Chromatin bridges,not micronuclei,activate cGAS after drug-induced mitotic errors in human cells

Mitotic errors can activate cyclic GMP–AMP synthase (cGAS) and induce type I interferon (IFN) signaling. Current models propose that chromosome segregation errors generate micronuclei whose rupture activates cGAS. We used a panel of antimitotic drugs to perturb mitosis in human fibroblasts and measured abnormal nuclear morphologies, cGAS localization, and IFN signaling in the subsequent interphase. Micronuclei consistently recruited cGAS without activating it. Instead, IFN signaling correlated with formation of cGAS-coated chromatin bridges that were selectively generated by microtubule stabilizers and MPS1 inhibitors. cGAS activation by chromatin bridges was suppressed by drugs that prevented cytokinesis. We confirmed cGAS activation by chromatin bridges in cancer lines that are unable to secrete IFN by measuring paracrine transfer of 2′3′-cGAMP to fibroblasts, and in mouse cells. We propose that cGAS is selectively activated by self-chromatin when it is stretched in chromatin bridges. Immunosurveillance of cells that fail mitosis, and antitumor actions of taxanes and MPS1 inhibitors, may depend on this effect.

Mitotic errors contribute to birth defects, aging, carcinogenesis, and cancer therapy. They occur at a low frequency in normal cells, a higher frequency in cancer cells, and a much higher frequency if mitosis occurs in the presence of chemotherapeutics (1, 2). The genetic consequences of mitotic errors include structural rearrangements such as chromothripsis (3) and numerical aberrations termed aneuploidy. A singular instance of structural or numerical defect can cause sustained genetic instability (4, 5). The cytological consequences of mitotic errors include micronuclei and chromatin bridges. Micronuclei exhibit abnormal nuclear transport and a high frequency of DNA damage in the subsequent cell cycle (6, 7). They are also prone to rupture, which exposes their chromatin to the cytoplasm (8). Chromatin bridges are caused by dicentric chromosomes, merotelic attachments, and catenations (9–11). They are typically resolved during anaphase (11) but can remain intact into the subsequent interphase when they become highly stretched due to tension from cell migration (10). Stretched chromatin bridges exhibit compromised nuclear envelopes, DNA damage, and ultimately break through actin-mediated traction forces or endonuclease activity (4, 10). Broken chromatin bridges retract into the primary nucleus or become encapsulated into micronuclei (12).In addition to genetic and cytological consequences, mitotic errors induce inflammation and immunosurveillance through the activation of the viral DNA sensor cyclic GMP–AMP synthase (cGAS) (13, 14). Upon binding to DNA, cGAS synthesizes 2′3''-cyclic GMP–AMP (cGAMP) which in turn activates STING followed by TBK1 and IRF3, ultimately leading to induction of type I interferon (IFN) expression and secretion (15). cGAS was originally proposed to discriminate viral from self-DNA by cytoplasmic localization of viral DNA (16). Additional regulatory mechanisms have now been identified which include cGAS inhibition by nucleosomes, competition with BANF1, and posttranslational modifications (17–20). Species differences between mouse and human cGAS exist and include different sensitivities to DNA substrate length and propensity to form condensates with DNA (21, 22). cGAMP can move between cells to activate STING in a paracrine manner in cell culture (23) and in tumors (24). This may allow efficient signal propagation from cancer cells that have evolved blocks to IFN secretion. IFN activates adaptive immune responses, which makes cGAS activation an attractive therapeutic strategy to sensitize tumors to immune checkpoint inhibitors (25, 26).Antimitotic drugs (A-Ms) are a class of cancer chemotherapeutics that perturb mitosis and greatly increase the frequency of mitotic errors (27). They are ideal tool compounds to investigate cGAS activation after mitotic failure because they induce chromosome missegregation in distinct manners that are independent of DNA damage. Taxanes are an important class of clinical A-Ms which stabilize microtubules (MTs) and induce solid-tumor regression. At saturating concentrations in cell culture, taxanes induce a prolonged mitotic arrest leading to cell death (28). Whether this mechanism is responsible for their tumor regression activity remains controversial (29, 30). Potent and specific A-Ms that target Aurora A kinase, Aurora B kinase, Polo-like kinase 1, and KIF11 were tested in cancer patients but found to lack tumor-regression activity for reasons that remain unclear (31, 32). We proposed that the special therapeutic activity of taxanes may depend on cGAS activation (33). Here, we test this idea by comparing the ability of different A-Ms to activate cGAS and correlating this with cytological defects. Unexpectedly, we found a key role for chromatin bridges in cGAS activation which could explain the higher clinical efficacy of taxanes.     

Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

The canonical Wnt signaling pathway plays essential roles in embryonic development and tissue homeostasis (1, 2). In particular, Wnt signaling governs stem cell maintenance and proliferation in many tissues, and its misregulation is a common cause of tumor initiation (3, 4).Wnt ligands bind Frizzled (FZD) receptors and the coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) (5). The activated receptor complexes cluster on Dishevelled (DVL) platforms and are internalized via caveolin into endosomes termed LRP6 signalosomes, which triggers sequential phosphorylation of LRP6 by GSK3β and CK1γ (6–10). LRP6 signalosomes recruit the β-catenin destruction complex, which contains the scaffold proteins AXIN1 and adenomatous polyposis coli, the kinases CK1α and GSK3β, and the E3 ubiquitin ligase β-TrCP (11). This recruitment inhibits GSK3β and releases β-TrCP, which leads to β-catenin stabilization and nuclear translocation in a IFT-A/KIF3A–dependent manner (12–16). LRP6 signalosomes mature into multivesicular bodies, sequestering the Wnt receptors together with GSK3β, thereby maintaining long-term activation of the Wnt pathway and promoting macropinocytosis (14, 17–21). In contrast to Wnt ligands, the Wnt inhibitor Dickkopf-related protein 1 (DKK1) induces the clathrin-dependent internalization and turnover of LRP5/6 and thereby abrogates canonical Wnt signaling (22).LRP6 signalosome formation peaks in mitosis (23, 24). On the one hand, the LRP6 competence to respond to Wnt ligands is promoted during G2/M by a priming phosphorylation at its intracellular domain by CDK14/16 and CCNY/CCNYL1 (24, 25). On the other hand, CDK1 phosphorylates and recruits B-cell CLL/lymphoma 9 (BCL9) to the mitotic LRP6 signalosomes (23). BCL9 protects the signalosome from clathrin-dependent turnover, thereby sustaining basal Wnt activity on the onset of mitosis.Mitotic Wnt signaling not only modulates β-catenin (24) but increasing evidence suggests that it promotes a complex posttranslational program during mitosis (26). For instance, we have shown that mitotic Wnt signaling promotes stabilization of proteins (Wnt/STOP), which is required for cell growth and ensures chromosome segregation in somatic and embryonic cells (23, 26–31). Particularly, basal Wnt/STOP activity maintains proper microtubule plus-end polymerization rates during mitosis, and its misregulation leads to whole chromosome missegregation (31, 32). Furthermore, mitotic Wnt signaling controls the orientation of the spindle (33) and promotes asymmetric division in stem cells through components of the LRP6 signalosome (34). Accordingly, several Wnt components functionally associate with centrosomes, kinetochores, and the spindle during mitosis (25, 33, 35, 36). Consequently, both aberrant up-regulation or down-regulation of Wnt signaling have been associated with chromosome instability (CIN) (31, 32, 35, 37), which is a hallmark of cancer (38). Despite the importance of these processes for tissue renewal and genome maintenance, the targets and specific functions of mitotic Wnt signaling remain largely uncharacterized.Kinesin family member 2A (KIF2A) is a member of the kinesin-13 group (KIF2A,B,C) of minus-end microtubule depolymerases (39–41). KIF2A is essential for the scaling of the spindle during early development (42) and plays critical roles in neurogenesis by modulating both cilium disassembly and neuronal wiring (43–47). In dividing cells, KIF2A was thought to be required for the assembly of a bipolar spindle due to a small interfering RNA (siRNA) off-target effect (48, 49). Current evidence supports a role of KIF2A in microtubule depolymerization at the spindle poles, which can generate pulling forces on attached kinetochores, thereby ensuring the congression, alignment, and segregation of chromosomes (50–56). Genetic depletion of KIF2A in mouse leads to neonatal lethality and to severe brain malformations, including microcephaly (43, 44, 57). KIF2A recruitment to microtubules is tightly coordinated by several protein kinases (45, 47, 50–52, 58–60). For instance, phosphorylation of KIF2A at several sites by Polo-like kinase 1 (PLK1) stimulates its recruitment to and activity at the spindle (45, 58, 61). On the other hand, Aurora kinase A and B inhibit KIF2A activity and restrict its subcellular localization during mitosis (50, 58, 60).Here, we show that mitotic Wnt signaling promotes chromosome congression and alignment in prometaphase by recruiting KIF2A to the spindle in both somatic cells and pluripotent stem cells. We found that KIF2A is recruited by the LRP6 signalosome during mitosis. Mechanistically, we identified that KIF2A clusters with DVL via the N-terminal and motor domains of the depolymerase. We show that Wnt signaling controls KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle poles. We propose that basal Wnt signaling ensures timely chromosome congression and alignment prior cell division by modulating the spindle minus-end depolymerization dynamics through KIF2A.     

