Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes

Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs fold and tether DNA, while Smc5/6 directly promotes DNA replication and repair. The functions of SMCs rely on their abilities to engage DNA, but how Smc5/6 binds and translocates on DNA remains largely unknown. Here, we present a 3.8 Å cryogenic electron microscopy (cryo-EM) structure of DNA-bound Saccharomyces cerevisiae Smc5/6 complex containing five of its core subunits, including Smc5, Smc6, and the Nse1-3-4 subcomplex. Intricate interactions among these subunits support the formation of a clamp that encircles the DNA double helix. The positively charged inner surface of the clamp contacts DNA in a nonsequence-specific manner involving numerous DNA binding residues from four subunits. The DNA duplex is held up by Smc5 and 6 head regions and positioned between their coiled-coil arm regions, reflecting an engaged-head and open-arm configuration. The Nse3 subunit secures the DNA from above, while the hook-shaped Nse4 kleisin forms a scaffold connecting DNA and all other subunits. The Smc5/6 DNA clamp shares similarities with DNA-clamps formed by other SMCs but also exhibits differences that reflect its unique functions. Mapping cross-linking mass spectrometry data derived from DNA-free Smc5/6 to the DNA-bound Smc5/6 structure identifies multi-subunit conformational changes that enable DNA capture. Finally, mutational data from cells reveal distinct DNA binding contributions from each subunit to Smc5/6 chromatin association and cell fitness. In summary, our integrative study illuminates how a unique SMC complex engages DNA in supporting genome regulation.

Structural maintenance of chromosomes (SMC) complexes are essential genome regulators in both prokaryotes and eukaryotes. In eukaryotes, the cohesin and condensin SMC complexes organize DNA, while the Smc5/6 complex (referred to as Smc5/6) directly regulates DNA replication and repair (1). At the structural level, SMC complexes share similarities while possessing unique attributes (1). Each complex contains a pair of SMC subunits and a set of non-SMC subunits. The SMC subunits define the tripartite filamentous architecture of the complex: their approximal 50-nm long coiled coil arm region connects their dimerized hinge and adenosine triphosphatase (ATPase) head regions (1). A non-SMC kleisin subunit uses its N- and C-terminal domains to link the head of one SMC to the head-proximal arm region (neck) of another SMC, forming a trimeric SMC-kleisin structure. In cohesin and condensin, two large U-shaped HEAT (Huntington, elongation factor 3, PR65/A, TOR) repeat HAWK (HEAT proteins associated with kleisins) subunits attach to the middle region of the kleisin. By contrast, the Smc5/6 kleisin (Nse4) binds to smaller WH (winged helix)-containing KITE (kleisin interacting tandem WH elements) subunits (Nse1 and Nse3) (2).SMC-mediated functions depend on interactions with DNA. Recent cryogenic electron microscopy (cryo-EM) structures of DNA-bound cohesin and condensin revealed that their HAWK subunits and the SMC head-neck regions form a clamp to enclose a single DNA double helix (3–7). DNA clamping can be critical for cohesin and condensin to extrude DNA loops for chromatin folding (5, 7–9). DNA loop extrusion additionally requires arm bending at a region called the elbow, which is found in both cohesin and condensin (5, 7–9). By contrast, a lack of arm bending in Smc5/6 was suggested by negative stain EM and cross-linking mass spectrometry (CLMS) data (10–14). Since Smc5/6 does not contain HAWK proteins nor shows arm-bending, it has remained unclear how Smc5/6 engages DNA to accomplish its multiple functions.Here we address the molecular mechanisms by which this unique SMC complex binds DNA using an integrative approach, coupling a cryo-EM-based structural characterization with CLMS analyses and functional investigation. Our atomic structure of a DNA-bound Saccharomyces cerevisiae Smc5/6 complex reveals that the head-neck Smc5-6 regions and the Nse1-3-4 subcomplex together form a clamp entrapping the DNA helix. The structure further reveals protein subunit folds and association, as well as how the subunits collaborate to entrap DNA. Comparison of CLMS analyses of DNA-free Smc5/6 with the structure of the DNA-bound Smc5/6 unveils large scale, multi-subunit conformational changes that enable Smc5/6 to encircle DNA. Finally, our mutational data suggest distinct contributions from each of the DNA binding regions to Smc5/6 chromatin association and cellular fitness. Comparison of our findings with those of other SMCs reveals that diverse SMC complexes use a similar DNA clamping strategy despite structural differences, and that Smc5/6 possesses unique features distinct from cohesin, condensin, and prokaryotic SMCs. Our work lays the foundation for an in-depth understanding of how Smc5/6 fulfills unique roles in genome protection.     

Clamping of DNA shuts the condensin neck gate

Condensin is a structural maintenance of chromosomes (SMC) complex needed for the compaction of DNA into chromatids during mitosis. Lengthwise DNA compaction by condensin is facilitated by ATPase-driven loop extrusion, a process that is believed to be the fundamental activity of most, if not all, SMC complexes. In order to obtain molecular insights, we obtained electron cryomicroscopy structures of yeast condensin in the presence of a slowly hydrolyzable ATP analog and linear as well as circular DNAs. The DNAs were shown to be “clamped” between the engaged heterodimeric SMC ATPase heads and the Ycs4 subunit, in a manner similar to previously reported DNA-bound SMC complex structures. Ycg1, the other non-SMC subunit, was only flexibly bound to the complex, while also binding DNA tightly and often remaining at a distance from the head module. In the clamped state, the DNA is encircled by the kleisin Brn1 and the two engaged head domains of Smc2 and Smc4. The Brn1/Smc2/Smc4 tripartite ring is closed at all interfaces, including at the neck of Smc2. We show that the neck gate opens upon head engagement in the absence of DNA, but it remains shut when DNA is present. Our work demonstrates that condensin and other SMC complexes go through similar conformations of the head modules during their ATPase cycle. In contrast, the behavior of the Ycg1 subunit in the condensin complex might indicate differences in the implementation of the extrusion reactions, and our findings will constrain further mechanistic models of loop extrusion by SMC complexes.

