Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1^−/− mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER^T2Ripk1^fl/fl mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1^fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1^fl/fl mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1^fl/fl Ripk3^−/− progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.The proinflammatory cytokine TNF stimulates receptor-interacting serine/threonine-protein kinase 1 (RIPK1) ubiquitination, NFκB and MAPK activation, and induction of apoptosis or necroptosis (1, 2). TNF signaling via TNF receptor 1 (TNFR1) is highly regulated and results in the recruitment of several adapter proteins including TNFR1-associated death domain (TRADD) protein, the E3 ubiquitin ligases cellular inhibitor of apoptosis protein-1 and -2 (cIAP1/2), and TNFR-associated factor 2 (TRAF2) or 5, and the serine threonine death domain-containing kinase RIPK1 (complex I) (1). We have demonstrated that the kinase activity of RIPK1 is not required for NFκB activation (3); rather, RIPK1 is modified by the addition of Lys63-linked and linear polyubiquitin chains (3–6). Polyubiquitinated RIPK1 then recruits NEMO/IκB kinase-γ (IKKγ) to mediate IKK activation and TAK1/TAB2/3 to mediate MAPK activation, resulting in antiapoptotic and proinflammatory gene expression (7, 8). Deubiquitination of RIPK1 by cylindromatosis (CYLD) results in the formation of a cytosolic complex containing TRADD, Fas-associated death domain protein (FADD), caspase-8, and RIPK1 (complex IIa) (2). Caspase-8 cleaves and inactivates RIPK1 and CYLD and stimulates apoptosis (9–11). In the absence of caspase-8 or the presence of caspase inhibitors, TNF family members and potentially other ligands stimulate the kinase activity of RIPK1 to induce necroptosis (9, 11–16). RIPK1 also is recruited to the Toll-like receptor adapter TRIF via the Rip homotypic interaction motif (RHIM) to mediate NFκB activation (17) and, under conditions of caspase-8 inhibition, initiates necroptosis (14, 16). Necrostatin-1 (Nec-1), an allosteric RIPK1 inhibitor, inhibits necroptosis induced by TNF or the TLR3 ligand poly I:C and abolishes the formation and activation of an RIPK1/3 complex (13–16, 18). Although the molecular details whereby RIPK1 initiates necroptosis are unclear, RIPK3 and the pseudo kinase MLKL appear to be required (2).Genetic studies in mice have revealed cross-regulation between the apoptotic and necroptotic pathways. For example, the FADD/caspase-8/FLICE-like inhibitory protein long form (FLIP_L) complex regulates RIPK1 and RIPK3 activity during development, because the embryonic lethality associated with a caspase-8 deficiency is completely rescued by the absence of RIPK3 (19, 20). Similarly, RIPK1 deficiency rescues FADD-associated embryonic lethality (21). Thus, in the absence of FADD or caspase-8, embryos succumb to RIPK1- and RIPK3-dependent necroptosis. However, Fadd^−/−/Ripk1^−/− mice, die perinatally (21, 22), as do Ripk1^−/− mice, revealing that RIPK1 has prosurvival roles beyond the regulation of the FADD/caspase-8/FLIP_L complex.We have demonstrated that complete RIPK1 deficiency results in increased TNF-induced cell death that can be rescued, in part, by the absence of the TNFR1 (22, 23). However, Ripk1^−/−Tnfr1^−/− animals still succumb (23), indicating that other death ligands/pathways contribute to the RIPK1-associated lethality. Consistent with this hypothesis, RIPK3 deficiency recently has been shown to rescue the perinatal lethality observed in Ripk1^−/−Tnfr1^−/− mice (24, 25). Similarly, combined caspase-8 and RIPK3 deficiency also rescues the RIPK1-associated lethality (24–26). Collectively, these genetic studies in mice reveal that the perinatal death of Ripk1^−/− mice reflects TNF-induced apoptosis and RIPK3-mediated necroptosis. The nature of the ligand(s) or the trigger(s) of RIPK3-mediated necroptosis in vivo remain unclear. However, Ripk1^−/− MEFs are prone to necroptosis induced by poly I:C or by treatment with type I or type II IFN (24, 25), suggesting that these pathways contribute. Although these studies reveal a regulatory role for RIPK1, the multiorgan cell death and inflammation observed in the complete and compound RIPK1-knockout strains have made it difficult to discern the specific tissues that require RIPK1 for survival.     

The K63 deubiquitinase CYLD modulates autism-like behaviors and hippocampal plasticity by regulating autophagy and mTOR signaling

Nondegradative ubiquitin chains attached to specific targets via Lysine 63 (K63) residues have emerged to play a fundamental role in synaptic function. The K63-specific deubiquitinase CYLD has been widely studied in immune cells and lately also in neurons. To better understand if CYLD plays a role in brain and synapse homeostasis, we analyzed the behavioral profile of CYLD-deficient mice. We found that the loss of CYLD results in major autism-like phenotypes including impaired social communication, increased repetitive behavior, and cognitive dysfunction. Furthermore, the absence of CYLD leads to a reduction in hippocampal network excitability, long-term potentiation, and pyramidal neuron spine numbers. By providing evidence that CYLD can modulate mechanistic target of rapamycin (mTOR) signaling and autophagy at the synapse, we propose that synaptic K63-linked ubiquitination processes could be fundamental in understanding the pathomechanisms underlying autism spectrum disorder.

