Iron robbery by intracellular pathogen via bacterial effector–induced ferritinophagy

Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3−induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia. Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

Ehrlichia spp., rickettsial obligatory intracellular bacteria cause tick-borne infectious diseases, which are greatly rising in worldwide prevalence (1, 2). Ehrlichia chaffeensis causes human monocytic ehrlichiosis, a severe flu-like illness accompanied by hematologic abnormalities and hepatitis, which can be fatal (2 to 5% mortality) (3–6). In humans, E. chaffeensis infects monocytes and macrophages and concocts unique membrane-bound compartments (inclusions). The inclusions have early endosome-like characteristics, including the presence of transferrin (Tf), transferrin receptor (TfR), and vacuolar-type H⁺-ATPase as well as the small GTPase RAB5 and its effectors, but the inclusions lack late endosomal or lysosomal markers or NADPH oxidase (7–9). Within the inclusions, Ehrlichia acquires all nutrients, including iron, for its reproduction to yield numerous mature infectious forms.Iron serves as a cofactor in many processes of bacteria and eukaryotes, including electron transfer, energy metabolism, oxygen transport, oxygen sensing, and DNA synthesis and repair (10). Ehrlichia is an obligate aerobe and is absolutely dependent on host iron for ATP synthesis via the electron transport chain, because its glycolytic pathway is incomplete and it lacks ATP−ADP translocase, unlike Rickettsia and Chlamydia (11). E. chaffeensis lacks the siderophore biosynthesis pathway and Fe³⁺ uptake regulator (11). Nonetheless, Ehrlichia acquires iron from the host cell labile cellular iron (LCI) pool, and pretreating human monocytes with the membrane-permeable iron chelator deferoxamine blocks E. chaffeensis infection (12). Ehrlichia enhances host cell iron uptake via up-regulating TfR messenger RNA (mRNA) (13) and acquires iron from the holoTf, as E. chaffeensis endosomes intersect with TfR-recycling endosomes and are slightly acidic—enough to release iron from holoTf (7). In fact, treatment of macrophages with interferon-γ down-regulates TfR mRNA and almost completely inhibits Ehrlichia infection, and addition of holoTf abrogates this inhibition (12). However, TfR mRNA levels return to basal level after 24 h postinfection when bacterial exponential growth begins (13), and treatment with interferon-γ can no longer inhibit infection at this point (12), suggesting that alternative or additional iron acquisition mechanisms exist to support exponential intracellular growth of Ehrlichia.The bacterial type IV secretion system (T4SS) translocates bacterial proteins and nucleoprotein complexes (called “effectors,” as they bring about responses) from bacteria to eukaryotic cells (14). Rickettsial organisms including E. chaffeensis have T4SS, sometimes referred to as T4aSS, similar to the virB/virD system of Agrobacterium tumefaciens (15, 16). The dot/icm system of Legionella pneumophila, sometimes referred to as T4bSS, secretes ∼300 effectors with redundant functions; hence, each effector can be knocked out, but the mutant lacks a phenotype (17). In contrast, the total number of T4aSS effectors is much lower [for example, fewer than six effectors exist in A. tumefaciens (15)], but each effector has a crucial role in infection/disease. To date, only a handful of T4SS effectors have been identified for rickettsial organisms, and even fewer have been functionally characterized (18–21). VirD4 is a well-established coupling protein involved in escorting translocated DNA and proteins in A. tumefaciens (15). By a bacterial two-hybrid screen using E. chaffeensis VirD4 as bait, we previously identified three Ehrlichia proteins that directly bind to Ehrlichia VirD4: ECH0825 (Ehrlichial translocated factor-1, Etf-1), ECH0261 (Etf-2), and ECH0767 (Etf-3) (22). All three were formerly annotated as hypothetical proteins, as they lack sequence similarity to previously known proteins or protein domains or motifs. Etf-1 has key roles in Ehrlichia infection of human cells by blocking host cell apoptosis and inducing RAB5-regulated autophagy for nutrient (amino acids) acquisition (9, 22, 23). Etf-2 is a unique RABGAP5 structural mimic that lacks GTPase-activating protein activity (24). Etf-2 directly binds RAB5-GTP on the Ehrlichia inclusion membrane and impedes the fusion of Ehrlichia-containing early endosomes with lysosomes (24). Whether Etf-3 is secreted or has any biological function is unknown. In the present study, we discovered that Etf-3 is a true T4SS effector that is secreted, binds tightly to ferritin, and induces ferritinophagy to provide free Fe²⁺ for intracellular Ehrlichia.     