Structural maintenance of chromosomes (SMC) complexes are drivers of chromosome dynamics in all domains of life. In eukaryotes, condensin organizes DNA into rod-shaped chromatids during mitosis, cohesin mediates sister chromatid cohesion and interphase chromosomal organization, and Smc5/6 is involved in DNA repair. In bacteria, SMC-ScpAB and MukBEF promote chromosome segregation by individualizing replicated chromosomes (1).Despite these divergent functions, SMC complexes share a common architecture, making it likely that they also function through common mechanisms. Several SMC complexes have been shown to enlarge DNA loops by a process known as loop extrusion, which is powered by their ATP binding cassette (ABC)-type ATPases (2). While loop extrusion by yeast condensin and human cohesin has been reconstituted in vitro (3–5), how they convert the energy from ATP binding and hydrolysis into movement along DNA is not clear.The core SMC complex is a heterotrimeric ring consisting of two SMC proteins and a kleisin, and in condensin these are Smc2, Smc4, and Brn1, respectively (Fig. 1A) (6). SMC proteins are highly elongated when fully extended, with a globular “hinge” domain at one apex and an ABC-type ATPase “head” domain at the other, separated by 50-nm antiparallel coiled-coil domains. The two SMC proteins, Smc2 and Smc4, come together stably via the hinge domains, forming heterodimers. The Brn1 N-terminal domain binds to the head proximal coiled coil of Smc2, the “neck,” and the C-terminal domain to the Smc4 head at a site called the “cap,” thereby creating a closed tripartite ring. To complete condensin, two HEAT repeat-containing proteins Associated With Kleisins (HAWKs), Ycs4 and Ycg1, stably bind to central regions of Brn1.Open in a separate windowFig. 1.(A) Architecture of the yeast condensin SMC complex. Adding DNA and an ATP analog leads to the formation of the clamped state, which is the subject of this study. The dotted rectangle marks the portion of the head module in the clamped state, of which the cryo-EM structures were solved. (B) Two-dimensional class averages of cryo-EM images of the condensin tetramer, containing subunits Smc2, Smc4, kleisin Brn1, and Ycs4. The dsDNA is clearly visible, whereas the coiled-coil arms of Smc2 and Smc4 and the hinge domains are highly flexible with respect to the well-resolved head module. (C) A 2.95-Å resolution cryo-EM map of the clamped condensin head module bound to DNA (Form I). (D) Cartoon representation of the atomic model built into and refined against the map shown in C. Various parts of the kleisin Brn1 are disordered.Previous electron cryomicroscopy (cryo-EM) structures of condensin showed that condensin switches between at least two states (7). In the absence of ATP, Ycs4 bound to the head domains of Smc2 and Smc4, while in contrast Ycg1 was mobile, presumably connected to the complex by a flexible region within Brn1. In the ATP-engaged structure, Ycs4 unbound and was mobile, whereas Ycg1 bound to the heads instead. Notably, the neck interface between Smc2 and Brn1, where the N-terminal portion of Brn1 binds to the coiled-coil neck of Smc2 near the ATPase head, is closed in the absence of ATP but was not resolved in the ATP-bound structure. Biochemical experiments demonstrated that opening of the neck interface between Smc2 and Brn1 is driven by ATP binding (8). A similar behavior was reported for cohesin, whose dissociation from DNA requires opening of the Smc3–kleisin (Scc1) neck interface, which depends on an accessory protein called Wapl and also ATP binding (9, 10).In vivo cysteine cross-linking analyses of cohesin (11, 12), SMC-ScpAB (13, 14), and MukBEF (15) have been used to reveal topological compartments in SMC complexes in which circular DNA can be entrapped (SI Appendix, Fig. S1A). These include the SMC-Kleisin (S-K) compartment and, when the head domains are engaged, the Engaged heads-Kleisin (E-K) and the Engaged heads-SMC (E-S) compartments. Sister chromatid cohesion is mediated by the coentrapment of sister chromatids within cohesin’s S-K compartment. In contrast, it is not currently clear in which compartment the DNAs reside during loop extrusion, or if in any (3, 16).Cryo-EM structures of cohesin in the ATP-bound engaged (E) state, and bound to one strand of DNA, revealed that DNA is “clamped” on top of the dimerized SMC ATPase domains by Scc2 and the closed neck of Smc3 (17–19). Analysis of the same cohesin–DNA interaction using in vitro cysteine cross-linking revealed that the DNA is not entrapped within the S-K ring but threads through both the E-S and E-K compartments (17). This strongly suggests that in the clamped state of cohesin the kleisin runs over the top of the DNA and that the DNA has not passed through one of the trimer interfaces to reach S-K. Furthermore, a recent structure of MukBEF in the ATP and DNA-bound state conclusively shows that the clamped DNA passes through the E-K and E-S compartments because the entire kleisin MukF is resolved (15). Interestingly, in this structure, the ν-SMC-kleisin neck interface is not completely closed; however, the MatP DNA-unloading protein was also bound.We aimed to investigate how condensin interacts with DNA in the ATP engaged state and how the DNA interaction might regulate the state of the neck interface. To address this, we started by using cryo-EM to solve the structure of condensin in the presence of DNA, ADP, and BeF₃.     

Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity

Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of some metazoan genes require the association of cohesins with chromosomes. Cohesins are deposited by a conserved heterodimeric loading complex composed of the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4 deposition complex regulates the spatiotemporal association of cohesin with chromosomes is not understood. We examined Scc2 chromatin association during the cell division cycle and found that the affinity of Scc2 for chromatin increases biphasically during the cell cycle, increasing first transiently in late G₁ phase and then again later in G₂/M. Inactivation of Scc2 following DNA replication reduces cellular viability, suggesting that this post S-phase increase in Scc2 chromatin binding affinity is biologically relevant. Interestingly, high and low Scc2 chromatin binding levels correlate strongly with the presence of full-length or amino-terminally cleaved forms of Scc2, respectively, and the appearance of the cleaved Scc2 species is promoted in vitro either by treatment with specific cell cycle-staged cellular extracts or by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2–Scc4 physical interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is defective for cohesin deposition. These observations suggest a previously unidentified mechanism for the spatiotemporal regulation of cohesin association with chromosomes through cell cycle regulation of Scc2 cohesin deposition activity by Scc2 dephosphorylation and cleavage.Multisubunit, ring-shaped cohesin complexes play key roles in chromosome morphogenesis that are required for faithful chromosome transmission to daughter cells. Newly replicated sister chromatids become tethered together by cohesins during S phase, which promotes chromosome biorientation on mitotic spindles (1). Cohesins also mediate efficient DNA double-strand break repair by homologous recombination (2, 3) and the formation or stabilization of chromatin loops that affect various nuclear processes, such as gene expression and Ig gene rearrangements (reviewed in refs. 4 and 5). Altered gene expression resulting from defective cohesin-mediated chromatin looping is likely responsible for the pathogenesis of Cornelia de Lange Syndrome (CdLS), a dominantly inherited human developmental disorder (6).Sister chromatid cohesion (Scc) proteins form a heterodimeric cohesin deposition complex, but the complex''s activity in deposition is not understood (7). Cohesins copurify with Scc2/Scc4, suggesting that Scc2/Scc4 plays a direct role in deposition (8–11). In the absence of either loader complex subunit, cohesin rings assemble, but fail to be deposited (7, 12, 13). ATP hydrolysis by cohesin’s structural maintenance of chromosome (SMC) subunits is required for cohesin loading, and deposition is inhibited when SMC hinge domains, which mediate Smc1/3 interactions within cohesin, are artificially tethered (8, 14, 15). Thus, Scc2/Scc4 may activate cohesin’s ATPase activity or facilitate a conformational change in cohesin structure that promotes its loading, perhaps by permitting transient hinge opening to allow chromatin to enter cohesin rings or by promoting cohesin oligomerization (14, 16).Factors that regulate Scc2/Scc4 chromatin association are only beginning to be elucidated. Interactions of Scc2 and Scc4 orthologs from Xenopus and humans, and their stable association with chromatin, require the amino termini of both proteins (10, 13, 17, 18). In contrast, the fission yeast Scc2 ortholog alone binds nonchromatinized DNA, but does not exhibit an expected preference for sequences shown to associate with Scc2/Scc4 in vivo (19). Xenopus Scc2/Scc4 chromatin association requires prereplication complexes and Drf1-dependent kinase (DDK) activity (10, 12, 20), although this scenario is not the case in budding yeast (21). Scc2/Scc4 interactions with histone deacetylases and an ATP-dependent chromatin remodeler suggest that underlying chromatin structure also influences Scc2/Scc4 chromatin association (22–26). Whether Scc2/Scc4 plays a role in chromatin remodeling or merely deposits cohesins at remodeled sites is unknown, however.The chromatin association of Scc2/Scc4 and its orthologs is also regulated temporally during the cell cycle, although the specifics of association vary across species. Scc2/Scc4 associates with chromatin in late mitosis of the previous cell cycle in metazoans (12, 13) and in late G₁ in budding yeast, but in all cases, this association precedes DNA replication initiation so that cohesins are deposited in time to tether newly replicated sister chromatids together. Surprisingly, budding yeast Scc2/Scc4 chromatin association is more robust in mitotically arrested cells than in G₁-staged cells. Reduced G₁ Scc2/Scc4 chromatin association is not due to the absence of either loader subunit, because Scc2 and Scc4 protein levels vary little during the cell cycle, or by a lack of assembled cohesin complexes in G₁, because Scc2/Scc4 chromatin association occurs independently of cohesins (18, 27, 28). Scc2/Scc4 removal from chromatin is also regulated and occurs during mitosis in Xenopus and, more specifically, during prophase in humans (12, 13). Although factors responsible for regulating Scc2/Scc4 chromatin association/dissociation during the cell cycle remain enigmatic, evidence that multiple Scc2 orthologs are phosphorylated suggests the intriguing possibility that Scc2 posttranslational modifications regulate Scc2/Scc4 chromatin association (29–31).Here, we describe our efforts to understand how budding yeast Scc2/Scc4 chromatin binding is regulated during the cell cycle. Our results demonstrate the existence of multiple Scc2 protein species in vivo and that a specific cleaved form of Scc2 accumulates at cell cycle periods when Scc2 chromatin binding is weak. The appearance of this cleaved Scc2 species is strongly correlated with Scc2 dephosphorylation, suggesting that the phosphorylation state of Scc2 is critical for the regulation of its stability. Scc2 cleavage is also correlated with the loss of Scc2–Scc4 interactions, and an scc2 truncation mutant that mimics cleaved Scc2 is defective in cohesin deposition. These observations suggest that Scc2–Scc4 interactions and, therefore, the function of the complex in cohesin deposition, may be influenced by dephosphorylation-induced Scc2 cleavage.     

Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD⁺. A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

The human ALC1 (Amplified in Liver Cancer 1) protein (also referred to as CHD1L or Chromodomain-Helicase-DNA-binding protein 1-Like) is a SNF2 family chromatin remodeling enzyme that functions together with the poly(ADP-ribose) polymerase PARP1 to catalyze ATP- and NAD⁺-dependent nucleosome remodeling. The ALC1 gene is amplified in a subset of hepatocellular carcinomas, and overexpression of the ALC1 protein leads to transformation of cultured cells and appearance of spontaneous tumors in mice (1, 2). Although the precise mechanism(s) by which ALC1 overexpression contributes to tumorigenesis remains unknown, ALC1 has been implicated in multiple DNA damage repair pathways (3–6). Several recent studies have shown that ALC1 overexpression confers resistance to PARP inhibitors used in treatment of DNA repair–deficient tumors, while loss or reduction of ALC1 expression renders cells exquisitely sensitive to these drugs (7–10). Hence, understanding the functional relationships between ALC1 and PARP1 is of considerable interest.We and others initially demonstrated that ALC1 has cryptic DNA-dependent ATPase and ATP-dependent nucleosome sliding activities that are strongly activated in the presence of PARP1 and NAD⁺ (3, 11), which PARP1 and other PARPs use as substrate for synthesis of poly(ADP-ribose) (PAR) (12). ALC1 is unique among SNF2 family members in containing a macrodomain. The macrodomain, located at the enzyme’s C terminus, binds PAR chains containing three or more ADP-ribose residues (3, 11, 13–15). ALC1 macrodomain mutations that abolish PAR binding block ALC1 ATPase and nucleosome remodeling, indicating that PAR binding by the macrodomain is important for ALC1 activation. Recent studies have led to a working model for how binding of PAR to ALC1 macrodomain contributes to nucleosome remodeling. According to this model, ALC1 SNF2 ATPase domain interacts with and is held in an inactive state by the macrodomain. Upon binding of PAR to the macrodomain, this interaction is released, leading to structural changes in the ALC1 ATPase domain that relieve autoinhibition (13, 16). In subsequent steps, nucleosome binding by ALC1 stabilizes the catalytically active conformation of the ATPase, and a linker region between the ATPase and macrodomains contacts an acidic patch on nucleosomes to couple ATP hydrolysis to nucleosome sliding (17).Although it is well established that one key role of PARP1 in ALC1-dependent nucleosome remodeling is to produce PAR, it is less clear whether it makes additional contributions. Recent findings indicating that free tri-ADP ribose is sufficient to activate ALC1 ATPase activity in the absence of PARP1 (13) suggest that the role of PARP1 might be limited merely to synthesizing PAR chains. In this case, PARP1 might act simply as a bystander in ALC1-dependent nucleosome remodeling. On the other hand, PARP1 possesses both nucleosome binding and histone chaperone activities (18). This, together with our previous evidence that ALC1 and PARP1 bind cooperatively to nucleosomes to form an ALC1–PARP1–nucleosome intermediate prior to remodeling (14), makes it tempting to speculate that PARP1 might play a more active role.In the course of experiments investigating the mechanism(s) by which PARP1 contributes to ALC1-dependent nucleosome remodeling, we identified PARP1 mutants capable of activating ALC1 ATPase, but defective in supporting ALC1-catalyzed nucleosome remodeling. By investigating the properties of these and additional PARP1 mutants, we show that both the PARP1 C-terminal ADP-ribosyl transferase domain and its N-terminal region, which contains nucleosome binding activity, play important roles in ALC1-dependent nucleosome remodeling. We report these findings, which bring to light a role for PARP1 in chromatin remodeling.     

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol (“reverse topology”), but they can also be directed toward the cytosol (“normal topology”). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA⁺ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B–IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B–IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B–IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

