Many Gram-negative bacteria utilize specialized secretion systems to inject proteins (effectors) directly into host cells. Little is known regarding how bacteria ensure that only small subsets of the thousands of proteins they encode are recognized as substrates of the secretion systems, limiting their identification through bioinformatic analyses. Many of these proteins require chaperones to direct their secretion. Here, using the newly described protein interaction platform assay, we demonstrate that type 3 secretion system class IB chaperones from one bacterium directly bind their own effectors as well as those from other species. In addition, we observe that expression of class IB homologs from seven species, including pathogens and endosymbionts, mediate the translocation of effectors from Shigella directly into host cells, demonstrating that class IB chaperones are often functionally interchangeable. Notably, class IB chaperones bind numerous effectors. However, as previously proposed, they are not promiscuous; rather they recognize a defined sequence that we designate the conserved chaperone-binding domain (CCBD) sequence [(LMIF)(1)XXX(IV)(5)XX(IV)(8)X(N)(10)]. This sequence is the first defined amino acid sequence to be identified for any interspecies bacterial secretion system, i.e., a system that delivers proteins directly into eukaryotic cells. This sequence provides a new means to identify substrates of type III secretion systems. Indeed, using a pattern search algorithm for the CCBD sequence, we have identified the first two probable effectors from an endosymbiont, Sodalis glossinidius.

Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.

Accurate control of the cell cycle is critical for development and tissue homeostasis, and requires precisely timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle in human cells. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and we identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase.

Faithful and timely repair of DNA double-strand breaks (DSBs) is fundamental for the maintenance of genomic integrity. Here, we demonstrate that the meiotic recombination co-factor MND1 facilitates the repair of DSBs in somatic cells. We show that MND1 localizes to DSBs, where it stimulates DNA repair through homologous recombination (HR). Importantly, MND1 is not involved in the response to replication-associated DSBs, implying that it is dispensable for HR-mediated repair of one-ended DSBs. Instead, we find that MND1 specifically plays a role in the response to two-ended DSBs that are induced by irradiation (IR) or various chemotherapeutic drugs. Surprisingly, we find that MND1 is specifically active in G2 phase, whereas it only marginally affects repair during S phase. MND1 localization to DSBs is dependent on resection of the DNA ends, and seemingly occurs through direct binding of MND1 to RAD51- coated ssDNA. Importantly, the lack of MND1-driven HR repair directly potentiates the toxicity of IR-induced damage, which could open new possibilities for therapeutic intervention, specifically in HR-proficient tumors.

Rare earth elements (REE) are essential ingredients in many modern technologies, yet their purification remains either environmentally harmful or economically unviable. Adsorption, or biosorption, of REE onto bacterial cell membranes offers a sustainable alternative to traditional solvent extraction methods. But in order for biosorption-based REE purification to compete economically, the capacity and specificity of biosorption sites must be enhanced. Although there have been some recent advances in characterizing the genetics of REE-biosorption, the variety and complexity of bacterial membrane surface sites make targeted genetic engineering difficult. Here, we propose using multiple rounds of in vivo random mutagenesis induced by the MP6 plasmid combined with plate-throughput REE-biosorption screening to improve a microbe's capacity and selectivity for biosorbing REE. We engineered a strain of Vibrio natriegens capable of biosorbing 210% more dysprosium compared to the wild-type and produced selectivity improvements of up to 50% between the lightest (lanthanum) and heaviest (lutetium) REE. We believe that mutations we observed in ABC transporters as well as a nonessential protein in the BAM outer membrane β-barrel protein insertion complex likely contribute to some─but almost certainly not all─of the biosorption changes we observed. Given the ease of finding significant biosorption mutants, these results highlight just how many genes likely contribute to biosorption as well as the power of random mutagenesis in identifying genes of interest and optimizing a biological system for a task.

Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance.

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

Activation of the DNA-damage response can lead to the induction of an arrest at various stages in the cell cycle. These arrests are reversible in nature, unless the damage is too excessive. Here we find that checkpoint reversibility is lost in cells that are in very late G2, but not yet fully committed to enter mitosis (antephase). We show that antephase cells exit the cell cycle and enter senescence at levels of DNA damage that induce a reversible arrest in early G2. We show that checkpoint reversibility critically depends on the presence of the APC/C inhibitor Emi1, which is degraded just before mitosis. Importantly, ablation of the cell cycle withdrawal mechanism in antephase promotes cell division in the presence of broken chromosomes. Thus, our data uncover a novel, but irreversible, DNA-damage response in antephase that is required to prevent the propagation of DNA damage during cell division.

Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency.

Rare earth elements (REE) are essential ingredients of sustainable energy technologies, but separation of individual REE is one of the hardest problems in chemistry today. Biosorption, where molecules adsorb to the surface of biological materials, offers a sustainable alternative to environmentally harmful solvent extractions currently used for separation of rare earth elements (REE). The REE-biosorption capability of some microorganisms allows for REE separations that, under specialized conditions, are already competitive with solvent extractions, suggesting that genetic engineering could allow it to leapfrog existing technologies. To identify targets for genomic improvement we screened 3,373 mutants from the whole genome knockout collection of the known REE-biosorbing microorganism Shewanella oneidensis MR-1. We found 130 genes that increased biosorption of the middle REE europium, and 112 that reduced it. We verified biosorption changes from the screen for a mixed solution of three REE (La, Eu, Yb) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in solution conditions with a range of ionic strengths and REE concentrations. We identified 18 gene ontologies and 13 gene operons that make up key systems that affect biosorption. We found, among other things, that disruptions of a key regulatory component of the arc system (hptA), which regulates cellular response to anoxic environments and polysaccharide biosynthesis related genes (wbpQ, wbnJ, SO_3183) consistently increase biosorption across all our solution conditions. Our largest total biosorption change comes from our SO_4685, a capsular polysaccharide (CPS) synthesis gene, disruption of which results in an up to 79% increase in biosorption; and nusA, a transcriptional termination/anti-termination protein, disruption of which results in an up to 35% decrease in biosorption. Knockouts of glnA, pyrD, and SO_3183 produce small but significant increases (≈ 1%) in relative biosorption affinity for ytterbium over lanthanum in multiple solution conditions tested, while many other genes we explored have more complex binding affinity changes. Modeling suggests that while these changes to lanthanide biosorption selectivity are small, they could already reduce the length of repeated enrichment process by up to 27%. This broad exploratory study begins to elucidate how genetics affect REE-biosorption by S. oneidensis, suggests new areas of investigation for better mechanistic understanding of the membrane chemistry involved in REE binding, and offer potential targets for improving biosorption and separation of REE by genetic engineering.