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

DNA double-strand breaks (DSBs) are potentially lethal lesions that threaten genomic integrity and cell viability. DSBs can occur spontaneously as a result of faulty DNA metabolism or by exposure to genotoxins. In eukaryotes, these DSBs have “dirty ends” that lack ligatable 3ʹ-hydroxyl/5ʹ-phosphate groups and are often firmly attached to proteins such as the Ku70-80 heterodimer and topoisomerases (1, 2). During meiosis, the topoisomerase-like Spo11 protein generates DSBs and remains covalently attached to the 5ʹ DNA ends (3). To enable further processing, these DSB ends must be converted to “clean” ends with 3ʹ-hydroxyl/5ʹ-phosphate groups properly exposed. This step is achieved by endonucleolytic cleavage, or clipping, by Mre11 (4–7).In mammals, the MRE11, RAD50, and NBS1 complex (Mre11-Rad50-Nbs1 [MRN]), together with CtIP, is involved in the clipping reaction. MRE11 is the nuclease subunit that has both endonuclease and 3′-to-5′ exonuclease activities, but only the former is essential for clipping (4, 8–12). RAD50, a member of the Structural Maintenance of Chromosomes protein family, binds to MRE11 to form an (MRE11)₂-(RAD50)₂ ring structure (MR complex) (13–15). NBS1 binds to the MR complex via MRE11 to form the MRN complex (16). Homologs of CtIP include Ctp1 in Schizosaccharomyces pombe and Sae2 in Saccharomyces cerevisiae (17–19). Upon phosphorylation, these proteins physically interact with their cognate MRN complex via the N-terminal forkhead-associated domain of NBS1, leading to activation of the MRE11 endonucleolytic clipping activity (20–24). However, the mechanistic details underlying this activation have not yet been determined.Through biochemical reconstitution using fission yeast proteins, we made three key findings regarding how Ctp1 activates MRN. First, MRN activation is mediated by Ctp1 phosphorylation, which promotes the direct association of Ctp1 with the Nbs1 subunit of MRN. Second, the highly conserved extreme C terminus of Ctp1 retains the ability to promote the endonuclease activity of MRN. Strikingly, a synthetic polypeptide comprising the 15 amino acids from the extreme C terminus of Ctp1 was sufficient for the full activation of MRN. Third, we verified the evolutionary significance of these findings by demonstrating that the conserved C-terminal polypeptide of CtIP can also stimulate the endonuclease activity of human MRN. Together, our results strongly suggest that the Ctp1-promoted MRN activation mechanism consists of at least two fundamentally separable elements: phosphorylation-induced Ctp1-MRN association and activation of MRN by the C-terminal peptide of Ctp1. Thus, recruitment of the Ctp1 C terminus to MRN is likely pivotal in this activation mechanism.     

The feeding system of Tiktaalik roseae: an intermediate between suction feeding and biting

Changes to feeding structures are a fundamental component of the vertebrate transition from water to land. Classically, this event has been characterized as a shift from an aquatic, suction-based mode of prey capture involving cranial kinesis to a biting-based feeding system utilizing a rigid skull capable of capturing prey on land. Here we show that a key intermediate, Tiktaalik roseae, was capable of cranial kinesis despite significant restructuring of the skull to facilitate biting and snapping. Lateral sliding joints between the cheek and dermal skull roof, as well as independent mobility between the hyomandibula and palatoquadrate, enable the suspensorium of T. roseae to expand laterally in a manner similar to modern alligator gars and polypterids. This movement can expand the spiracular and opercular cavities during feeding and respiration, which would direct fluid through the feeding apparatus. Detailed analysis of the sutural morphology of T. roseae suggests that the ability to laterally expand the cheek and palate was maintained during the fish-to-tetrapod transition, implying that limited cranial kinesis was plesiomorphic to the earliest limbed vertebrates. Furthermore, recent kinematic studies of feeding in gars demonstrate that prey capture with lateral snapping can synergistically combine both biting and suction, rather than trading off one for the other. A “gar-like” stage in early tetrapod evolution might have been an important intermediate step in the evolution of terrestrial feeding systems by maintaining suction-generation capabilities while simultaneously elaborating a mechanism for biting-based prey capture.