The endosomal sorting complexes required for transport (ESCRT) proteins are an ancient and conserved membrane remodeling machinery, present in two of the three domains of life, the Archaea and Eukaryota. In humans, the ESCRTs are involved in myriad cell biological processes (1, 2) ranging from multivesicular body biogenesis (3), cytokinetic abscission (4), membrane repair (5–8), and exosome (9) and HIV-1 release (10, 11). The underlying commonality of most of these processes is that they are topologically equivalent, with scission occurring on the cytosolic and inner surface of a narrow membrane neck (“reverse topology”). The ESCRTs consist of ALIX, ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, and VPS4 (12, 13). The ESCRT-III proteins (14) are most directly involved in catalyzing membrane scission (15, 16). These ESCRTs are first recruited to the neck, then the AAA⁺ ATPase VPS4 (17) is finally recruited to ESCRT-III–enriched sites prior to scission (18). VPS4 forms a hexamer (19, 20) that interacts with ESCRT-III through its N-terminal microtubule-interacting and trafficking (MIT) domain binding to the exposed C-terminal MIT domain interacting motif (MIM) domains found in some ESCRT-III proteins (21, 22). ESCRT-III together with VPS4 constitutes the minimal module to drive scission of vesicles that bud away from the cytosol (16). While ESCRTs are best known for reverse-topology membrane scission, a subset of ESCRTs, CHMP1B and IST1, can also coat the outer surface of membrane tubes, leading to a dramatic constriction in the tube (23, 24). This process is implicated in tubular endosomal traffic from the endoplasmic reticulum (ER) to lysosomes (25–27) and lipid droplets to peroxisomes (28) and the release of newly formed peroxisomes from the ER (29). These observations suggested that CHMP1B and IST1 could carry out normal topology scission; however, direct observation of this type of scission has not been reported.Twelve different ESCRT-III proteins are found in humans, which can combine in various compositions that nucleate and grow on membranes of various curvatures (23, 30, 31). ESCRT-III proteins are monomeric and are highly basic and share similar secondary core structures containing five helices (32). These proteins are in an autoinhibited closed conformation in solution (33, 34). Activation can be triggered upon binding to membranes or upstream activators, or artificially through truncation of their C-terminal elements (33, 34). Upon activation, ESCRT-III proteins polymerize into spirals (35–37) and helical tubes (24, 38). Incubation of CHMP1B with liposomes leads to formation of protein-coated tubules in vitro as shown using cryoelectron microscopy (cryo-EM) (23). This positively curved coat was initially unexpected in the ESCRT field, but subsequently has been observed more generally with combinations of CHMP2, 3, and 4 (39, 40).In reverse-topology scission, the ATPase activity of VPS4 is essential for the remodeling of ESCRT-III assemblies that lead to membrane constriction and scission (16). Polymerization of CHMP4 is considered a major driver of scission; however, this is held in check by capping with CHMP2 (41). VPS4 can solubilize CHMP2 subunits, allowing CHMP4 growth to progress, leading to scission (42). VPS4 binds to most of the MIM-containing CHMPs, including IST1 and CHMP1; however, CHMP2 is not known to have a role in normal topology scission, and CHMP1 and IST1 are not known to engage in capping. Therefore, it is not clear whether the VPS4-driven decapping mechanism established in reverse topology membrane scission has any role in normal topology scission.Spastin belongs to the same meiotic clade of AAA⁺ ATPases as VPS4 and is best known as a microtubule-severing enzyme (43, 44). Mutations of the spastin (SPG4 and SPAST) gene are the main causes in patients suffering from hereditary spastic paraplegia (45). The spastin linkage to CHMP1B and IST1 is involved in the scission of recycling endosomal tubules (27). Disruption of any of these interactions increases endosomal tubulation, mistrafficking of cargoes, and dysregulation of proper lysosomal functions (25–27). It has remained unclear whether spastin can substitute for the possible scission or disassembly functions of VPS4 with respect to CHMP1B- and IST1-containing membrane tubes, in addition to its canonical microtubule-severing activity.Recent cryo-EM reconstructions on synthetic liposomes showed that the polymerization of IST1 on the exterior of CHMP1B-coated tubes leads to a remodeling of the CHMP1B coat and tightly constricts the membrane but does not lead to membrane scission (24). Here, we used a powerful technique to assay ESCRT membrane scission in vitro by combining optical tweezers and fluorescence microscopy to visualize membrane nanotubes pulled from giant unilamellar vesicles (GUVs) (46, 47) and characterize the effect in addition of various ATPases on ESCRT dynamics. This assay allows for the formation of a positively curved membrane that mimics the membrane tubules where CHMP1B and IST1 bind. Using this highly sensitive and flexible system, we were able to reconstitute the scission reaction and delineate the roles of CHMP1B, IST1, VPS4, and spastin in normal topology scission.     

