Abscission is the final step of cell division when the cytokinetic furrow ingresses completely, leading to midbody formation and plasma membrane fission [1]. In human cells, the Aurora-B-driven abscission checkpoint delays cytokinesis until any residual chromatin spanning the midbody is removed [2-5]. If this does not occur efficiently, uneven segregation of daughter genomes can occur. The mechanism by which the abscission checkpoint becomes satisfied to permit cytokinesis is poorly defined. Here, we identify RIF1 and its binding partner, protein phosphatase 1 (PP1), as being critical for regulation of abscission timing in human cells. We show that RIF1 promotes cytokinesis through recruitment of PP1 to the midbody, which then counteracts Aurora B kinase activity, leading to dephosphorylation of a regulator of abscission timing, CHMP4C [6-10]. Although RIF1 binds to unresolved DNA bridges that persist into telophase [11], we show that this cytokinetic function of the RIF1-PP1 axis is not limited to instances where cell division is perturbed by the presence of bridges. Nevertheless, we show that altering the balance of the opposing Aurora B kinase and PP1 phosphatase activities makes cells unresponsive to DNA bridges and sensitizes cells to agents that induce bridge formation. Our data define a new mechanism for regulation of abscission timing and emphasize how antagonism between kinases and phosphatases is a widespread mechanism for determining the timing of mitotic transactions. Because cancer cells experiencing oncogene-induced replication stress generate excessive mitotic DNA bridging [12], targeting this new regulatory pathway could be a promising therapeutic strategy.

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and shows a broad clinical presentation ranging from asymptomatic infection to fatal disease. A very prominent feature associated with severe COVID-19 is T cell lymphopenia. However, homeostatic and functional properties of T cells are ill-defined in COVID-19.

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

Antibodies reactive to human leukocyte antigens (HLA) represent a barrier for patients awaiting transplantation. Based on reactivity patterns in single-antigen bead (SAB) assays, various epitope matching algorithms have been proposed to improve transplant outcomes. However, some antibody reactivities cannot be explained by amino acid motifs, leading to uncertainty about their clinical relevance. Antibodies against the HLA class II molecule, DQβ0603:DQα0103, present in some candidates, represent one such example. Here, we show that peptides derived from amino acids 119-148 of the HLA class I heavy chain are bound to DQβ0603:DQα0103 proteins and contribute to antibody reactivity through an HLA-DM-dependent process. Moreover, antibody reactivity is impacted by the specific amino acid sequence presented. In summary, we demonstrate that polymorphic HLA class I peptides, bound to HLA class II proteins, can directly or indirectly be part of the antibody binding epitope. Our findings have potential important implications for the field of transplant immunology and for our understanding of adaptive immunity.

The spindle assembly checkpoint (SAC) ensures proper chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Checkpoint signalling requires the kinetochore localization of the Mad1-Mad2 complex that in more complex eukaryotes depends on the Rod-Zwilch-ZW10 (RZZ) complex. The kinetochore protein Zwint has been proposed to be the kinetochore receptor for RZZ, but here we show that Bub1 and not Zwint is required for RZZ recruitment. We find that the middle region of Bub1 encompassing a domain essential for SAC signalling contributes to RZZ localization. In addition, we show that a distinct region in Bub1 mediates kinetochore localization of BubR1 through direct binding, but surprisingly removal of this region increases checkpoint strength. Our work thus uncovers how Bub1 coordinates checkpoint signalling by distinct domains for RZZ and BubR1 recruitment and suggests that Bub1 localizes antagonistic checkpoint activities.

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

Vaccines against SARS-CoV-2 have been rapidly approved. Although pivotal studies were conducted in healthy volunteers, little information is available on the safety and efficacy of mRNA vaccines in immunocompromised patients, including recipients of allogeneic hematopoietic cell transplantation (allo-HCT). Here we used a novel assay to analyze patient- and transplantation-related factors and their influence on immune responses to SARS-CoV-2 vaccination over an extended period (up to 6 months) in a large and homogenous group of allo-HCT recipients at a single center in Switzerland. We examined longitudinal antibody responses to SARS-CoV-2 vaccination with BNT162b2 (BioNTech/Pfizer) and mRNA-1273 (Moderna) in 110 allo-HCT recipients and 86 healthy controls. Seroprofiling recording IgG, IgA, and IgM reactivity against SARS-CoV-2 antigens (receptor-binding domain, spike glycoprotein subunits S1 and S2, and nucleocapsid protein) was performed before vaccination, before the second dose, and at 1, 3, and 6 months after the second dose. Patients were stratified to 3 groups: 3 to 6 months post-allo-HCT, 6 to 12 months post-allo-HCT, and >12 months post-allo-HCT. Patients in the 3 to 6 months and 6 to 12 months post-allo-HCT groups developed significantly lower antibody titers after vaccination compared with patients in the >12 months post-allo-HCT group and healthy controls (P < .001). Within the cohort of allo-HCT recipients, patients age >65 years (P = .030), those receiving immunosuppression for prevention or treatment of graft-versus-host disease (GVHD) (P = .033), and patients with relapsed disease (P = .014) displayed low humoral immune responses to the vaccine. In contrast, the intensity of the conditioning regimen, underlying disease (myeloid/lymphoid/other), and presence of chronic GVHD had no impact on antibody levels. Antibody titers achieved the highest levels at 1 month after the second dose of the vaccine but waned substantially in all transplantation groups and healthy controls over time. This analysis of long-term vaccine antibody response is of critical importance to allo-HCT recipients and transplant physicians to guide treatment decisions regarding revaccination and social behavior during the SARS-CoV-2 pandemic.