Although suction feeding is a primary mode of prey capture among aquatic vertebrates (1), it is physically impractical on land due to the lower viscosity of air as compared to water (2–4). Terrestrial-feeding vertebrates must resort to other means, such as biting or tongue capture, to procure food (2). Naturally, researchers seeking to understand shifts in feeding strategies in tetrapodomorph vertebrates during the water-to-land transition have focused primarily on whether feeding systems in fossil forms showed adaptations for either suction or biting (2, 5). Generally, plesiomorphic “fish-like” morphology is interpreted as a means to create suction during the feeding cycle, and derived “tetrapod-like” morphology is interpreted as suggestive of biting (5–8). Suction feeding in fish is typically associated with jointed, kinetic skulls that allow for large volumetric expansion to draw in food (1, 9). In contrast, many lineages of modern tetrapods have consolidated skulls, such as mammals, crocodilians, and amphibians, thought to strengthen the skull for biting (9–12). While there is evidence for kinetic joints in the palate and skull roof of multiple early stem tetrapods (6, 13–16), it is uncertain if they represent plesiomorphic holdovers of limited fish-like cranial kinesis (13, 17, 18) or were independently derived mechanisms to improve biting capabilities on land (17, 19).A central challenge of paleontology has been to understand how, and when, transitions in the feeding system of early terrestrial vertebrates occurred. Late Devonian finned tetrapodomorphs, typified by Eusthenopteron foordi, have expansive, kinetic skulls with open sutures, robust gill covers, large hyomandibulae, tall palatal elements, and a jointed neurocranium all thought to be features that play a role in suction feeding (5, 9, 20). In contrast, the Late Devonian limbed tetrapodomorph Acanthostega gunnari has a flat skull, interdigitating sutures between the bones of the skull roof, absent gill covers, reduced hyomandibulae, horizontal palatal elements, and a consolidated neurocranium that are hypothesized to be derived adaptations for biting (5, 6, 21, 22). Analyses of tetrapodomorph lower jaws have produced equivocal results, noting few differences between presumed aquatic and terrestrial forms (7, 8). These results suggest that either a fish-like suction-based feeding mechanism was maintained well into the Carboniferous (7, 8, 23) or that a biting-based feeding mechanism had evolved in water prior to the origin of terrestrial tetrapods (24).To understand how feeding modes shifted among tetrapodomorphs and assess the origin of novel feeding mechanisms in the tetrapod lineage, we use high-resolution microcomputed tomography (µCT) to analyze multiple specimens of a well-preserved elpistostegalian-grade tetrapodomorph, Tiktaalik roseae, and compare the anatomy resolved from those µCT scans to features of other extinct tetrapodomorphs and extant fishes with analogous features. T. roseae is a tetrapodomorph from the Upper Devonian (Frasnian, ∼375 Mya) of Arctic Canada (Ellesmere Island, Nunavut Territory) (25, 26) that, according to most-recent phylogenies (27, 28), is representative of the outgroup of limbed vertebrates (tetrapods). Although plesiomorphic in lower jaw morphology (7, 8, 29), elpistostegalian-grade tetrapodomorphs (a group also including Panderichthys rhombolepis and Elpistostege watsoni) represent a period of rapid cranial evolution that could nevertheless suggest shifts in feeding strategies (5, 26, 30, 31). µCT was performed on four specimens of T. roseae from the Nunavut Fossil Vertebrate Collection (NUFV) consisting of three-dimensionally (3D) preserved palatal material in articulation with the cranium, as well as individual bones from multiple disarticulated specimens (SI Appendix, Table S1). Sutural cross-sections were compared with homologous sutures reported for E. foordi (5, 20) and A. gunnari (5, 21). Cranial joints were compared with possible modern analogs, alligator gar (Atractosteus spatula) (32–34) and ornate bichir (Polypterus ornatipinnis) (5, 32, 35), which were selected on the basis of convergent feeding morphologies with T. roseae. Finally, joints between the palate, hyomandibula, and braincase were modeled with the same kinematic range of motion as reported in A. spatula for comparison purposes (34).     

Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea, morbidity, and mortality in low- and middle-income countries (1), while enterohemorrhagic E. coli (EHEC) is a major cause of food poisoning in industrial countries (2). The ability of EPEC and EHEC to colonize the intestinal epithelium is encoded on a pathogenicity island named the locus of enterocyte effacement, which encodes gene regulators, the outer membrane adhesin intimin, structural components of a type III secretion system (T3SS), translocon components, chaperones, effectors, and an ATPase, which energizes protein translocation (3).T3SS is a common virulence factor among Gram-negative pathogens of humans, animals, and plants, including Salmonella enterica serovars, Shigella, Chlamydia, and Yersinia spp., Pseudomonas aeruginosa, and Pseudomonas syringae (4). The overall architecture of the multisubunit T3SS injectisome, which spans the entire cell envelope, is highly conserved among the different pathogens. It comprises several substructures, including a cytosolic C-ring and an ATPase complex (EscN in EPEC), a basal body consisting of a series of ring structures embedded in the bacterial inner and outer membranes (including a Secretin, EscC) and a periplasmic rod (EscI) which connects the inner membrane rings with a hollow extracellular needle projection (EscF). In EPEC, the EscF needle is 8–9 nm in diameter and 23 nm in length (5–7). In most pathogenic bacteria having a T3SS, the function of the needle is to connect the basal body to a translocation pore in the plasma membrane of the eukaryotic cell (3, 4).Electron microscopic observations of EPEC and EHEC have shown an ∼12-nm-diameter helical tube, made of the secreted translocator protein EspA (8–10), which serves as an extension to the needle, enclosing a central channel of ∼25 Å diameter (11, 12). Around 12 EspA filaments are elaborated on individual EPEC bacteria (8); when grown in vitro, EspA can vary in length and can reach 600 nm (7). Our current understanding is that the EPEC and EHEC EspA filaments evolved as an adaptation to their environment, where the needle alone would not be long enough to traverse the intestinal mucus layer. EspA filaments, like the needle, share similar helical symmetry parameters with flagellar structures and are elongated by addition of EspA subunits to the tip of the growing filament, the same mode of elongation that occurs in flagella filaments (13). Functionally, the filaments form a long flexible helical conduit which connects the tip of the needle with the translocation pore (made in EPEC by EspB and EspD), thus mediating effector translocation (3). Indeed, EspA has been shown to interact with both EspB and EspD (14, 15). In addition to their protein translocation activity, EspA filaments are important adhesins, mediating binding to both epithelial cells and edible leaves (16, 17). In the absence of EspA filaments, effectors can be secreted but not translocated; accordingly, the virulence of an espA deletion is highly attenuated in animal models (18). Moreover, EspA filaments are major antigens in vivo; antibodies against EspA were found in both human colostrum of mothers in Brazil and in serum from culture-positive patients infected with EHEC (19, 20). In animal models, IgG antibodies against EspA play a major role in clearing the pathogen (21).EspA alone is sufficient to form filamentous structures (22). Similarly to flagellar biosynthesis (23), EspA coiled-coil interactions between N- and C-terminal α-helical segments are required for assembly of the filament (24, 25). Monomeric EspA subunits are maintained in the cytosol via interactions with the chaperone CesAB (26), which has also been called CesA (22). CesAB is essential for stability of EspA within the bacterial cell prior to secretion. A cesAB deletion cannot secrete EspA or assemble EspA filaments (26). Crystallographic analysis of the CesAB–EspA complex at 2.8 Å resolution (22) showed that the EspA α-helices are also involved in extensive coiled-coil interactions with CesAB (22). Due to disorder in the parts of EspA not directly interacting with CesAB, only 72 of the 192 EspA residues were visualized in this complex. Importantly, the ATPase EscN selectively interacts with the CesAB–EspA complex; abrogation of this interaction attenuates EspA secretion and infection (27).Like flagella, EspA filaments show antigenic polymorphism, as EspA from different EPEC and EHEC clones show no immunological cross-reactivity (10). We have previously identified a surface-exposed hypervariable domain that contains the immunodominant EspA epitope (28). By exchanging the hypervariable domains of EspA(EPEC) and EspA(EHEC) we swapped the antigenic specificity of the EspA filaments (26). As with the Salmonella flagellin D3 domain (29), which is known to tolerate insertions of natural and artificial amino acid sequences (30), we were able to insert short peptides into the surface-exposed, hypervariable region of EspA (28).While EspA was first identified in 1996 (31), and the EspA filaments were first described in 1998 (8), low-resolution structures (at ∼15–25 Å resolution) were reported about 15 y ago (11, 12). The aim of this study was to obtain a high-resolution cryoelectron microscopy (cryo-EM) structure of the EPEC EspA filament.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