Polo kinase Cdc5 is a central regulator of meiosis I

During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome segregation. CDC5 also establishes conditions for centromeric cohesin removal during meiosis II by promoting the degradation of Spo13, a protein that protects centromeric cohesin during meiosis I. Despite CDC5’s central role in meiosis I, the protein kinase is dispensable during meiosis II and does not even phosphorylate its meiosis I targets during the second meiotic division. We conclude that Cdc5 has evolved into a master regulator of the unique meiosis I chromosome segregation pattern.Polo kinases are central regulators of chromosome segregation and control multiple mitotic events (1). Budding yeast contains a single Polo kinase, CDC5. Unlike in higher eukaryotes, budding yeast CDC5 primarily regulates postmetaphase events, its essential function being to trigger exit from mitosis (2). CDC5 also contributes to the efficient inactivation of cohesins, the protein complexes that hold sister chromatids together until the onset of chromosome segregation. Cdc5 phosphorylates the cohesin subunit Mcd1/Scc1 to facilitate its cleavage by the protease separase (3).CDC5 also regulates the specialized cell division that gives rise to gametes, known as meiosis (4). During meiosis, two consecutive rounds of chromosome segregation follow one round of DNA replication. During meiosis I, homologous chromosomes segregate; during meiosis II, sister chromatids separate (5). The chromosome segregation machinery is modified in three ways to facilitate the unusual meiosis I division. First, the combination of homologous recombination and cohesin complexes distal to the resulting cross-overs mediate the physical linkage of homologous chromosomes, which is essential for their accurate segregation during meiosis I. Second, sister chromatids of each homolog must be segregated to the same pole rather than to opposite poles, as they are during mitosis. The fusion of sister kinetochores by co-orientation factors (the monopolin complex in yeast) facilitates the attachment of microtubules emanating from one spindle pole. Third, cohesin complexes must be lost in a stepwise manner from chromosomes. During meiosis I cohesin complexes are lost from chromosome arms to bring about the segregation of homologous chromosomes (6). The residual cohesins at centromeres facilitate the accurate segregation of sister chromatids during meiosis II. Cdc5 has been implicated in the execution of all three meiosis I-specific events. CDC5 is required for the resolution of double Holliday junctions during homologous recombination (7, 8). Cdc5 also controls the co-orientation of sister chromatids by promoting the association of the monopolin complex with kinetochores (7, 9). Finally, CDC5 has been implicated in regulating the stepwise loss of cohesins (7, 9, 10). Phosphorylation of the cohesin subunit Rec8, a meiosis-specific cohesin subunit that replaces Scc1/Mcd1 in the meiotic cohesin complex, controls the stepwise loss of cohesins from chromosomes. Rec8 phosphorylation is critical for its proteolytic cleavage and removal from chromosome arms during meiosis I (10, 11). Maintaining Rec8 in a dephosphorylated form around centromeric regions protects it from cleavage. This is accomplished by Sgo1, a shugoshin/MEI-S332 family member that recruits protein phosphatase 2A to centromeric regions (12). Our studies have implicated Cdc5 as one, but not the only, protein kinase phosphorylating Rec8 to target it for proteolytic cleavage by separase (10).In addition to controlling meiosis I-specific events, CDC5 also regulates general cell-cycle functions during meiosis I that it does not affect during mitosis. During meiosis I, CDC5 controls separase activity. Degradation of the separase inhibitor securin (Pds1 in yeast) liberates separase to trigger anaphase (5). During meiosis I, but not during mitosis, CDC5 is required for Pds1 degradation (7, 9). How Cdc5 takes on new functions during meiosis I is not understood. Similarly, little is known about whether and how Cdc5 functions during meiosis II because cells depleted for Cdc5 arrest in metaphase I (7, 9).Here we show that CDC5 controls cohesin removal in multiple ways. CDC5-dependent phosphorylation of Rec8 is essential for efficient Rec8 cleavage. Furthermore, Cdc5 triggers the degradation of Spo13, thereby contributing to the dismantling of the cohesin-protective domain around centromeres. Our data further show that despite its central role in meiosis I chromosome segregation, CDC5 is dispensable during meiosis II and does not phosphorylate its meiosis I targets during meiosis II. Our findings indicate that the evolution of additional CDC5 functions is a central aspect of establishing the unique meiotic chromosome segregation pattern.     

Eicosanoid regulation of debris-stimulated metastasis

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

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

Topological braiding and virtual particles on the cell membrane

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

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

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

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

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

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

Parallel hippocampal-parietal circuits for self- and goal-oriented processing

The hippocampus is critically important for a diverse range of cognitive processes, such as episodic memory, prospective memory, affective processing, and spatial navigation. Using individual-specific precision functional mapping of resting-state functional MRI data, we found the anterior hippocampus (head and body) to be preferentially functionally connected to the default mode network (DMN), as expected. The hippocampal tail, however, was strongly preferentially functionally connected to the parietal memory network (PMN), which supports goal-oriented cognition and stimulus recognition. This anterior–posterior dichotomy of resting-state functional connectivity was well-matched by differences in task deactivations and anatomical segmentations of the hippocampus. Task deactivations were localized to the hippocampal head and body (DMN), relatively sparing the tail (PMN). The functional dichotomization of the hippocampus into anterior DMN-connected and posterior PMN-connected parcels suggests parallel but distinct circuits between the hippocampus and medial parietal cortex for self- versus goal-oriented processing.