CYLD is a deubiquitinating (DUB) enzyme first identified as being mutated in familial cylindromatosis, an autosomal dominant genetic predisposition to multiple tumors called cylindromas (1, 2). CYLD is located in the cytoplasm, and its C-terminal catalytic domain mediates the cleavage of tetra-ubiquitin to tri-, di-, and monoubiquitin with a preference for Lysine 63 (K63)– or Met1-linked polyubiquitin chains from several substrates (3). The N terminus comprises three cytoskeletal-associated protein–glycine-rich (CAP–Gly) domains, which can bind microtubules facilitating cytoskeleton formation (4).Although CYLD is highly expressed in the brain (1, 5), surprisingly only few data are available on its role there. Specifically, CYLD was identified in the postsynaptic density (PSD) as detected by mass spectrometry analysis, immunoblotting, and immunoelectron microscopy (6, 7). In addition, immunogold labeling of dissociated hippocampal cultures under basal and depolarizing conditions showed that CYLD expression significantly increases at PSDs upon neuronal depolarization (7). It was further shown that CYLD recruitment to the PSD is dependent on the activation of Ca²⁺/calmodulin-dependent protein kinase II upon membrane depolarization and N-methyl-D-aspartate (NMDA) receptor activation (8). Thus, CYLD is recruited to synapses in an activity-dependent manner and involved in the regulation of several signaling pathways. In addition, CYLD was shown to control synapse organization by localizing and compartmentalizing specific synapse targets by deubiquitinating PSD-95, which functions as a major scaffold protein organizing the structure of the PSD (9). Moreover, CYLD was shown to positively regulate dendritic growth by promoting both α-tubulin acetylation in mouse hippocampal neurons and through the interaction of its first CAP–Gly domain with microtubules (10).Recently, several neuropsychiatric disorders such as autism spectrum disorder (ASD) have been linked to molecular changes in synaptic connections. Interestingly, the proteome analysis of striatal synaptosomes from two mutant lines for Shank3, a major ASD candidate gene, revealed that the amount of synaptic CYLD is significantly increased and reduced in the striatum of Shank3 overexpressing and Shank3-deficient mice, respectively (11, 12).In this study, we investigated the role of CYLD in synaptic dysfunction related to ASD. We show that Cyld^−/− mice exhibit major autism-like phenotypes including impaired social communication, increased repetitive behavior, and cognitive dysfunction. In addition, we associate the behavioral phenotypes with reduced basal synaptic transmission, impaired network excitability, and reduced long-term potentiation (LTP) in Cyld^−/− hippocampi. Furthermore, the presence of K63-linked polyubiquitin substrates at the synapse and the abundance of CYLD in the PSD fraction suggest that CYLD is a primary DUB for multiple synaptic proteins. Based on our data, we suggest that CYLD controls mechanistic target of rapamycin (mTOR) signaling and the regulation of autophagic processes. Indeed, the deletion of Cyld resulted in increased hippocampal mTOR activity correlating with decreased levels of the autophagy marker LC3B-II within hippocampal synaptosomes.     

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1–caspase-8 death–inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase–dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1β release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1β neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