Structure,self-assembly,and properties of a truncated reflectin variant

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between the protein’s structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

Materials from naturally occurring and recombinant proteins are frequently employed for the study of fundamental biological processes and leveraged for applications in fields as diverse as electronics, optics, bioengineering, medicine, and fashion (1–13). Such broad utility is enabled by the numerous advantageous characteristics of protein-based materials, which include sequence modularity, controllable self-assembly, stimuli-responsiveness, straightforward processability, inherent biological compatibility, and customizable functionality (1–13). Within this context, unique structural proteins known as reflectins have recently attracted substantial attention because of their key roles in the fascinating color-changing capabilities of cephalopods, such as the squid shown in Fig. 1A, and have furthermore demonstrated their utility for unconventional biophotonic and bioelectronic technologies (11–40). For example, in vivo, Bragg stack-like ultrastructures from reflectin-based high refractive index lamellae (membrane-enclosed platelets) are responsible for the angle-dependent narrowband reflectance (iridescence) of squid iridophores, as shown in Fig. 1B (15–20). Analogously, folded membranes containing distributed reflectin-based particle arrangements within sheath cells lead to the mechanically actuated iridescence of squid chromatophore organs, as shown in Fig. 1C (15, 16, 21, 22). Moreover, in vitro, films processed from squid reflectins not only exhibit proton conductivities on par with some state-of-the-art artificial materials (23–27) but also support the growth of murine and human neural stem cells (28, 29). Additionally, morphologically variable coatings assembled from different reflectin isoforms can enable the functionality of chemically and electrically actuated color-changing devices, dynamic near-infrared camouflage platforms, and stimuli-responsive photonic architectures (27, 30–34). When considered together, these discoveries and demonstrations constitute compelling motivation for the continued exploration of reflectins as model biomaterials.Open in a separate windowFig. 1.(A) A camera image of a D. pealeii squid for which the skin contains light-reflecting cells called iridophores (bright spots) and pigmented organs called chromatophores (colored spots). Image credit: Roger T. Hanlon (photographer). (B) An illustration of an iridophore (Left), which shows internal Bragg stack-like ultrastructures from reflectin-based lamellae (i.e., membrane-enclosed platelets) (Inset). (C) An illustration of a chromatophore organ (Left), which shows arrangements of reflectin-based particles within the sheath cells (Inset). (D) The logo of the 28-residue-long N-terminal motif (RMN), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (E) The logo of the 28-residue-long internal motif (RMI), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (F) The logo of the 21-residue-long C-terminal motif (RMC), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (G) The amino acid sequence of full-length D. pealeii reflectin A1, which contains a single RMN motif (gray oval) and five RMI motifs (orange ovals). (H) An illustration of the selection of the prototypical truncated reflectin variant (denoted as RfA1TV) from full-length D. pealeii reflectin A1.Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures (30, 31, 35–39). For example, fibers drawn from full-length Euprymna scolopes reflectin 1a and films processed from truncated E. scolopes reflectin 1a were shown to possess secondary structural elements (i.e., α-helices or β-sheets) (30, 31). In addition, precipitated nanoparticles and drop-cast films from full-length Doryteuthis pealeii reflectin A1 have exhibited β-character, which was seemingly associated with their conserved motifs (35, 36). Moreover, nanoparticles assembled from both full-length and truncated Sepia officinalis reflectin 2 variants have demonstrated signatures consistent with β-sheet or α-helical secondary structure, albeit in the presence of surfactants (38). However, such studies were made exceedingly challenging by reflectins’ atypical primary sequences enriched in aromatic and charged residues, documented extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for poorly controlled aggregation (12, 14, 15, 30–32, 34–39). Consequently, the reported efforts have all suffered from multiple drawbacks, including the need for organic solvents or denaturants, the evaluation of only polydisperse or aggregated (rather than monomeric) proteins, a lack of consensus among different experimental techniques, inadequate resolution that precluded molecular-level insight, imperfect agreement between computational predictions and experimental observations, and/or the absence of conclusive correlations between structure and optical functionality. As such, there has emerged an exciting opportunity for investigating reflectins’ molecular structures, which remain poorly understood and the subject of some debate.Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a robust methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between its structural characteristics and optical properties. We first rationally select a prototypical reflectin variant expected to recapitulate the behavior of its parent protein by using a bioinformatics-guided approach. We next map the conformational and energetic landscape accessible to our selected protein by means of all-atom molecular dynamics (MD) simulations. We in turn produce our truncated reflectin variant with and without isotopic labeling, develop solution conditions that maintain the protein in a monomeric state, and characterize the variant’s size and shape with small-angle X-ray scattering (SAXS). We subsequently resolve our protein’s dynamic secondary and tertiary structures and evaluate its backbone conformational fluctuations with NMR spectroscopy. Finally, we demonstrate a straightforward mechanical agitation-based approach to controlling our truncated reflectin variant’s secondary structure, hierarchical self-assembly, and bulk refractive index distribution. Overall, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and appear poised to inform new directions across biochemistry, cellular biology, bioengineering, and optics.     