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing COVID-19 pandemic, a key question has focused on which SARS-CoV-2-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Here, using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor sequencing, we longitudinally characterized individual SARS-CoV-2-specific CD8+ T cells of patients with COVID-19 from acute infection to 1 year into recovery and found a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting 1 year after acute infection express CD45RA, IL-7 receptor-α and T cell factor 1, but they maintain low expression of CCR7, thus resembling CD45RA+ effector memory T cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones that give rise to long-lived cells, whereas prolonged proliferation and mechanistic target of rapamycin signalling are associated with clonal disappearance from the blood. Collectively, we describe a transcriptional signature that marks long-lived, circulating human memory CD8+ T cells following an acute viral infection.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

Transplantation of solid organs can be life-saving in patients with end-stage organ failure, however, graft rejection remains a major challenge. In this study, by pre-conditioning with interleukin-2 (IL-2)/anti-IL-2 antibody complex treatment biased toward IL-2 receptor α, we achieved acceptance of fully mismatched orthotopic lung allografts that remained morphologically and functionally intact for more than 90 days in immunocompetent mice. These allografts are tolerated by the actions of forkhead box p3 (Foxp3)+ regulatory T (Treg) cells that home to the lung allografts. Although counts of circulating Treg cells rapidly return to baseline following cessation of IL-2 treatment, Foxp3+ Treg cells persist in peribronchial and peribronchiolar areas of the grafted lungs, forming organized clusters reminiscent of inducible tertiary lymphoid structures (iTLS). These iTLS in lung allografts are made of Foxp3+ Treg cells, conventional T cells, and B cells, as evidenced by using microscopy-based distribution and neighborhood analyses. Foxp3-transgenic mice with inducible and selective deletion of Foxp3+ cells are unable to form iTLS in lung allografts, and these mice acutely reject lung allografts. Collectively, we report that short-term, high-intensity and biased IL-2 pre-conditioning facilitates acceptance of vascularized and ventilated lung allografts without the need of immunosuppression, by inducing Foxp3-controlled iTLS formation within allografts.

Phosphorylation is a ubiquitous post-translation modification that regulates protein function by promoting, inhibiting or modulating protein-protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide-phage display library to screen for phosphosites that modulate short linear motif-based interactions. The peptidome covers ~13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild-type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif-mediated interactions. Affinity measurements confirmed the phospho-modulation of 14 out of 18 tested interactions. We performed a detailed follow-up on a phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP), demonstrating the essentiality of the phospho-dependency to the mitotic function of HURP. Structural characterisation of the clathrin-HURP complex elucidated the molecular basis for the phospho-dependency. Our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated interactions required for cellular function.

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modelling with comprehensive high resolution mutational scanning, we show that α-helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α-helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting complex by binding to an α-helical recruitment module in RBM7. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 in health and disease.

During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.

The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes-UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C.

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis.

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel, SUMOylation of so-far elusive upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response. We show that HERC2 and RNF168 are novel DNA damage-dependent SUMOylation targets in human cells. In response to DSBs, both HERC2 and RNF168 were specifically modified with SUMO1 at DSB sites in a manner dependent on the SUMO E3 ligase PIAS4. SUMOylation of HERC2 was required for its DSB-induced association with RNF8 and for stabilizing the RNF8-Ubc13 complex. We also demonstrate that the ZZ Zinc finger in HERC2 defined a novel SUMO-specific binding module, which together with its concomitant SUMOylation and T4827 phosphorylation promoted binding to RNF8. Our findings provide novel insight into the regulatory complexity of how ubiquitylation and SUMOylation cooperate to orchestrate protein interactions with DSB repair foci.

Several autoimmune features occur during coronavirus disease 2019 (COVID-19), with possible implications for disease course, immunity, and autoimmune pathology. In this study, we longitudinally screened for clinically relevant systemic autoantibodies to assess their prevalence, temporal trajectory, and association with immunity, comorbidities, and severity of COVID-19.