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

Constraining the volatile content of magmas is critical to our understanding of eruptive processes and their deep Earth cycling essential to planetary habitability [R. Dasgupta, M. M. Hirschmann, Earth Planet. Sci. Lett. 298, 1 (2010)]. Yet, much of the work thus far on magmatic volatiles has been dedicated to understanding their cycling through subduction zones. Further, studies of intraplate mafic volcanism have disproportionately focused on Hawaii [P. E. Wieser et al., Geochem. Geophys. Geosyst. 22, e2020GC009364 (2021)], making assessments of the overall role of intraplate volcanoes in the global volatile cycles a challenge. Additionally, while mafic volcanoes are the most common landform on Earth and the Solar System [C. A. Wood, J. Volcanol. Geotherm. Res. 7, 387-413 (1980)], they tend to be overlooked in favor of silicic volcanoes when it comes to their potential for explosivity. Here, we report primitive (olivine-hosted, with host Magnesium number - Mg# 78 to 88%) melt inclusion (MI) data from Fogo volcano, Cabo Verde, that suggest that oceanic intraplate silica-undersaturated explosive eruptions sample volatile-rich sources. Primitive MI (melt Mg# 70 to 71%) data suggest that these melts are oxidized (NiNiO to NiNiO+1) and very high in volatiles (up to 2 wt% CO2, 2.8 wt% H2O, 6,000 ppm S, 1,900 ppm F, and 1,100 ppm Cl) making Fogo a global endmember. Storage depths calculated from these high volatile contents also imply that magma storage at Fogo occurs at mantle depths (~20 to 30 km) and that these eruptions are fed from the mantle. Our results suggest that oceanic intraplate mafic eruptions are sustained from the mantle by high volatile concentrations inherited from their source and that deep CO2 exsolution (here up to ~800 MPa) drives their ascent and explosivity.

Recent geochemical evidence confirms the oxidized nature of arc magmas, but the underlying processes that regulate the redox state of the subarc mantle remain yet to be determined. We established a link between deep subduction-related fluids derived from dehydration of serpentinite ± altered oceanic crust (AOC) using B isotopes and B/Nb as fluid proxies, and the oxidized nature of arc magmas as indicated by Cu enrichment during magma evolution and V/Yb. Our results suggest that arc magmas derived from source regions influenced by a greater serpentinite (±AOC) fluid component record higher oxygen fugacity. The incorporation of this component into the subarc mantle is controlled by the subduction system’s thermodynamic conditions and geometry. Our results suggest that the redox state of the subarc mantle is not homogeneous globally: Primitive arc magmas associated with flat, warm subduction are less oxidized overall than those generated in steep, cold subduction zones.

The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized.

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.

DNA in micronuclei is likely to get damaged. When shattered DNA from micronuclei gets reincorporated into the primary nucleus, aberrant rearrangements can take place, a phenomenon referred to as chromothripsis. Here, we investigated how chromatids from micronuclei act in subsequent divisions and how this affects their fate. We observed that the majority of chromatids derived from micronuclei fail to establish a proper kinetochore in mitosis, which is associated with problems in chromosome alignment, segregation and spindle assembly checkpoint activation. Remarkably, we found that, upon their formation, micronuclei already display decreased levels of important kinetochore assembly factors. Importantly, these defects favour the exclusion of the micronucleus over the reintegration into the primary nucleus over several divisions. Interestingly, the defects observed in micronuclei are likely overcome once micronuclei are reincorporated into the primary nuclei, as they further propagate normally. We conclude that the formation of a separate small nuclear entity represents a mechanism for the cell to delay the stable propagation of excess chromosome(s) and/or damaged DNA, by inducing kinetochore defects.

Activation of the DNA-damage checkpoint culminates in the inhibition of cyclin-dependent kinase (Cdk) complexes to prevent cell-cycle progression. We have shown recently that Cdk activity is required for activation of the Forkhead transcription factor FoxM1, an important regulator of gene expression in the G2 phase of the cell cycle. Here, we show that FoxM1 is transcriptionally active during a DNA-damage-induced G2 arrest and is essential for checkpoint recovery. Paradoxically, Cdk activity, although reduced after checkpoint activation, is required to maintain FoxM1-dependent transcription during the arrest and for expression of pro-mitotic targets such as cyclin A, cyclin B and Plk1. Indeed, we find that cells need to retain sufficient levels of Cdk activity during the DNA-damage response to maintain cellular competence to recover from a DNA-damaging insult.

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Substantial accumulation of single-stranded DNA during DNA replication activates the DNA replication stress checkpoint response that slows progression from S/G2 to M phase to protect genomic integrity. Whether and how mild replication stress restricts proliferation remains controversial. Here, we identify a cell cycle exit mechanism that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the anaphase promoting complex/cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead box O (FOXO)-mediated inhibition of its transcription factor E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible and thereby play an important role in maintaining genomic stability.

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.