Photosynthesis tunes quantum-mechanical mixing of electronic and vibrational states to steer exciton energy transfer

Photosynthetic species evolved to protect their light-harvesting apparatus from photoxidative damage driven by intracellular redox conditions or environmental conditions. The Fenna–Matthews–Olson (FMO) pigment–protein complex from green sulfur bacteria exhibits redox-dependent quenching behavior partially due to two internal cysteine residues. Here, we show evidence that a photosynthetic complex exploits the quantum mechanics of vibronic mixing to activate an oxidative photoprotective mechanism. We use two-dimensional electronic spectroscopy (2DES) to capture energy transfer dynamics in wild-type and cysteine-deficient FMO mutant proteins under both reducing and oxidizing conditions. Under reducing conditions, we find equal energy transfer through the exciton 4–1 and 4–2-1 pathways because the exciton 4–1 energy gap is vibronically coupled with a bacteriochlorophyll-a vibrational mode. Under oxidizing conditions, however, the resonance of the exciton 4–1 energy gap is detuned from the vibrational mode, causing excitons to preferentially steer through the indirect 4–2-1 pathway to increase the likelihood of exciton quenching. We use a Redfield model to show that the complex achieves this effect by tuning the site III energy via the redox state of its internal cysteine residues. This result shows how pigment–protein complexes exploit the quantum mechanics of vibronic coupling to steer energy transfer.