The hippocampus is critically important for a diverse range of cognitive processes, such as episodic and prospective memory, affective processing, and spatial navigation (1–7). The hippocampus’ diverse functions rely on its pattern of connectivity (8). Atypical cortico-hippocampal functional connectivity is associated with cognitive and affective deficits (9–12). A precise understanding of the functional organization of the hippocampus is crucial for understanding the neurobiology underlying hippocampally related diseases.The hippocampus seems to exhibit functional heterogeneity along its longitudinal axis (anterior–posterior in humans; ventral–dorsal in rodents). Studies of the rodent hippocampus have demonstrated modular differentiation along its longitudinal axis in patterns of gene expression, function, and anatomical projections (2, 13, 14). The rodent ventral hippocampus (anterior in humans) plays a role in the modulation of stress and affect (2, 4), whereas the dorsal hippocampus (posterior in humans) is important for spatial navigation. Hippocampal place field representation sizes in rodent models also follow a ventral–dorsal gradient reflecting large-to-small spatial resolution (13, 14). The ventral hippocampus in rats is anatomically interconnected with the amygdala, temporal pole, and ventromedial prefrontal cortex (4, 15), while the dorsal hippocampus is connected with the anterior cingulate and retrosplenial cortex (4, 15).In humans, evidence for structural differentiation between the anterior and posterior hippocampus is provided by age and Alzheimer’s disease–related hippocampal volume reduction differences (16) and diffusion tractography (17). Functional MRI (fMRI) research has suggested an anterior–posterior gradient in coarse-to-fine mnemonic spatiotemporal representations (18), such that anterior hippocampus supports schematics, while specific details associated with a given event are represented in posterior hippocampus (6, 7). Similarly, other studies have suggested anterior–posterior hippocampal differences in pattern completion (i.e., integrating indirectly related events) and pattern separation (i.e., discriminating between separate but similar events) (19).Resting-state functional connectivity (RSFC) studies in humans have provided additional insights into the hippocampal connectivity that underlies hippocampus-mediated cognition. RSFC exploits the phenomenon that even in the absence of overt tasks, spatially separated but functionally related regions exhibit correlations in blood oxygen level–dependent (BOLD) signal (20–24). Group-averaged RSFC studies have found the hippocampus to be functionally connected to the default mode network (DMN) (25–28). The DMN is deactivated by attention-demanding tasks and thought to be important for self-referential processes, such as autobiographical memory, introspection, emotional processing, and motivation (26). Other group-averaged RSFC studies have reported the anterior hippocampus to be preferentially functionally connected to anterior parts of the DMN, while the posterior hippocampus was more strongly connected to the posterior DMN via the perirhinal and parahippocampal gyri (29–32).Recent precision functional mapping studies have highlighted that RSFC group-averaging approaches obscure individual differences in network architecture in both the cortex and subcortical structures (33–41). The large amounts of RSFC data utilized (>300 min per subject) in precision functional mapping improve the signal-to-noise ratio and allow for the replicable detection of additional functional neuroanatomical detail in the cerebral cortex (34), cerebellum (33), basal ganglia, thalamus (35, 42), and amygdala (36). In a small, deep-lying structure like the hippocampus, group-averaging RSFC data may be even more problematic.The medial parietal cortex is one of the main targets of hippocampal anatomical and functional connectivity (4, 15, 43–47) and was previously considered part of the DMN (25–28). The medial parietal cortex encompasses the swath of posterior midline neocortex between motor and visual regions. It includes the retrosplenial cortex, posterior cingulate, and precuneus (Brodmann Area 7, 23, 26, 29, 30, and 31). More recent studies revealed that parts of the medial parietal cortex belong to the parietal memory network (PMN) and the contextual association network (CAN) (23, 34, 39, 41, 48, 49). The PMN and CAN are immediately adjacent to the DMN in medial parietal cortex and therefore easily confounded in group-averaged data. The CAN (34, 41) corresponds to Braga et al.’s DMN subnetwork B (39). The identification of multiple different networks (DMN, PMN, CAN, and FPN [fronto-parietal network]) in medial parietal cortex reflects the ongoing recognition of novel networks, subnetworks, and organizational principles driven by precision functional mapping (23, 34, 39, 41, 50–52).The DMN, PMN, and CAN are all thought to be important for memory. The DMN and PMN have been associated with different aspects of episodic memory processing. Autobiographical retrieval (i.e., memory over a lifetime) preferentially increases activity in the DMN, whereas memory for recently experienced events preferentially engages the PMN (27, 48, 53, 54). During explicit memory tasks, activity within the PMN decreases in response to novel stimuli but increases in response to familiar stimuli, such that increased activity seems to reflect attention to internal memory representations during retrieval (48, 55). The CAN processes associations between objects or places and their scenes (41, 56).Here, we utilized precision functional mapping to examine individual-specific, hippocampal-cortical functional connectivity. We utilized both the Midnight Scan Club (MSC) dataset (n = 10 participants; 300 min. of resting-state fMRI data/subject) (34) and additional extremely highly sampled, higher-resolution resting-state fMRI data (2.6 mm isotropic voxels; 2,610 min; MSC06-Rep) from an independent dataset (57, 58). We generated individual-specific RSFC parcellations of the hippocampus, drawing on several advantages over group-averaging, including the following: (1) higher signal-to-noise ratio in deeper subcortical structures without blurring individual differences in network features and (2) more precise definition of individual-specific cortical functional network maps (i.e., DMN, PMN, CAN, and FPN).     