Gasdermins are a family of recently described pore-forming proteins and are emerging as key drivers of cell death and inflammation. Gasdermins comprise a cytotoxic N-terminal domain connected to an inhibitory carboxyl-terminal domain and are activated upon proteolytic cleavage (1, 2). This cleavage event releases the cytotoxic N-terminal fragment, which creates membrane pores and triggers a form of lytic cell death called pyroptosis (3–6). Gasdermin D (GSDMD) is arguably the best characterized family member to date and is activated upon proteolysis by caspase-1, 4, 5, 8, and 11 and serine proteases (7–14). Active GSDMD promotes host defense by eliminating the replicating niche of intracellular pathogens (15) and inducing the extrusion of antimicrobial neutrophil extracellular traps (NETs) (16). In addition, GSDMD pores act as a conduit for bioactive IL-1β release (17–19), a potent proinflammatory cytokine that similarly requires proteolytic cleavage by caspase-1 or -8 to gain biological activity (20). By contrast, gasdermin E (GSMDE [also known as DFNA5]) is activated by apoptotic caspase-3 and 7 and granzyme B, which drives tumor cell pyroptosis and anti-tumor immunity (21–23). The physiological function of GSDME in primary immune cells and its potential role in host defense remain unresolved and have not been reported.Pathogenic Yersinia are a group of Gram-negative extracellular bacteria that causes disease ranging from gastroenteritis (Yersinia pseudotuberculosis) to plague (Y. pestis). A major mechanism by which pathogenic Yersinia establish systemic infection is by injecting the effector protein YopJ, an acetyltransferase that blocks transforming growth factor beta-activated kinase 1 (TAK1), to inhibit host innate immune signaling and proinflammatory cytokine production (24). To counteract this, detection of YopJ activity by myeloid cells induces the assembly of a cytoplasmic death–inducing complex that comprises receptor-interacting serine/threonine protein kinase 1 (RIPK1), fas-associated protein with death domain, and caspase-8 (24–26). During in vivo infection, RIPK1/caspase-8–dependent cell death in myeloid cells restricts bacterial dissemination and replication at distal sites by inducing proinflammatory cytokine production from uninfected bystander cells (24). More recently, GSDMD was identified as a caspase-8 substrate during Yersinia infection that drives antimicrobial defense in vivo (11, 12, 27). However, whether RIPK1 activates additional substrates to restrict Yersinia infection is unclear and is a focus of this study. Here, we identify GSDME as a substrate activated downstream of RIPK1 that confers host resistance against Yersinia. Gsdme-deficient mice failed to control bacterial replication in the spleen and liver and consequently are more susceptible to Yersinia infection than wild-type (WT) animals. Mechanistically, our data reveal that RIPK1 promotes caspase-3–dependent GSDME activation and IL-1β release in neutrophils, but not macrophages. Neutralization of IL-1β impaired bacterial clearance in WT, but not Gsdme^−/−, animals, indicating that IL-1β is mainly secreted through GSDME pores during Yersinia challenge in vivo.     

ZBP1 promotes inflammatory responses downstream of TLR3/TLR4 via timely delivery of RIPK1 to TRIF

ZBP1 is widely recognized as a mediator of cell death for its role in initiating necroptotic, apoptotic, and pyroptotic cell death pathways in response to diverse pathogenic infection. Herein, we characterize an unanticipated role for ZBP1 in promoting inflammatory responses to bacterial lipopolysaccharide (LPS) or double-stranded RNA (dsRNA). In response to both stimuli, ZBP1 promotes the timely delivery of RIPK1 to the Toll-like receptor (TLR)3/4 adaptor TRIF and M1-ubiquitination of RIPK1, which sustains activation of inflammatory signaling cascades downstream of RIPK1. Strikingly, ZBP1-mediated regulation of these pathways is important in vivo, as Zbp1^−/− mice exhibited resistance to LPS-induced septic shock, revealed by prolonged survival and delayed onset of hypothermia due to decreased inflammatory responses and subsequent cell death. Further findings revealed that ZBP1 promotes sustained inflammatory responses by mediating the kinetics of proinflammatory “TRIFosome” complex formation, thus having a profound impact downstream of TLR activation. Given the well-characterized role of ZBP1 as a viral sensor, our results exemplify previously unappreciated crosstalk between the pathways that regulate host responses to bacteria and viruses, with ZBP1 acting as a crucial bridge between the two.

ZBP1 (Z-DNA-binding protein 1) is a cytosolic nucleic acid sensor and RHIM (receptor interacting protein [RIP] homotypic interaction motif) domain–containing protein that has been studied extensively in the context of RIPK3 (RIP kinase)-dependent necroptosis (1–4). As such, ZBP1 uses its two Z-DNA-binding domains to recognize viruses such as murine cytomegalovirus and influenza A virus (IAV) (2, 4–6), driving RHIM-mediated interactions between ZBP1 and RIPK3 and the activation of mixed lineage kinase domain like pseudokinase. This activation cascade ultimately induces necroptosis, as well as the assembly of a RIPK1/Fas receptor-associated death domain protein (FADD)/caspase-8 (CASP8)-containing complex that drives apoptosis in response to viral infection (1). In response to IAV infection, ZBP1 also promotes RIPK3-independent apoptosis, dependent on the direct recruitment of RIPK1 and activation of FADD and CASP8 (1, 7). ZBP1-mediated induction of necroptosis can also be unleashed during mammalian development if RIPK1 is deleted or mutated. Indeed, mutation of the RHIM domain of RIPK1 results in embryonic lethality in mice that can be rescued by the additional deletion of ZBP1 (8, 9).Recently, we reported that when the host inflammatory response is inhibited, ZBP1 initiates CASP8-mediated, RIPK1-dependent pyroptosis in response to bacterial lipopolysaccharide (LPS) (10–12). That is, ZBP1 promoted formation of a prodeath complex (or TRIFosome) downstream of the RHIM domain-containing protein and Toll-like receptor 4 (TLR4) adaptor TRIF (TIR domain containing adaptor protein-inducing interferon [IFN] β), suggesting that ZBP1 might also regulate LPS-induced inflammatory responses downstream of TRIF. Herein, we demonstrate that ZBP1 is important for the core functions of the TLR4 and TLR3 pathways by promoting the production of proinflammatory cytokines in response to LPS or polyinosine-polycytidylic acid (poly(I:C)) in vitro and in vivo. Via RHIM-dependent interactions, ZBP1 tunes the timing and magnitude of the inflammatory response by regulating the kinetics of proinflammatory complex formation and activation of mitogen activated protein kinase (MAPK), nuclear factor-κappa B (NF-κB), and IRF3-mediated signaling cascades. Importantly, deficiency in ZBP1 promotes resistance to LPS-induced septic shock in vivo by damping serum and tissue-specific inflammatory responses and cell death, mirroring the decrease and delay in the inflammatory response observed in vitro in the absence of ZBP1. Together with our previously reported role for ZBP1 in the regulation of the prodeath TRIFosome (10) complex, these data suggest that a similar complex is assembled in the proinflammatory context, thus presenting the TRIFosome as a universal regulator of cell death and inflammatory responses.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal endocytosis