Eicosanoid regulation of debris-stimulated metastasis

Cancer therapy reduces tumor burden via tumor cell death (“debris”), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E₂ receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death and the most rapidly increasing cancer in the United States (1). Pancreatic cancer is the fourth leading cause of cancer-related deaths (2). Both of these cancer types are associated with a poor prognosis (1, 2). Despite the effectiveness of chemotherapy as a frontline cancer treatment, accumulating evidence from animal models suggests that chemotherapy may stimulate tumor growth and metastasis (3–22). The Révész effect, described in 1956, demonstrates that tumor cell death (“debris”) generated by cancer therapy, such as radiation, accelerates tumor engraftment (23). Follow-up studies have confirmed the Révész effect, whereby radiation-generated debris stimulates tumor growth via a proinflammatory response (24–29). Dead cell–derived mediators also stimulate tumor cell growth (30, 31). Notably, large numbers of cells are known to die in established tumors (32), which can lead to endogenous tumor-promoting debris in the tumor microenvironment (8, 33–35).Chemotherapy-generated tumor cell debris (e.g., apoptotic and necrotic cells) promotes tumor growth and metastasis via several mechanisms, including: 1) triggering a storm of proinflammatory and proangiogenic eicosanoids and cytokines (8, 9, 33, 35–38); 2) hijacking tumor-associated macrophages (TAMs) (37, 39); 3) inactivating M1-like TAMs (37); and 4) inducing immunosuppression and limiting antitumor immunity (40–42). Importantly, a metastatic phenotype and poor survival in cancer patients can be predicted by high levels of tumor cell debris (43–48). Thus, every attempt to induce tumor cell death is a double-edged sword as the resulting debris stimulates the growth of surviving tumor cells (8, 25, 33, 34, 35, 37, 38, 49–53). Tumor cells that survive treatment with chemotherapy or radiation undergo tumor cell repopulation (29). Yet, no strategy currently exists to stimulate the clearance or resolution of therapy-induced tumor cell debris and inflammation in cancer patients (35, 54).The failure to resolve inflammation-associated debris critically drives the pathogenesis of many human diseases, including cancer (8, 35, 55). Inflammation is regulated by a balance between inflammation-initiating eicosanoids (e.g., prostaglandins, leukotrienes, and thromboxanes) and specialized proresolving lipid autacoid mediators (SPMs; e.g., resolvins and lipoxins), which are endogenously produced in multiple tissues throughout the human body (56). Notably, arachidonic acid metabolites, collectively called eicosanoids, are potent mediators of inflammation and cancer metastasis (57, 58). Epoxyeicosatrienoic acids (EETs, also named EpETrEs), key eicosanoid regulators of angiogenesis, also stimulate inflammation resolution via macrophage-mediated phagocytosis of cell debris (59–64). Because EETs are rapidly metabolized by soluble epoxide hydrolase (sEH) to the less active dihydroxyeicosatrienoic acids (DiHETEs) (62), inhibition of sEH stabilizes EETs (62, 65). Indeed, sEH is a key therapeutic target for pain, as well as neurodegenerative and inflammatory diseases, including cancer (33, 35, 65–74). Thus, sEH regulates inflammatory responses (62). Importantly, sEH inhibition reduces the circulating levels and the expression of pancreatic mRNA of inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in experimental acute pancreatitis in mice (75). Chronic pancreatitis is essential for the induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice, suggesting that inflammation is a critical driver of pancreatic cancer (76, 77). Potent, selective inhibitors of sEH have been demonstrated to suppress human cancers (e.g., glioblastoma) and inflammation-induced carcinogenesis (67, 71). Similarly, inhibition of sEH can suppress inflammatory bowel disease-induced carcinogenesis and inflammation-associated pancreatic cancer (74, 78). In addition, a dual inhibitor of c-RAF and sEH suppresses chronic pancreatitis and murine pancreatic intraepithelial neoplasia in mutant K-Ras–initiated carcinogenesis (72, 73). Likewise, dual cyclooxygenase-2 (COX-2)/sEH inhibitors (e.g., PTUPB) potentiate the antitumor activity of chemotherapy and suppress primary tumor growth and metastasis via inflammation resolution (33, 35, 66, 70).Cancer therapy-induced debris can stimulate tumor growth and metastasis via prostaglandin E₂ (PGE₂) in the tumor microenvironment (25, 35, 79). PGE₂ exerts its biological activity via four G protein-coupled receptors: EP1, EP2, EP3, and EP4 (80). Among these, EP4 is upregulated in both tumor cells and immune cells (e.g., macrophages) and exhibits protumorigenic activity in many human malignancies (e.g., breast, prostate, colon, ovarian, and lung) by regulating angiogenesis, lymphangiogenesis, liver metastasis, and lymphatic metastasis (81–85). Interestingly, PGE₂ impairs macrophage phagocytosis of pathogens via EP4 receptor activation (86–88). Moreover, EP4 stimulates cancer proliferation, migration, invasion, and metastasis (89). EP4 gene silencing inhibits metastatic potential in vivo in preclinical models of breast, prostate, colon, and lung cancer (85, 90). Additionally, EP4 antagonists can suppress proinflammatory cytokines (e.g., C-C motif chemokine ligand 2 [CCL2], IL-6, and C-X-C chemokine motif 8 [CXCL8]), reduce inflammation-dependent bone metastasis, and diminish immunosuppression, while restoring antitumor immunity (91–93). In a clinical study, the EP4 antagonist E7046 increased the levels of T cells and tumor-infiltrating M2 macrophages in patients with advanced malignancies (94). Intriguingly, EP4 antagonists enhance the tumor response to chemotherapy by inducing extracellular vesicle-mediated clearance of cancer cells (95). Notably, EP4 antagonists reverse chemotherapy resistance or enhance immune-based therapies in various tumor types, including lymphoma, colorectal cancer, and lung cancer (80, 93, 96). Thus, targeting the EP4 receptor may be a strategy to suppress debris-stimulated tumor growth and metastasis.Here, we demonstrate that tumor cell debris generated by chemotherapy (e.g., gemcitabine) stimulates primary hepato-pancreatic cancer growth and metastasis when coinjected with a subthreshold (nontumorigenic) inoculum of tumor cells. Chemotherapy-generated debris upregulated sEH and EP4, which triggered a macrophage-derived storm of proinflammatory and proangiogenic mediators. Inhibitors of sEH and EP4 antagonists promoted inflammation resolution through macrophage phagocytosis of tumor cell debris and reduced proinflammatory eicosanoid and cytokine production in the tumor microenvironment. Altogether, our data show that the combined pharmacological abrogation of sEH and EP4 can prevent hepato-pancreatic cancer and metastatic progression.     

Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B–Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.Greatwall was originally identified in Drosophila as a modulator of Polo activity and a protein required for DNA condensation and normal progression through mitosis (1–4). Biochemical assays in Xenopus extracts have demonstrated that Greatwall is able to inhibit PP2A–B55 phosphatase complexes by phosphorylating the cAMP-regulated phosphoprotein Arpp19 and α-endosulfine, thus participating in the maintenance of the mitotic state (5–8). The control of PP2A through the Greatwall-dependent phosphorylation of Arpp19/Ensa proteins has also been supported by genetic studies in Drosophila (9, 10). The mammalian ortholog of Greatwall, also known as microtubule-associated serine/threonine kinase-like protein (Mastl), also participates in the maintenance of the mitotic state by inhibiting PP2A phosphatases (11, 12). Inhibition of Greatwall is required for the activation of PP2A–B55α,δ complexes during mitotic exit (13), thus suggesting the relevance of this pathway in maintaining the mitotic state (4). How Greatwall activity and function is regulated is not well established. Several evidences support a role for cyclin-dependent kinase (Cdk)-dependent phosphorylation in the activation of Greatwall, and several phosphorylation sites for multiple kinases have been mapped (14, 15). In addition, although Greatwall is mostly nuclear in interphase (3, 11), the cellular and molecular basis of the control of its dynamic intracellular trafficking and its activity remains largely unknown.We show in this work that the murine Greatwall ortholog, encoded by the Mastl gene, is essential for mouse development and cell cycle progression. Greatwall-null cultures, however, display normal kinetics during the G₂/M transition, suggesting that this protein is not required for mitotic entry. Greatwall is exported to the cytoplasm before nuclear envelope breakdown (NEB) but, interestingly, this export follows nuclear import of cyclin B–Cdk1. The lack of Greatwall activity results in defects in chromosome condensation after NEB, and these defects can be rescued by concomitant ablation of B55 proteins. Our results imply that cells are subjected to a mitotic stress resulting from NEB, a moment in which nuclear chromatin becomes exposed to cytoplasmic phosphatases. We therefore propose that Greatwall shuttles to the cytoplasm before NEB and prevents mitotic collapse by inhibiting the PP2A–B55-dependent dephosphorylation of Cdk substrates.     