Photosynthetic organisms convert solar photons into chemical energy by taking advantage of the quantum mechanical nature of their molecular systems and the chemistry of their environment (1–4). Antenna complexes, composed of one or more pigment–protein complexes, facilitate the first steps in the photosynthesis process: They absorb photons and determine which proportion of excitations to move to reaction centers, where charge separation occurs (4). In oxic environments, excitations can generate highly reactive singlet oxygen species. These pigment–protein complexes can quench excess excitations in these environments with molecular moieties such as quinones and cysteine residues (1, 5–7).The Fenna–Matthews–Olson (FMO) complex, a trimer of pigment–protein complexes found in the green sulfur bacterium Chlorobaculum tepidum (8), has emerged as a model system to study the photophysical properties of photosynthetic antenna complexes (9–19). Each subunit in the FMO complex contains eight bacteriochlorophyll-a site molecules (Protein Data Bank, ID code: 3ENI) that are coupled to form a basis of eight partially delocalized excited states called excitons (Fig. 1) (20–23). Previous experiments on FMO have observed the presence of long-lived coherences in nonlinear spectroscopic signals at both cryogenic and physiological temperatures (11, 13). The coherent signals are thought to arise from some combination of electronic (24–26), vibrational (16–18), and vibronic (27) coherences in the system (28–30). One previous study reported that the coherent signals in FMO remain unchanged upon mutagenesis of the protein, suggesting that the signals are ground state vibrational coherences (17). Others discuss the role of vibronic coupling, where electronic and nuclear degrees of freedom become coupled (29). Other dimeric model systems have demonstrated the regimes in which these vibronically coupled states produce coherent or incoherent transport and vibronic coherences (31–33). Recent spectroscopic data has suggested that vibronic coupling plays a role in driving efficient energy transfer through photosynthetic complexes (27, 31, 33, 34), but to date there is no direct experimental evidence suggesting that biological systems use vibronic coupling as part of their biological function.Open in a separate windowFig. 1.(Left) Numbered sites and sidechains of cysteines C353 and C49 in the FMO pigment–protein complex (PDB ID code: 3ENI) (20). (Right) Site densities for excitons 4, 2, and 1 in reducing conditions with the energy transfer branching ratios for the WT oxidized and reduced protein. The saturation of pigments in each exciton denotes the relative contribution number to the exciton. The C353 residue is located near excitons 4 and 2, which have most electron density along one side of the complex, and other redox-active residues such as the Trp/Tyr chain. C353 and C49 surround site III, which contains the majority of exciton 1 density. Excitons 2 and 4 are generally delocalized over sites IV, V, and VII.It has been shown that redox conditions affect excited state properties in pigment-protein complexes, yet little is known about the underlying microscopic mechanisms for these effects (1, 9). Many commonly studied light-harvesting complexes—including the FMO complex (20), light-harvesting complex 2 (LH2) (35), the PC645 phycobiliprotein (36), and the cyanobacterial antenna complex isiA (37)—contain redox-active cysteine residues in close proximity to their chromophores. As the natural low light environment of C. tepidum does not necessitate photoprotective responses to light quantity and quality, its primary photoprotective mechanism concerns its response to oxidative stress. C. tepidum is an obligate anaerobe, but the presence of many active anoxygenic genes such as sodB for superoxide dismutase and roo for rubredoxin oxygen oxidoreductase (38) suggests that it is frequently exposed to molecular oxygen (7, 39). Using time-resolved fluorescence measurements, Orf et al. demonstrated that two cysteine residues in the FMO complex, C49 and C353, quench excitons under oxidizing conditions (1), which could protect the excitation from generating reactive oxygen species (7, 40–42). In two-dimensional electronic spectroscopy (2DES) experiments, Allodi et al. showed that redox conditions in both the wild-type and C49A/C353A double-mutant proteins affect the ultrafast dynamics through the FMO complex (9, 43). The recent discovery that many proteins across the evolutionary landscape possess chains of tryptophan and tyrosine residues provides evidence that these redox-active residues may link the internal protein behavior with the chemistry of the surrounding environment (41, 43).In this paper, we present data showing that pigment–protein complexes tune the vibronic coupling of their chromophores and that the absence of this vibronic coupling activates an oxidative photoprotective mechanism. We use 2DES to show that a pair of cysteine residues in FMO, C49 and C353, can steer excitations toward quenching sites in oxic environments. The measured reaction rate constants demonstrate unusual nonmonotonic behavior. We then use a Redfield model to determine how the exciton energy transfer (EET) time constants arise from changing chlorophyll site energies and their system-bath couplings (44, 45). The analysis reveals that the cysteine residues tune the resonance between exciton 4–1 energy gap and an intramolecular chlorophyll vibration in reducing conditions to induce vibronic coupling and detune the resonance in oxidizing conditions. This redox-dependent modulation of the vibronic coupling steers excitations through different pathways in the complex to change the likelihood that they interact with exciton quenchers.     

Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.