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca²⁺ plays critical roles in exocytosis–endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca²⁺ in endocytosis have been reported; however, Ca²⁺ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca²⁺ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis–endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca²⁺-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca²⁺-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca²⁺ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.

Endocytosis and subsequent vesicle recycling are spatiotemporally coupled to exocytosis, which is critical for neurons and endocrinal cells to maintain the integrity of plasma membrane architecture, intracellular homeostasis, and sustained neurotransmission (1–3). In addition to triggering vesicular exocytosis, neural activity/Ca²⁺ also play an executive role in the coupling of endocytosis to exocytosis (1, 2, 4–6). Following a pioneering study 40 y ago (7), extensive studies have been conducted and showed that Ca²⁺ triggers and facilitates vesicle endocytosis in neurons and nonneuronal secretory cells (1, 8–11). Accumulating evidence also shows that intracellular Ca²⁺ may inhibit endocytosis (12–15), which has been challenged greatly due to the apparently lower occurrences in few preparations and the missing underlining mechanisms, making the endocytic role of Ca²⁺ a four-decades–long dispute (1, 2, 4, 6).Machineries and regulators involved in exocytosis–endocytosis coupling have been extensively studied for over 30 y. The soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) and synaptophysin play critical dual roles in exocytosis and endocytosis during neurotransmission (2, 3, 16, 17). Calmodulin and synaptotagmin-1 (Syt1) are currently known primary Ca²⁺ sensors facilitating endocytosis (1, 9, 16, 18, 19). Ca²⁺/calmodulin activate calcineurin, which dephosphorylates endocytic proteins (e.g., dynamin, synaptojanin, and amphiphysin) to facilitate clathrin-mediated endocytosis (CME) and clathrin-independent fast endocytosis (1, 2). Syt1 is a dual Ca²⁺ sensor for both exocytosis and endocytosis (5, 16, 18–20). It promotes CME through binding with the endocytic adaptors adaptor protein-2 (AP-2) and stonin-2 (21–24). In contrast to the well-established Ca²⁺ sensors that promote endocytosis, the mechanism of Ca²⁺-dependent inhibition in endocytosis remains unknown.CME is the classical but slow endocytosis pathway for vesicle retrieval under resting conditions or in response to mild stimulation, while the accumulated Ca²⁺ also triggers calmodulin/calcineurin-dependent bulk endocytosis, which takes up a large area of plasma membrane to fulfill the urgent requirement for high-speed vesicle exocytosis (1–3). They cooperate with kiss-and-run and ultrafast endocytosis to ensure both sufficient and precise membrane retrieval following exocytosis (3, 25–27). These endocytic pathways are all initiated from membrane invagination and are critically controlled by neural activity. However, how the switch between different endocytic modes is precisely determined remains largely unknown.Here, by combining electrophysiological recordings, confocal live imaging, superresolution stimulated emission depletion (STED) imaging, in vitro liposome manipulation, and electron microscope imaging of individual endocytic vesicles, we define Syt1 as a primary and bidirectional Ca²⁺ sensor for endocytosis, which promotes CME but inhibits bulk endocytosis, probably by mediating membrane remodeling. The balance between the facilitatory and inhibitory effects of Syt1 on endocytosis offers a fine-tuning mechanism to ensure both efficient and precise coupling of endocytosis to exocytosis. By including a non-Ca²⁺–binding Syt as the constitutive brake, this work also explains the four-decades–long puzzle about the positive and negative Ca²⁺ effects on endocytosis.     

DNA damage–induced phosphorylation of CtIP at a conserved ATM/ATR site T855 promotes lymphomagenesis in mice

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)–mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ–mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4^−/−Tp53^−/− mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs^−/−Tp53^−/− mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ–mediated chromosomal translocation.