Numb-deficient satellite cells have regeneration and proliferation defects

The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.Satellite cells represent a muscle-specific stem cell population that allows for muscle growth postnatally and is necessary for muscle repair (1). In response to muscle-fiber damage, quiescent satellite cells that lie along the myofibers under the plasmalemma are activated and proliferate. Proliferating satellite cells have a binary fate decision to make—they can differentiate into myoblasts and intercalate into myofibers by fusion to repair the damaged muscle or they can renew the satellite cell population and return to a quiescent state (2–4). Quiescent satellite cells express paired box 7 (Pax7), but low or undetectable levels of the myogenic regulatory factors Myf5 and MyoD (5, 6). Activated satellite cells robustly express Pax7 and MyoD/Myf5, but a subset will subsequently down-regulate the myogenic regulatory factors in the process of satellite cell self-renewal (7). Recent studies have demonstrated that, in vivo, Pax7-positive cells are necessary for muscle repair (8, 9).Notch signaling is an important regulator of satellite cell function; it is implicated in satellite cell activation, proliferation (2, 10, 11), and maintenance of quiescence (12, 13). Expression of constitutively active Notch1 results in maintenance of Pax7 expression and down-regulation of Myod/Myf5 whereas inhibition of Notch signaling leads to myogenic differentiation (10, 14). In fact, conditional ablation of Rbpj embryonically results in hypotrophic muscle (15), and, if ablated in the adult, satellite cells undergo spontaneous activation and precocious differentiation with a failure of self-renewal (12, 13). In adult muscle, the Notch ligand, Delta-like1 (Dll1), is expressed on satellite cells, myofibers, and newly differentiating myoblasts and is necessary for repair (10, 11, 16). In aged muscle, impairment of regeneration is due, in part, to a failure of Dll1 expression (17).Numb en`s four proteins with molecular masses of 65, 66, 71, and 72 kDa by alternative splicing of two exons (18, 19). The Numb proteins are cytoplasmic adaptors that direct ubiquitination and degradation of Notch1 by recruiting the E3 ubiquitin ligase Itch to the receptor (18–22). Numb is a cell-fate determinant that mediates asymmetric cell division, leading to selective Notch inhibition in one daughter cell and its subsequent differentiation whereas the other daughter has active Notch signaling and remains proliferative (10). Embryonically, Numb is expressed in the myotome whereas Notch1 is limited to the dermomyotome (23, 24). This pattern suggests that the expression of Numb in one daughter cell allows entry into the myogenic lineage. Indeed, overexpression of Numb embryonically increases the number of myogenic progenitors in the somite (25, 26).Numb expression increases during the activation and proliferative expansion of satellite cells, becoming asymmetrically segregated in transit-amplifying cells and leading to asymmetric cell divisions (10, 27). These observations led to a model in which Numb inhibits Notch signaling in one daughter satellite cell, allowing it to undergo myogenic differentiation. The molecular switch that controls the decision of satellite cell progeny to continue proliferating or to differentiate is not well understood. This process seems to be controlled by a decrease of Notch signaling due to increased expression of Numb and an increase in Wnt signaling (10–14, 17, 28). In these studies, we examined the role of Numb in satellite cell function by genetic deletion of Numb from myogenic progenitors and satellite cells. Our observations reveal that Numb is necessary for satellite cell-mediated repair. Furthermore, Numb-deficient satellite cells have an unexpected proliferation defect due to an up-regulation of Myostatin. These data indicate a unique role for Numb in regulating the activation and proliferation of satellite cells.     

Topological braiding and virtual particles on the cell membrane

Braiding of topological structures in complex matter fields provides a robust framework for encoding and processing information, and it has been extensively studied in the context of topological quantum computation. In living systems, topological defects are crucial for the localization and organization of biochemical signaling waves, but their braiding dynamics remain unexplored. Here, we show that the spiral wave cores, which organize the Rho-GTP protein signaling dynamics and force generation on the membrane of starfish egg cells, undergo spontaneous braiding dynamics. Experimentally measured world line braiding exponents and topological entropy correlate with cellular activity and agree with predictions from a generic field theory. Our analysis further reveals the creation and annihilation of virtual quasi-particle excitations during defect scattering events, suggesting phenomenological parallels between quantum and living matter.

Braiding confers remarkable robustness to static and dynamic structures, from plaited hair and fabrics (1) to the entangled world lines of classical (2) and quantum particles (3). Stabilized by an inherent topological protection, braided threads, ropes, and wires have long been used to transmit forces and shield signals (4). Over the last decade, dynamic braiding processes (5–7) have attracted major interest in soft matter (8, 9) and quantum physics (3) as promising candidates for robust information storage and processing (10, 11). A widely studied application is topological quantum algorithms that perform computations by braiding the world lines of two-dimensional (2D) quasiparticle excitations (3, 10, 11). Of similar importance to information processing in living systems—albeit much less well understood—are the braiding dynamics of chemical spiral wave signals on cell membranes, which control a wide range of developmental and physiological functions, including cell division (12), cardiac rhythm (13–16), and brain activity (17). These spiral waves belong to a rapidly expanding class of recently discovered biological phenomena (18, 19) in which topological structures serve as robust organizers of essential life processes.Similar to quantum states, biochemical spiral wave patterns can be described by complex wave functions (20), with spiral cores acting as topologically protected 2D quasiparticles (21). Although modern live-cell imaging now enables the direct observation of membrane spiral waves (22), their braiding dynamics have remained unexplored due to insufficient spatiotemporal resolution. Identifying the dynamic similarities and differences between 2D biochemical and quantum excitations poses a theoretically and practically relevant challenge, since optogenetic advances (23, 24) promise unprecedented control over cell signaling and hence biological computation. A particularly interesting open question in this context is whether fundamental quantum mechanical particle−particle interactions, symmetries (25), and scattering phenomena find counterparts in biological signaling processes. Our combined experimental and theoretical results below show that the self-braiding events of biochemical spiral wave cores on the cell membranes can exhibit virtual particle pair creation and annihilation and bosonic exchange symmetry, revealing profound parallels between defect dynamics and information transport in living and quantum matter.Driven by recent experimental progress (18, 22, 26–28), the exploration of topological defects in synthetic and natural active matter has become a rapidly expanding area of research (29–38). In living systems, energy conversion of ATP at the microscale leads to the emergence of complex biochemical and biophysical signaling patterns at the mesoscale and macroscale (22, 27, 39). Such nonequilibrium patterns often display rich topological textures and dynamics (32, 33, 40, 41), arising from the defects’ self-propulsion (29) and interactions (30, 31). Owing to their robustness and slow dynamics, topological excitations can act as stabilizers and organizers of active force generation (18), biological functions (19), and information flows. Recent work determined the topological entropy associated with the braiding of defects in active nematic liquid crystals (37). By contrast, the relation between spontaneous topological defect braiding and information loss in cell membrane signaling processes (22) has remained relatively unexplored.To investigate the braiding dynamics of biochemical spiral waves in living cells, we compared here experimental observations of Rho-GTP activation waves on starfish oocyte membranes (22) with predictions of a generic continuum theory (20). Rho-GTP is a highly conserved signaling protein pivotal in regulating cellular division (42) and mechanics (43) across a wide variety of eukaryotic species (44). Since the biological functions of Rho-GTP have been widely investigated previously (45), we focused here on the topological characterization of the biochemical signaling dynamics through braiding analysis of defect world lines and entropic information measures, to identify similarities and differences with wave propagation and particle scattering dynamics in quantum systems. Overcoming previous observational and algorithmic limitations, we achieved the spatiotemporal resolution required for dynamical analysis by combining in vivo imaging with spectral signal representation, quantitative mathematical modeling, and large-scale computational parameter estimations (Materials and Methods) (46).     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

