the nuclear periphery by lamin B1 may be a mechanism by which the nuclear envelope can regulate gene expression and contribute to the cellular response to stress, development, and aging.
Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects whose complex multigenic causation has hampered efforts to delineate their molecular basis. The effect of putative modifier genes in determining NTD susceptibility may be investigated in mouse models, particularly those that display partial penetrance such as curly tail, a strain in which NTDs result from a hypomorphic allele of the grainyhead-like-3 gene. Through proteomic analysis, we found that the curly tail genetic background harbours a polymorphic variant of lamin B1, lacking one of a series of nine glutamic acid residues. Lamins are intermediate filament proteins of the nuclear lamina with multiple functions that influence nuclear structure, cell cycle properties, and transcriptional regulation. Fluorescence loss in photobleaching showed that the variant lamin B1 exhibited reduced stability in the nuclear lamina. Genetic analysis demonstrated that the variant also affects neural tube closure: the frequency of spina bifida and anencephaly was reduced three-fold when wild-type lamin B1 was bred into the curly tail strain background. Cultured fibroblasts expressing variant lamin B1 show significantly increased nuclear dysmorphology and diminished proliferative capacity, as well as premature senescence, associated with reduced expression of cyclins and Smc2, and increased expression of p16. The cellular basis of spinal NTDs in curly tail embryos involves a proliferation defect localised to the hindgut epithelium, and S-phase progression was diminished in the hindgut of embryos expressing variant lamin B1. These observations indicate a mechanistic link between altered lamin B1 function, exacerbation of the Grhl3-mediated cell proliferation defect, and enhanced susceptibility to NTDs. We conclude that lamin B1 is a modifier gene of major effect for NTDs resulting from loss of Grhl3 function, a role that is likely mediated via the key function of lamin B1 in maintaining integrity of the nuclear envelope and ensuring normal cell cycle progression.
Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni- or bidirectional. We also asked whether GPIHBP1 and LPL are transported across cells in vesicles and whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent.
Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimers disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. ?-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-? peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD.
Regulatory T (Treg) cells are critically important for the maintenance of immunological tolerance. Both centrally arising natural nTreg cells and those emerging in the periphery in response to TGF-?, iTreg cells, play a role in the control of unwanted immune responses. Treg cells adopt multiple mechanisms to inhibit effector T cells, yet it is unclear whether these mechanisms are shared by nTreg cells and iTreg cells alike. Here, we show that iTreg cells, like nTreg cells, are able to out-compete naïve T cells in clustering around dendritic cells (DCs). However, using both a tamoxifen-responsive inducible Foxp3 retroviral construct and TGF-?-induced iTreg cells from hCD2-Foxp3 knock in reporter mice, we show that it is prior antigen-induced activation rather than Foxp3 expression per se that determines the ability of iTreg cells to competitively cluster around DCs. We found no difference in the capacity of iTreg cells to displace naïve T cells around DCs to that of Tr1, Th1, Th2, or Th9 cells. An important difference was, however, that clustering of iTreg cells around DCs, just as for naïve T cells, did not effectively activate DCs.
The aggregation of proteins or peptides into amyloid fibrils is a hallmark of protein misfolding diseases (e.g., Alzheimers disease) and is under intense investigation. Many of the experiments performed are in vitro in nature and the samples under study are ordinarily exposed to diverse interfaces, e.g., the container wall and air. This naturally raises the question of how important interfacial effects are to amyloidogenesis. Indeed, it has already been recognized that many amyloid-forming peptides are surface-active. Moreover, it has recently been demonstrated that the presence of a hydrophobic interface can promote amyloid fibrillization, although the underlying mechanism is still unclear. Here, we combine theory, surface property measurements, and amyloid fibrillogenesis assays on islet amyloid polypeptide and amyloid-? peptide to demonstrate why, at experimentally relevant concentrations, the surface activity of the amyloid-forming peptides leads to enriched fibrillization at an air-water interface. Our findings indicate that the key that links these two seemingly different phenomena is the surface-active nature of the amyloid-forming species, which renders the surface concentration much higher than the corresponding critical fibrillar concentration. This subsequently leads to a substantial increase in fibrillization.
Inhibitor of apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP (X-linked IAP) regulate apoptosis and cytokine receptor signalling, but their overlapping functions make it difficult to distinguish their individual roles. To do so, we deleted the genes for IAPs separately and in combination. While lack of any one of the IAPs produced no overt phenotype in mice, deletion of cIap1 with cIap2 or Xiap resulted in mid-embryonic lethality. In contrast, Xiap(-/-)cIap2(-/-) mice were viable. The death of cIap2(-/-)cIap1(-/-) double mutants was rescued to birth by deletion of tumour necrosis factor (TNF) receptor 1, but not TNFR2 genes. Remarkably, hemizygosity for receptor-interacting protein kinase 1 (Ripk1) allowed Xiap(-/-)cIap1(-/-) double mutants to survive past birth, and prolonged cIap2(-/-)cIap1(-/-) embryonic survival. Similarly, deletion of Ripk3 was able to rescue the mid-gestation defect of cIap2(-/-)cIap1(-/-) embryos, as these embryos survived to E15.5. cIAPs are therefore required during development to limit activity of RIP kinases in the TNF receptor 1 signalling pathway.
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