B cell lymphomas often carry oncogenic chromosomal translocations involving the immunoglobulin (Ig) genes, where programmed DNA double-strand breaks (DSBs) are created during the assembly and modifications of the Ig loci (1). The classical nonhomologous end-joining (cNHEJ) pathway of DSB repair is exclusively required for the assembly of functional Ig genes by V(D)J recombination. However, significant (up to 25 to 50%) class switch recombination (CSR) on the Ig heavy chain (IgH) can be achieved in cNHEJ-deficient cells via alternative end-joining (A-EJ), a distinct DSB repair pathway that preferentially uses microhomologies (MHs) at the junctions (2–6). In addition to CSR, the A-EJ pathway can also generate chromosomal translocations in reporter assays (7–9). DNA end resection that generates 3′ single-stranded DNA (ssDNA) overhangs (10) promotes A-EJ by exposing the flanking MHs. However, whether end resection is necessary for A-EJ–mediated oncogenic translocation and lymphomagenesis in vivo remains unknown.The C-terminal–binding protein (CtBP)-interacting protein (CtIP), like its yeast ortholog Sae2, initiates DNA end resection together with the MRE11-RAD50-NBS1 (MRN) nuclease complex (11). By virtue of its resection activity, CtIP was implicated in A-EJ (7–9). CtIP expression and protein levels are higher in S and G2 phases and lower in the G1 phase (12, 13). Like the MRN complex, CtIP is essential for murine development (14) and the proliferation of normal lymphocytes (4, 15, 16), rendering it difficult to examine its role during oncogenesis using null or conditional-null alleles. CtIP is phosphorylated by CDK at T847 in the S and G2 phases of the cell cycle (17) and by ATM and ATR kinases at T859 (T855 in mouse) and other sites upon DNA damage (18–21). While T847 phosphorylation of CtIP is essential for murine development (16), mice carrying an alanine substitution at the T855 phosphorylation site of CtIP (Ctip^T855A) develop normally with mild end resection defects (4, 15). Moreover, Ctip^T855A/T855A mice display normal lymphocyte development and proliferation (4, 15), providing a tool to test how CtIP and T855 phosphorylation contribute to chromosomal translocation and lymphomagenesis in vivo.The cNHEJ/Tp53 double-deficient mice routinely succumb to pro–B cell lymphomas bearing A-EJ–mediated IgH-Myc translocation and coamplification (22–24), providing an ideal model to examine the role of CtIP and end resection in A-EJ–mediated lymphomagenesis. Mechanistically, the initial translocation joins a RAG-initiated IgH DSB on chromosome 12 with sequences downstream of the c-Myc oncogene on chromosome 15 to form a dicentric (12, 15) chromosome (22). The dicentric intermediate breaks during mitosis, and the chromosome that contains the IgH-Myc translocation is joined with its sister to form a new dicentric chromosome, thereby initiating a breakage-fusion-bridge (BFB) cycle and eventually leading to a coamplification of IgH-Myc translocation under proliferation selection (1, 22). Since this occurs in cNHEJ-deficient cells, the initial translocation and the dicentric formation are both mediated by the A-EJ pathway. Tp53 deficiency is critical for the tolerance of the genomic instability and subsequent overexpression of the Myc oncogene. In addition to Xrcc4/Tp53-deficient mice, other cNHEJ/Tp53-deficient models, including Artemis- and DNA-PKcs–deficient mice, also develop pro–B cell lymphomas with IgH-Myc coamplification (24–27), although the exact organization of amplicons remains undetermined. In addition to these experimental pro–B cell lymphoma models, BFB cycles also underlie tumor initiation and drug resistance in other human cancers (28, 29).Here, we examine how CtIP-mediated end resection contributes to A-EJ–mediated chromosomal translocations and lymphomagenesis by characterizing cNHEJ/Tp53 double-deficient mice with or without the CtIP-T855A mutation. The results showed that the CtIP-T855A mutation causes neonatal lethality in Xrcc4^−/−Tp53^−/− mice without apparent lymphomas or hematopoietic failure. Instead, the CtIP-T855A mutation exacerbates the reduced mitotic index in Xrcc4^−/−Tp53^−/− olfactory neurons. In contrast, CtIP^T855A/T855AKu70^−/− mice are viable, although small even with wild-type (WT) Tp53 status. Moreover, the CtIP-T855A mutation delays lymphomagenesis and alters the tumor spectrum of DNA-PKcs^−/−Tp53^−/− mice. Yet, high-throughput genome-wide translocation sequencing (HTGTS) of RAG- and endonuclease-initiated DSBs shows that CtIP T855 phosphorylation is not required for hairpin opening or the initiation of end resection but consistently reduces the extent of end resection. Correspondingly, the CtIP-T855A mutation attenuates splenomegaly in the λ-Myc transgenic mouse model, suggesting a role for T855 phosphorylation in lymphomagenesis beyond translocation. In this context, CtIP^T855A/T855A cells show no measurable defects in replication fork stability but consistent defects in IR-induced G2/M checkpoint maintenance.     