NMDARs in granule cells contribute to parallel fiber–Purkinje cell synaptic plasticity and motor learning

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

The ability of an organism to adjust its behavior to environmental demands depends on its capacity to learn and execute coordinated movements. The cerebellum plays a central role in this process by optimizing motor programs through trial-and-error learning (1). Within the cerebellum, the synaptic output from granule cells (GCs) to Purkinje cells (PCs) shapes computational operations during basal motor function and serves as a substrate for motor learning (2). Several forms of motor learning depend on changes in the strength of the parallel fiber (PF), the axon of GCs, to the PC synapse (3, 4).In the mammalian forebrain, synaptic plasticity typically relies on postsynaptic N-methyl-D-aspartate receptor (NMDAR) activation, which alters AMPA receptor (AMPAR) turnover at the postsynaptic site (5). However, this may not extend to the cerebellar synapse between GCs and PCs, since no functional postsynaptic NMDARs have been identified in young or adult rodents (6, 7). Pharmacological approaches, however, have shown that both long-term depression (LTD) and long-term potentiation (LTP) induction depend on NMDAR activation at the PF-PC synapse in young rodents (8–12). Hence, the alternative mechanisms for NMDAR-dependent synaptic modulation may involve presynaptic NMDARs activation [(12–15); for review: refs. 16 and 17]. Indeed, cell-specific deletion of NMDARs in GCs abolishes LTP in young rodents (12). In addition to NMDARs, PF-PC synaptic plasticity also requires nitric-oxide (NO) signaling (18–20). As nitric-oxide synthase (NOS) is expressed in GCs, but not in PCs (21), the activation of presynaptic NMDARs might allow Ca²⁺ influx that activates NO synthesis, which in turn may act upon the PCs. However, in the mature cerebellum, the existence of presynaptic NMDARs on PFs and the role of NO in PF-PC plasticity remains a matter of debate. Previously, we have proposed that the activation of putatively presynaptic NMDARs in young rodents is necessary for inducing PF-PC synaptic plasticity without affecting transmitter release (8, 9, 11, 12). More recently, it has been shown that a subset of PFs express presynaptic NMDARs containing GluN2A subunits and that these receptors are functional (11, 12). Thus, in contrast to their role at other synapses, at least in young rodent, presynaptic NMDARs as part of the PF-PC synapses might act via the production of NO to induce postsynaptic plasticity, without altering neurotransmitter release (9, 11, 12, 18–22). However, a causal link between NMDARs activation in PFs, NO synthesis, and synaptic plasticity induction is still missing.In the cerebral cortex, the expression of presynaptic NMDARs is developmentally regulated (23, 24). However, little is known about the presence and function of presynaptic NMDARs in adult tissue. In the adult cerebellum, PCs only express postsynaptic NMDARs at their synapse with climbing fibers (CFs) (25). It has been proposed that the activation of these receptors could have heterosynaptic effects during PF-PC LTD. This mechanism would explain why LTD in adults depends on NMDARs. According to this model, presynaptic NMDARs would be a transient feature of developing tissue and not necessary for induction of synaptic plasticity and motor learning in adult animals (25).Here, we combine electron microscopy, two-photon calcium imaging, synaptic plasticity experiments, and behavioral measurements to show that presynaptic NMDARs are not developmentally regulated but are required for cerebellar motor learning in adults. We demonstrate that presynaptic NMDARs are present and functional in PFs of mature rodents. By specifically deleting the NMDAR subunit GluN1 either in the post- (PC) or the presynaptic cells (GCs), we demonstrate that NMDAR activation in GCs plays a key role in bidirectional synaptic plasticity and in vestibulo-ocular reflex (VOR) adaptation, an important paradigm for testing cerebellar motor learning (26–28). In contrast, NMDARs in PCs are neither involved in PF-PC synaptic plasticity nor required for cerebellar motor learning.     

Live imaging of remyelination in the adult mouse corpus callosum

Oligodendrocyte precursor cells (OPCs) retain the capacity to remyelinate axons in the corpus callosum (CC) upon demyelination. However, the dynamics of OPC activation, mode of cell division, migration, and differentiation on a single-cell level remain poorly understood due to the lack of longitudinal observations of individual cells within the injured brain. After inducing focal demyelination with lysophosphatidylcholin in the CC of adult mice, we used two-photon microscopy to follow for up to 2 mo OPCs and their differentiating progeny, genetically labeled through conditional recombination driven by the regulatory elements of the gene Achaete-scute homolog 1. OPCs underwent several rounds of symmetric and asymmetric cell divisions, producing a subset of daughter cells that differentiates into myelinating oligodendrocytes. While OPCs continue to proliferate, differentiation into myelinating oligodendrocytes declines with time, and death of OPC-derived daughter cells increases. Thus, chronic in vivo imaging delineates the cellular principles leading to remyelination in the adult brain, providing a framework for the development of strategies to enhance endogenous brain repair in acute and chronic demyelinating disease.