Neurobiological basis of head motion in brain imaging

Individual differences in brain metrics, especially connectivity measured with functional MRI, can correlate with differences in motion during data collection. The assumption has been that motion causes artifactual differences in brain connectivity that must and can be corrected. Here we propose that differences in brain connectivity can also represent a neurobiological trait that predisposes to differences in motion. We support this possibility with an analysis of intra- versus intersubject differences in connectivity comparing high- to low-motion subgroups. Intersubject analysis identified a correlate of head motion consisting of reduced distant functional connectivity primarily in the default network in individuals with high head motion. Similar connectivity differences were not found in analysis of intrasubject data. Instead, this correlate of head motion was a stable property in individuals across time. These findings suggest that motion-associated differences in brain connectivity cannot fully be attributed to motion artifacts but rather also reflect individual variability in functional organization.Head motion has long been known as a confounding factor in brain imaging including MRI (1, 2), PET (3, 4), single-photon emission computerized tomography (5, 6), and near infrared spectroscopy (7), but has raised particular concerns recently following the growing prominence of resting-state functional connectivity MRI. Studies found that head motion can vary considerably across individuals and often demonstrates systematic group effects when contrasting different populations, especially in neurodevelopmental (8–10), aging (11, 12), and neuropsychiatric studies (13). Some recent work reported that head motion augmented local coupling of the blood oxygenation level-dependent (BOLD) signal but reduced distant coupling (14–16). These correlations between connectivity measures and head motion have raised appropriate concern that previously observed differences in connectivity are due to artifact induced by differences in head motion. For example, developmental changes in functional connectivity might also be predicted by head motion (15). The assumption has been that head motion causes distorted connectivity measurements that must be addressed through improved motion-correction techniques (15). However, this correlation could be driven by causal factors in the other direction. Specifically, individual differences in brain connectivity could determine how well a subject can lie still in the scanner. This is not unreasonable as individual differences in structural connectivity can predict trait anxiety and can be related to attention deficits (17, 18) and individual differences in resting-state functional MRI (fMRI) measures may relate to various behavioral differences, including impulsivity (19–22). In such a scenario, certain intersubject differences in connectivity measures could persist even after the most rigorous motion correction, as has been suggested in several earlier studies (23, 24).To explore the relation between head motion and brain connectivity, we examined functional connectivity in different subject groups selected on the basis of head motion parameters from a large database of 3,000+ participants, many of whom were scanned multiple times. These cohorts allowed us to compare intersubject and intrasubject differences in connectivity in high- versus low-motion scans. If motion causes connectivity differences, these should be similar both inter- and intrasubject. However, if connectivity differences include a stable trait that predisposes to head motion, then these differences should be present between subjects but not within subjects.     

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

Molecular chaperones are involved in the maintenance of protein homeostasis by aiding correct protein folding (1). Yet severe stress conditions induce excessive protein misfolding and aggregation (2). Upon stress relief, the return to the proteostasis is mediated by the Hsp70 chaperone with cochaperones, including J-domain proteins (JDPs/Hsp40s), which together restore the native state of misfolded polypeptides trapped in aggregates (3–5). The JDP–Hsp70 system acts alone in metazoans or in cooperation with an Hsp100 disaggregase in most other eukaryotes and bacteria (5, 6).Protein disaggregation and refolding starts with a recognition of misfolded polypeptides within an aggregate by a JDP, and then, its J-domain interacts with the nucleotide-binding domain of Hsp70, inducing ATP hydrolysis which triggers the closure of the Hsp70’s substrate-binding domain over the aggregated substrate (7, 8). The aggregate-bound Hsp70 interacts with an Hsp100 disaggregase, and this interaction allosterically activates Hsp100 and tethers it to the aggregate (9–16). Subsequently, in an ATP-driven process, Hsp100 disentangles and translocates polypeptides from aggregates (17–21), which enables their correct refolding, spontaneous or with an assistance of Hsp70 and its cochaperones (22, 23).JDPs are the major regulators of the Hsp70 activity and substrate specificity (3, 24, 25). In yeast Saccharomyces cerevisiae, a general Hsp70 chaperone, Ssa1, is recruited to protein disaggregation by two main cytosolic JDPs, Ydj1 and Sis1, assigned to the Class A and Class B, respectively (3, 4, 26). Both Ydj1 and Sis1 comprise a helical, highly conserved J-domain, a flexible, mostly unstructured G/F region, two beta-barrel peptide-binding domains, CTDI and CTDII, and a C-terminal dimerization domain (27–33). Ydj1 additionally features a Zn-binding domain located in the first part of the CTDI region of the protein, which is distinctive for the Class A JDPs (32, 34).Despite the structural similarities, the two JDPs are functionally nonredundant. Sis1 is essential, and Ydj1 is required for growth above 34 °C (26, 27, 35, 36). Overexpression of Sis1 suppresses the phenotype caused by the deletion of YDJ1, while Ydj1 overexpression is not sufficient to suppress the deletion of SIS1 (26, 27, 35–37). The two JDPs show different specificities toward amorphous and amyloid aggregates (35, 38) and different populations of amorphous aggregates formed in vitro (4, 24).Recent reports shed more light on the JDPs’ divergence. Both JDPs form homodimers, which differ in the structural orientation of the J-domain: In Sis1, the J-domain is restrained from Hsp70 binding by the interaction with the Helix 5 in the G/F region (26, 33, 39–41). Such autoinhibition, which also occurs in most human Class B JDPs, is released through the interaction with the C-terminal EEVD motif of Hsp70 (33, 42). This regulation is important for the disassembly of amyloid fibrils by the human JDP–Hsp70 system (43), but its role in the handling of stress-related, amorphous aggregates is not clear. Despite the breadth of data on Hsp70 mechanisms, we still lack understanding of how the disparate features of the JDPs impact Hsp70 functioning in protein disaggregation.Here, we investigate individual steps of protein disaggregation in the context of functional differences between Sis1 and Ydj1. Using various biochemical approaches, we show that the two JDPs drive different modes of Ssa1 binding to aggregated substrates, which dictate diverse kinetics of their disaggregation by Hsp104. The distinctive performance of Sis1 is associated with its interaction with the C terminus of Hsp70. Our results suggest that the bivalent interaction with the Class B JDP conditions aggregate remodeling by the Hsp70 system, resulting in enhanced Hsp104-dependent protein recovery. Our data indicate a mechanism by which the Class A and B JDPs contribute to the disaggregation efficacy in a complex and divergent manner.