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition

The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1^−/− mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like–mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1^−/−Casp8^−/−Rip3^+/− mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.Receptor interacting protein (RIP) kinase RIP1 (RIPK1) functions as an essential adapter in a number of innate immune signal transduction pathways, including those initiated by Toll-like receptor (TLR)3, TLR4, and retinoic acid-inducible gene 1 (RIG-I)-like receptors, in addition to death receptors (1–4). Signaling via these pathways bifurcates at the level of RIP1 to produce opposing outcomes, a prosurvival inflammatory response counterbalanced by extrinsic cell death signaling that drives either apoptosis or necroptosis. Despite the normal development of many organs and neuromuscular architecture, RIP1-null mice die within a few days of birth with signs of edema as well as significant levels of cell death within lymphoid tissues, particularly immature thymocytes (5). Although TNF-signaling contributes to this perinatal death (6) and implicates the prosurvival role of RIP1 in activating nuclear factor κB (NF-κB) (5), the precise mechanism responsible for developmental failure of RIP1-deficient mice remains unresolved. It seems likely that dysregulation of additional signaling pathways contributes to this phenotype, given that deficiency in TNF receptor 1 (TNFR1) only modestly extends the lifespan of RIP1-null mice and deficiency in TNFR2 only rescues thymocytes from death (7).RIP1 orchestrates assembly of distinct signaling platforms via two C-terminal protein–protein binding domains: a death domain and a RIP homotypic interaction motif (RHIM) (3, 4). This unique architecture facilitates convergent death domain-dependent and RHIM-dependent pathways. RIP1 partners with death domain-containing proteins, particularly fas-associated death domain protein (FADD), as well as RHIM-containing proteins, such as the pronecrotic kinase RIP3 and the TLR3/TLR4 adapter TIR-domain–containing adapter-inducing IFN (TRIF) (8, 9). RIP1 is essential for TNF-induced necroptosis but dispensable for other forms of RIP3 kinase-dependent death (10, 11). Oligomerization of RIP1 through either domain promotes activation of its N-terminal serine/threonine kinase and triggers either of two distinct cell death pathways: (i) apoptosis following assembly of a cytosolic FADD–Casp8–cellular FLICE-like inhibitory protein (cFLIP)-containing complex or (ii) necroptosis via RIP3-dependent, mixed lineage kinase domain-like (MLKL)-mediated membrane permeabilization (1–4).In addition to death, RIP1 activation downstream of either TNFR1 or TNFR2 facilitates prosurvival NF-κB gene expression contingent on the balance of ubiquitination and deubiquitination (12). In this context, deubiquitination converts RIP1 into a death-inducing adapter within the TNFR-signaling complex (12). RIP1 remains a component of a death receptor-free cytosolic complex, termed complex II (also called the ripoptosome) (1–3), together with FADD, Casp8, and cFLIP where cFLIP levels control Casp8 activation (13) and death (14). When Casp8 or FADD are absent or Casp8 activity is inhibited (14–17), RIP1 mediates RHIM-dependent recruitment of RIP3. Then, RIP1 kinase activity facilitates RIP3 kinase-dependent phosphorylation of MLKL to drive necroptosis (18, 19). Importantly, basal Casp8 activity conferred by cFLIP blocks this process (14), and in vivo, this translates into a unique requirement for Casp8 to prevent RIP3-dependent embryonic lethality and tissue inflammation triggered by Casp8 or FADD compromise (14–17). Recently, the importance of Casp8 suppression of necroptosis has been extended to diverse innate signaling pathways, including those activated by TLR3 as well as type I or II interferon (IFN) (11, 20, 21), broadening a concept that first emerged in death receptor signaling (3, 4). Once TLR3 becomes activated, the adapter protein TRIF recruits RIP1 or RIP3 via RHIM interactions (8). In this context, the RIP1 death domain ensures the suppression of necrotic death by recruiting FADD, Casp8, and cFLIP. Necroptosis is unleashed whenever Casp8 or FADD is compromised. Likewise, IFN activation of protein kinase R sets up a similar relationship with the FADD–Casp8–cFLIP–RIP1 complex (21). Thus, innate immunity elicits dueling signals that both potentiate and suppress programmed necrosis.In this study, we implicate multiple innate immune signaling pathways in the death of RIP1-deficient mice. Once dysregulated by disruption of RIP1, RIP3-mediated necroptosis and Casp8-dependent apoptosis contribute to death at the time of birth. Our observations bring to light the consequences of diverse innate immune stimuli arising from TNF, IFN, and/or nucleic acids that play out during mammalian parturition. RIP1 plays a vital role suppressing cell death consequences of this innate signaling. RIP3 and Casp8 must be eliminated to rescue RIP1-null mice from perinatal death and produce fully viable, fertile, and immunocompetent triple-knockout (TKO) mice.     

Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling

Toll-like receptor signaling and subsequent activation of NF-κB– and MAPK-dependent genes during infection play an important role in antimicrobial host defense. The YopJ protein of pathogenic Yersinia species inhibits NF-κB and MAPK signaling, resulting in blockade of NF-κB–dependent cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8–mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.The innate immune response forms the first line of defense against pathogens. Microbial infection triggers the activation of pattern recognition receptors, such as Toll-like receptors (TLRs) on the cell surface or cytosolic nucleotide binding domain leucine-rich repeat family proteins (NLRs) (1). TLRs induce NF-κB and MAPK signaling to direct immune gene expression, whereas certain NLRs direct the assembly of multiprotein complexes known as inflammasomes that provide platforms for caspase-1 or -11 activation (2). Active caspase-1 and -11 mediate cleavage and secretion of the IL-1 family of proteins and a proinflammatory cell death termed pyroptosis. However, microbial pathogens can interfere with various aspects of innate immune signaling, and the mechanisms that mediate effective immune responses against such pathogens remain poorly understood. Pathogenic Yersiniae cause diseases from gastroenteritis to plague and inject a virulence factor known as YopJ, which inhibits NF-κB and MAPK signaling pathways in target cells (2–4). YopJ activity inhibits proinflammatory cytokine production (4) and induces target cell death (5). YopJ activity induces processing of multiple caspases, including caspases-8, -3, -7, and -1 (6–8). Nevertheless, Yersinia-infected cells exhibit properties of both apoptosis and necrosis (9, 10), and no specific cellular factors have been identified as being absolutely required for YopJ-induced caspase activation and cell death. We previously found that the inflammasome proteins NLR CARD 4 (NLRC4), NLR Pyrin 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC), are dispensable for YopJ-induced caspase-1 processing and cell death (11). Thus, additional pathways likely mediate YopJ-induced caspase-1 activation and cell death.Death receptors, such as TNF receptor and Fas, mediate caspase-8–dependent apoptosis via a death-inducing signaling complex containing receptor-interacting serine/threonine kinase 1 (RIPK1), caspase-8, and Fas-associated death domain (FADD) (12, 13). Whether these proteins are required for Yersinia-induced cell death, and whether this death contributes to antibacterial immune responses, is not known. The Ripoptosome complex, which contains RIPK1, FADD, caspase-8, as well as RIPK3 and cFLIP, regulates apoptosis, programmed necrosis, and survival in response to various stimuli including signaling by the TLR adaptor TRIF (14, 15). Because YopJ-induced cell death is inhibited in the absence of either TLR4 or TRIF (17) we sought to determine whether YopJ-dependent cell death and caspase-1 activation is regulated by caspase-8 or RIPK3 and to define the role of YopJ-dependent cell death in host defense. Here, we describe a previously unappreciated requirement for RIPK1, FADD, and caspase-8, but not RIPK3, in YopJ-induced caspase-1 activation and cell death. Critically, loss of caspase-8 in the hematopoietic compartment resulted in a failure of innate immune cells to produce proinflammatory cytokines in response to Yersinia infection and severely compromised resistance against Yersinia infection. Our data suggest that caspase-8–mediated cell death in response to blockade of NF-κB/MAPKs by YopJ allows for activation of host defense against Yersinia infection. This cell death may thus enable the immune system to override inhibition of immune signaling by microbial pathogens.     

Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.