In the central nervous system (CNS), oligodendrocytes form myelin sheaths around axons, allowing for the rapid transduction of electrical impulses and providing metabolic support (1, 2). Myelin remodeling occurs throughout life, is regulated by neuronal activity, and has been associated with learning and circuit plasticity in mice and humans (3–10). However, a number of neurological diseases, multiple sclerosis (MS) among others, leads to impaired oligodendrocyte function and an eventual loss of myelin sheaths, causing demyelination (1, 11). Demyelination is associated with progressive axonal degeneration and neurological decline (2). Whereas substantial progress has been made over the last decade to reduce myelin loss in acute phases of demyelinating diseases such as MS, regenerative approaches to enhance remyelination remain scant (1, 12, 13). In rodents, remyelination is achieved via activation of oligodendrocyte precursor cells (OPCs) that are capable of generating new functional oligodendrocytes (1, 14–17). Snapshot-based genetic lineage tracing experiments showed that OPCs expand within areas of damage through a combination of migration and proliferation in response to demyelinating injury (1, 18). Recruited OPCs differentiate into oligodendrocytes and myelin sheaths are eventually restored within lesions. Remyelination also occurs in humans in the context of demyelinating diseases, in which differentiated oligodendrocytes possibly also contribute to myelin repair (4, 19, 20). However, myelin recovery in humans often remains incomplete. Thus, new therapeutic approaches to enhance myelin repair are needed (1).Understanding OPC dynamics on a single-cell level could facilitate the development of remyelination-promoting strategies with the aim to prevent disease progression or to ameliorate symptoms. Recently, OPC kinetics, oligodendrocyte differentiation, and myelin formation in the healthy and injured adult mouse brain have begun to be studied in superficial layers of the cortex using in vivo two-photon imaging (7, 9, 21–23). However, myelination and remyelination in gray and white matter follows different dynamics (24). The clonal behavior of OPCs during remyelination in gray and white matter structures remains largely unknown (1). For example, the mode of cell division, self-renewal potential, and successive rounds of division of individual OPCs is unclear. We here used in vivo two-photon imaging to analyze OPC behavior and subsequent remyelination in the corpus callosum (CC), a white matter area connecting the two brain hemispheres that is commonly affected in demyelinating disease (11). Combining lysophosphatidylcholin (LPC, also called lysolecithin)–induced demyelination and repeated in vivo imaging (25, 26), we characterize the cellular principles of OPC behavior upon demyelination, allowing for insights that may guide future strategies to enhance regenerative brain repair.     

Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H₂O₂-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.Exposure to ionizing irradiation (IR) has dire consequences for the cell, because it causes the formation of radicals and reactive oxygen species (ROS) that can oxidize and damage cellular components including proteins and DNA (1), whereas protection from IR-induced radical-mediated protein oxidation can lead to significant radio resistance (2). At the DNA level, IR leads to single-stranded and double-stranded DNA breaks (DSBs), with the latter being a severe threat to cellular survival (3). To cope with DSBs that may arise physiologically and/or by genotoxic environmental impacts such as IR, the cell repairs DSBs by two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), which predominate in the G1 and G2 phase of the cell cycle, respectively, and underlie cell-cycle-dependent sensitivities to IR exposure (4, 5). In the G2 phase and in the first meiotic prophase, DSB repair is mediated by HR, in yeast meiosis addressing ∼150 DSBs (6, 7) that are formed by the Topo2-related endonuclease/transesterase enzyme SPO11 (8). Absence of DSBs and the resulting compromised spore viability (9) can be partially rescued by ionizing irradiation (10). In all, yeast cells exhibit a high resistance to IR (11–13) which is also true for cells in prophase I (10, 14). In the meiosis of numerous species, programmed Spo11-induced DSBs are instrumental for homologous chromosome search and pairing and provide the substrate for HR, generating two outcomes: noncrossovers (NCO) and crossovers (CO) that allow for homolog segregation in the meiosis I division (reviewed by refs. 15 and 16). For CO to occur, homologous chromosomes need to encounter and pair lengthwise (synapse) during first meiotic prophase (see ref. 17). Homolog pairing occurs after completion of premeiotic DNA replication. Live cell studies in the synaptic meiosis of the yeast Saccharomyces cerevisiae (2n = 32) have shown that meiotic telomeres (18), chromosomes, and bivalents undergo a striking mobility throughout the entire prophase I (19–21), which contrasts with the relative immobility of pachytene bivalents in mammalian prophase I (22). It has been found that rapid and continuous telomere and chromosome movements in budding yeast meiocytes depend on actin polymerization (18–20) and an intact meiotic telomere complex (21, 23, 24). Besides a general mobility of chromosomes throughout prophase I, single bivalents are capable to rapidly move away and return to the motile chromosome mass, a behavior termed “maverick” formation (19) or rapid chromosome movements (20, 21).Exposure to ionizing radiation induces a plethora of physicochemical effects in the irradiated cells including DNA damage (1, 3). Extensive research addressing the adverse effects of IR exposure using yeast as a model system had largely been directed toward mutation induction, DSB repair, and cell cycle effects (e.g., 11–13, 25, 26). Meiotic yeast cells exposed to 50–80 krad (500–800 Gy) X or γ irradiation have been shown to exhibit a profound reduction in cell survival, particularly when exposed in the G1 cell cycle phase that lacks a sister chromatid for repair (4). Irradiated meiotic yeast cells exhibit mutations and chromosome missegregation at meiosis I, leading to reduced sporulation (5, 10, 27). While previous studies addressed late deterministic effects in irradiated yeast cells such as DNA repair, mutations, and cell survival, we were interested in the immediate consequences of IR exposure on motile meiotic chromosomes. Bivalent mobility can be expected to promote chromosomal rearrangements, if it continues after the formation of ectopic unregulated DSBs. Chromosomal translocations have, for instance, been observed after irradiation of mitotic budding yeast cells (28) and of meiotic prophase cells of mice (29). Furthermore, meiotic chromosome mobility has been proposed to be involved in regulating (adverse) chromosomal interactions (30). To study the consequences of IR exposure on meiotic chromosome mobility we followed live bivalent movements in X-irradiated and nonirradiated yeast cells expressing the GFP-tagged version of the synaptonemal complex protein ZIP1 (19) undergoing sporulation.     

Optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus controls locomotion in a mouse model of Parkinson’s disease

In Parkinson’s disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits, such as gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to the decreased activity in the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion. Clinicians are exploring the deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of PD, based on the bilateral striatal injection of 6-hydroxydopamine, which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that the limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in intact animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open avenues to develop the targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy, or optogenetics.

In Parkinson’s disease (PD), midbrain dopaminergic (DA) cells are lost, resulting in motor dysfunction, including severe locomotor deficits (e.g., gait freezing, akinesia, and falls) (1). Growing evidence indicates that part of these deficits can be attributed to changes in the mesencephalic locomotor region (MLR) (refs. 2–7; for review, see ref. 8). This brainstem region plays a key role in locomotor control by sending projections to reticulospinal neurons that carry the locomotor drive to the spinal cord in vertebrates (lamprey: refs. 9 and 10; salamander: refs. 11 and 12; and mouse: refs. 13 to 19; for review, see ref. 20). The DA neurons of the substantia nigra pars compacta (SNc) indirectly control MLR activity through the basal ganglia (15, 21, 22). In parallel, the MLR receives direct DA projections from the SNc (23–26) and from the zona incerta (27). The DA innervation of the MLR degenerates in a monkey model of PD (28). Therefore, the loss of DA cells in PD has major effects on MLR activity. In PD, locomotor deficits are associated with MLR cell loss, abnormal neural activity, altered connectivity, and metabolic deficits, likely resulting in a loss of amplification of the locomotor commands (for review, see ref. 8). Accordingly, motor arrests and gait freezing are associated with a decrease in MLR activity in PD (ref. 29; for review, see ref. 8).One approach to improve locomotor function in PD would be to increase MLR activity. L-DOPA, the gold standard drug used to improve motor symptoms in PD, increases MLR activity and this likely contributes to the locomotor benefits (30). The MLR has been proposed to contribute to the locomotor benefits of deep brain stimulation (DBS) of the subthalamic nucleus (31–33), which has direct and indirect projections to the MLR (refs. 34, 35; for review, see ref. 20). However, the benefits of L-DOPA and subthalamic DBS on locomotor deficits may wane over time, highlighting the need to find new therapeutic approaches (36, 37). Since 2005, the MLR has been explored as a DBS target (38). The results vary, from modest to promising (39, 40). However, the best target in the MLR in PD conditions is not yet identified. The MLR is a heterogeneous structure, with the cuneiform nucleus (CnF) controlling the largest range of locomotor speeds, and the pedunculopontine nucleus (PPN) controlling slow speeds, posture and in some cases locomotor arrests (refs. 15, 17–19; for review, see ref. 20). Human DBS protocols targeted the PPN, but left the CnF unexplored, despite its major importance in locomotor control in animal research (19, 41, 42). In humans, a recent anatomical analysis of DBS electrode position relative to the pontomesencephalic junction suggests that some of the beneficial effects attributed to the PPN could be due to CnF activation (43).To add further complexity, three main cell types are present in the MLR: glutamatergic, GABAergic, and cholinergic cells. It is still unknown which cell type is the best target to improve locomotor function in PD conditions. Optogenetic studies uncovered that glutamatergic cells in the CnF play a key role in generating the locomotor drive for a wide range of speeds (15, 17–19). Glutamatergic cells of the PPN control slower speeds (17, 18) and, in some cases, evoke locomotor arrests (18, 44, 45). The GABAergic cells in the CnF and PPN stop locomotion likely by inhibiting glutamatergic cells (15, 17). The role of the PPN cholinergic cells is not resolved, as their activation can increase or decrease locomotion (refs. 15, 17, 18; for review, see ref. 20). Clinically, DBS likely stimulates all cells around the electrode, including the GABAergic cells that stop locomotion, and this could contribute to the variability of outcomes.Here, we aimed at identifying a relevant target in the MLR to improve the locomotor function in parkinsonian conditions. We hypothesized that the selective activation of CnF glutamatergic neurons should improve locomotor function in a mouse model of PD. We induced parkinsonian conditions in mice by bilaterally injecting into the striatum the neurotoxin 6-hydroxydopamine (6-OHDA), which is well known to damage the nigrostriatal DA pathway and to induce a dramatic decrease in locomotor activity (e.g., refs. 22 and 46). Using in vivo optogenetics in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice), we show that the photostimulation of glutamatergic neurons in the CnF robustly initiated locomotion, reduced immobility, increased the time spent in locomotion, and precisely controlled locomotor speed. Our results should help in defining therapeutic strategies aimed at specifically activating CnF glutamatergic neurons to improve locomotor function in PD using optimized DBS protocols, pharmacotherapy, or future optogenetic tools for human use.     

CD81 costimulation skews CAR transduction toward naive T cells

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells.

Genetic manipulation of T cells has enabled adoptive T cell therapy to be translated to the clinic (1–10). Chimeric antigen receptor (CAR) therapy has evoked recent enthusiasm upon mediating curative outcomes in aggressive, refractory B cell malignancies (1–7, 11–15), leading to Food and Drug Administration approval (16–18). The process of ex vivo transduction and expansion of T cells to express CARs influences the phenotype, function, and ultimate fate of the final CAR T cell product (19–23). Preclinical data in animal models indicate that selecting specific T cell subsets for CAR transduction improves efficacy (21, 22, 24–26). Naive-derived T cells have been shown to exhibit greater replicative capacity, persistence, and antitumor function, compared with both effector- and memory-derived T cells (19, 20, 27). Naive CD4⁺ T cells, specifically, have a critical role in enhancing the cytotoxic effect of the CD8⁺ cooperating central memory cell subset (21). Furthermore, the translational CAR experience demonstrates that the presence of cells consistent with the naive and early memory phenotype in premanufactured T cell products correlates with successful clinical responses in both pediatric and adult B cell leukemia (28–30). Here, we explore if selective activation of naive T cells can result in skewing of transduction toward this specific T cell subset without the need for physical subset sorting.CAR constructs rely on intrinsic costimulatory signals, such as the intracellular domains of CD28 or 41BB, for efficacy (1–9). Here we focus on exogenous costimulatory signals necessary to induce proliferation and permit viral-mediated gene transfer. Prior to CAR transduction and antigen encounter, the majority of T cells are in a state of rest. Resting T cells mandate primary and costimulatory signals for activation (31, 32). CD28 is best known for its ability to costimulate T cells (33–38) and along with CD3 activation renders T cells susceptible to viral transduction (1, 39). CD81 is a member of the tetraspanin family that physically associates with CD4 and CD8 on the surface of T cells. CD81 was shown to have independent costimulatory properties and, when used with anti-CD3 and -CD28 antibodies, preferentially activates naive T cells as compared with effector and memory T cells, despite conserved surface CD81 expression across T cell subsets (40). Tetraspanins have no known cell-surface ligands, and therefore antibodies are used to engage and stimulate them. CD81 is the only tetraspanin whose complete three-dimensional structure has been solved (41). Moreover, the crystal structure of 5A6, the anti-CD81 antibody used in our study, in complex with CD81 has also been most recently solved (42). These authors demonstrate that engagement by this antibody changes the conformation of the large extracellular loop of the CD81 molecule. This conformational change may affect the interaction of CD81 with its associated CD4 and CD8 molecules.Here, we costimulate purified T cells with CD81 as a proof of principle to illustrate that the in vitro activation window prior to CAR transduction can be leveraged to favor transduction of a specific T cell subset.     

Kirigami-based metastructures with programmable multistability

Multistability plays an important role in advanced engineering applications such as metastructures, deployable structures, and reconfigurable robotics. However, most existing multistability design is based on the two-dimensional (2D)/3D series or parallel combinations of bistable unit cells, which are derived from snap-through instability, nonrigid foldable origami structures, and compliant mechanism, due to the lack of a generic multistable unit cell. Here, we develop a tristable kirigami cuboid by creating a set of elastic joints only effective in a specific motion range which integrates the elastic sheets and switchable hinge axes inspired by the kinematic behaviors of a kirigami cuboid with thick facets. The energy barriers between the stable states can be programmed by the geometric design parameters and material properties of the elastic joints. Taking the tristable cuboid as a unit cell, we construct a family of metastructures with multiple stable states. The number of stable states, the combination of unit stable states, and their transform sequences can be programmed by the number of unit cells, unit design parameters, and loading modes and loading sequences. We also apply this tristable cuboid to the design of frequency reconfigurable antenna with three programmable working frequencies, which demonstrates that such versatile multistability and structural diversity facilitate the development of multifunctional materials and devices.

Multistability is a characteristic of structures with more than one stable equilibrium configuration, which can realize the rapid structural reconfiguration to meet certain functional requirements. Recently, multistable structures have been used to design mechanical structural materials with shape reconfiguration (1–3) and negative stiffness (4) for trapping elastic strain energy (5), energy absorption (6–8), and ternary logic operation (9); robots (10–14) for simplifying actuators, reducing power consumption, and improving the locomotion speed and motion integration; soft media (15) and mechanical diodes (16, 17) for the propagation of mechanical signals; devices for mechanical memory storage (18); deployable structures for self-locked configuration (19, 20) and rapid deployment (21); and other potential applications (22).However, most existing multistability is based on the two-dimensional (2D)/3D series or parallel combinations of bistable unit cells, which are derived from snap-through instability (1, 8, 17, 23–27), nonrigid foldable origami structures (28–32), and compliant mechanisms including rigid origami (33–37). Among them, the snap-through instable beam or structure is the most commonly used fundamental unit in construction with planer motifs or spatial topologies to form 1D, 2D, and 3D multistable structures with unidirectional (1, 24), bidirectional, and multidirectional multistability (2, 4, 6, 9), such as the multistable 1D cylindrical structures, 2D square lattices, and 3D cubic/octahedral lattices (9). Recently, nonrigid origami structure is an emerging resource for designing bistable units based on the elastic deformation of origami facets, such as the Kresling pattern (11, 18, 20, 38) and the hypar pattern (30, 39). Multiple Kresling units can be assembled in series to construct multistable structures (18, 31), and multiple hypar-origami units can be tessellated in plane to be a multistable metasurface (30). Meanwhile, compliant mechanisms derived from mechanisms by introducing spring hinges with compliant segments (34, 36) or torsional springs (40) to store energy have been used to propose bistable unit cells, such as four-bar developable mechanisms (37), Sarrus linkages (41), twisting and rotational mechanisms (42, 43), rotating polygon embedded magnets (44–46), the waterbomb unit (34), and the Miura-ori unit (47, 48). The Miura-ori units have been stacked to be multilayer multistable structures (16, 33, 47).Besides few tristable units with nonzero energy stable states (32, 41), there is no generic tristable or multistable structure which itself is a basic unit rather than constructing with bistable units. On the other hand, most of the bistable unit cells are accompanied by large deformation on beams or facets, while few are derived from the design of joints. One such example is quadrastable overconstrained spatial Sarrus mechanisms with compliant joints (41), whose stable states are also nonzero energy ones, except the initial fabrication state. Therefore, in this paper, we are aiming to develop a generic tristable kirigami cuboid with a set of specially designed elastic joints based on its kinematic behaviors. By combining the tristable kirigami cuboid in series, multistable structures with programmable stable configurations, transformation sequence, and stiffness are constructed. This work paves the way to design multistable metastructures, which facilitates the development of functional materials and devices.