The precise inheritance of genetic information relies on the accurate segregation of chromosomes in mitosis and meiosis. Kinetochores are large protein complexes assembled on centromeres and play a crucial role in chromosome segregation. The kinetochore links the chromosome to microtubule polymers, drives the movement of chromosomes, and ensures correct microtubule–kinetochores attachment (1–3). The kinetochore assembly is thus tightly regulated. Yet, the mechanism by which kinetochores are precisely assembled through the cell cycle remains poorly understood.The kinetochore comprises an outer region and an inner region. The outer kinetochore interacts with microtubules and is assembled on the platform of the inner kinetochore. The inner kinetochore consists of a complex of 14 to 16 subunits known as the constitutive centromere–associated network (CCAN) that is directly built on centromeric chromatin (4–6). In centromeres, the histone H3 variant, CENP-A, replaces the canonical histone H3 to form CENP-A–containing nucleosomes (7–9). Most eukaryotes contain large complex regional centromeres where CENP-A–containing nucleosomes are interspersed with canonical H3–containing nucleosomes (10–12). Regional centromeres are epigenetically specified by CENP-A (12–14). But how CENP-A– and histone H3–containing nucleosomes are balanced in centromeres remains unclear.CENP-T, an integral component of CCAN, is also a histone fold–containing protein. CENP-T provides a platform for the assembly of the Ndc80 complex (Ndc80C), an essential outer kinetochore component, during mitosis (5, 15–18). Ndc80C acts as the interface between microtubules and kinetochores and mediates the microtubule attachments (19, 20). The long N terminus of CENP-T contains a conserved Ndc80 receptor motif. The motif forms an alpha-helix that directly interacts with the Spc24-Spc25 heterodimer in Ndc80C (15, 16). The motif can be phosphorylated by cyclin-dependent kinase 1 (CDK1) to stabilize the interaction between CENP-T and Ndc80C (16, 21–24). However, how CENP-T is regulated through the cell cycle to mediate the assembly of Ndc80C is still not well understood.CENP-T has been shown to interact with three other histone fold–containing proteins, CENP-W, CENP-S, and CENP-X, to form the heterotetrameric nucleosome-like structure in vitro (25, 26). The CENP-T-W-S-X complex directly associates with centromeric DNA. The DNA binding activity of the complex is important for kinetochore formation (5, 25). Interestingly, the complex also directly associates with histone H3, not with CENP-A (5, 27), suggesting that CENP-T particles and the CENP-A nucleosome occupy different positions in centromeres. How the spatial relationship between the CENP-A nucleosome and CENP-T particles in centromeres is regulated remains unclear.The fission yeast Schizosaccharomyces pombe contains large regional centromeres and is considered to be a model system for centromere study. The CENP-A homolog, Cnp1, is enriched in centromere cores, which are surrounded by pericentromeric heterochromatin (28–30). CENP-A^Cnp1 nucleosomes nucleate kinetochore assembly. Mislocalization of CENP-A^Cnp1 results in severe chromosome segregation defects in fission yeast (28, 31–34). Fission yeast also contains the CENP-T homolog, Cnp20, which associates with centromeres throughout the cell cycle. The same as in higher eukaryotes, CENP-T^Cnp20 in S. pombe is essential for viability (35).Recently, Ccp1, a nucleosome assembly protein (NAP) family protein, has been shown to play an important role in antagonizing the loading of CENP-A in fission yeast (36). Ccp1 forms a homodimer and is enriched at centromeres. Ccp1 acts as a key player in balancing CENP-A and histone H3 levels in the region (36). How Ccp1 regulates the CENP-A level in centromeres remains elusive. Interestingly, its centromere localization is cell cycle regulated. Ccp1 is dissociated from centromeres at the onset of mitosis and reassociates with centromeres at the end of mitosis (36, 37). The biological importance of the cell cycle–dependent interaction between Ccp1 and centromeres is unknown.Here using mass spectrometry, we found that Ccp1 interacts directly with CENP-T^Cnp20 in fission yeast. We further identified a conserved Ccp1-interaction motif (CIM) at the N terminus of CENP-T^Cnp20, which is adjacent to the Ndc80 receptor motif. We demonstrated that CIM is important for Ccp1 localization. Furthermore, our data suggested that CDK1-mediated phosphorylation of the CIM motif at the onset of mitosis dissociates Ccp1 from CENP-T^Cnp20, allowing proper positioning of Ndc80C. Ccp1 associates with centromeres during mitosis in the phospho-null mutant of the CIM domain, leading to mislocalization of Ndc80C and severe chromosome segregation defects. Our study uncovers a previously unrecognized mechanism regulating kinetochore organization in regional centromeres.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Synergistic regulation of nonbinary molecular switches by protonation and light

We report a molecular switching ensemble whose states may be regulated in synergistic fashion by both protonation and photoirradiation. This allows hierarchical control in both a kinetic and thermodynamic sense. These pseudorotaxane-based molecular devices exploit the so-called Texas-sized molecular box (cyclo[2]-(2,6-di(1H-imidazol-1-yl)pyridine)[2](1,4-dimethylenebenzene); 1⁴⁺, studied as its tetrakis-PF₆ ⁻ salt) as the wheel component. Anions of azobenzene-4,4′-dicarboxylic acid (2H⁺•2) or 4,4′-stilbenedicarboxylic acid (2H⁺•3) serve as the threading rod elements. The various forms of 2 and 3 (neutral, monoprotonated, and diprotonated) interact differently with 1⁴⁺, as do the photoinduced cis or trans forms of these classic photoactive guests. The net result is a multimodal molecular switch that can be regulated in synergistic fashion through protonation/deprotonation and photoirradiation. The degree of guest protonation is the dominating control factor, with light acting as a secondary regulatory stimulus. The present dual input strategy provides a complement to more traditional orthogonal stimulus-based approaches to molecular switching and allows for the creation of nonbinary stimulus-responsive functional materials.

Multifactor regulation of biomolecular machines is essential to their ability to carry out various biological functions (1 –11). Construction of artificial molecular devices with multifactor regulation features may allow us to understand and simulate biological systems more effectively (12 –31). However, creating and controlling such synthetic constructs remains challenging (16, 32 –37). Most known systems involving multifactor regulation, including most so-called molecular switches and logic devices (38 –43), have been predicated on an orthogonal strategy wherein the different control factors that determine the distribution of accessible states do not affect one another (20, 44 –56). However, in principle, a greater level of control can be achieved by using two separate regulatory inputs that operate in synergistic fashion. Ideally, this could lead to hierarchical control where different states are specifically accessed by means of appropriately selected nonorthogonal inputs. However, to our knowledge, only a limited number of reports detailing controlled hierarchical systems have appeared (57). Furthermore, the balance between specific effects (e.g., kinetics vs. thermodynamics) under conditions of stimulus regulation is still far from fully understood (54). There is thus a need for new systems that can provide further insights into the underlying design determinants. Here we report a set of pseudorotaxane molecular shuttles that act as multimodal chemical switches subject to hierarchical control.