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Shima, T.

Publications and source records attributed to Shima, T..

18 recordsLinked to original sources

Analysis of oogenesis defects in Dyro mutant of Drosophila melanogaster.

Dyro mutant is female sterile, and oogenesis is aborted during stage8-9 of oogenesis. We investigated detail of the oogenesis defect of Dyro mutant. At first, we confirmed loss of Dyro caused female sterility by genetic rescue experiment. Then, we performed genetic mosaic analysis and found Dyro expression in germ cell is important for oogenesis. In Dyro mutant, inhibition of programmed cell death suppressed cell death of germ cells during oogenesis but failed to rescue fertility. It indicates that abortion of oogenesis is not because of mis-regulation of cell death signal but there is oogenesis defect which activates Caspase signaling pathway. Then, we observed Dyro mutant and looking for defects which may trigger cell death of germ cells in Dyro mutant. We found oogenesis abortion timing is similar to yolk protein mutant but different from amino acid starvation. It suggests that nutrient signal defect does not triggers cell death in Dyro mutant. We carefully observed the defect of Dyro mutant ovaries and found abnormal morphology of nucleolus and chromosome in nurse cells. It seems chromosome in Dyro mutant is thick and nucleolus is limited in small space between thick chromosomes in Dyro mutant nurse cells. Other defect we found is aggregated protein accumulation in germ cells. These data suggest that Dyro has important role in mid-oogenesis stage germ cell and loss of Dyro causes defect in nuclear of nurse cells which may leads to abortion of oogenesis.

developmental biology↗

Muscle-derived miR-200a-3p through light-intensity exercise may contribute to improve memory dysfunction in type 2 diabetic mice

BackgroundMemory dysfunction associated with type 2 diabetes mellitus (T2DM) poses a considerable threat to overall well-being. Engaging in light-intensity exercise has been shown to exert favorable effects on hippocampal function and molecular profiles, including Mct2 mRNA and miR-200a-3p. Nonetheless, a comprehensive understanding of the mechanisms underlying the positive impact of light-intensity exercise remains elusive. Here, we assessed the influence of exosomal miR-200a-3p secretion from gastrocnemius muscles in T2DM mice undergoing light-intensity exercise intervention, focusing on its potential to ameliorate memory dysfunction. Basic proceduresWe initially assessed the effects of light-intensity exercise (7.0 m/min for healthy mice, 5.0 m/min for ob/ob mice, 30 min/day, five days/week, over a four-week period) on memory function, hippocampal mRNA associated with memory function, and the secretion of exosomal miR-200a-3p from their gastrocnemius muscle. Subsequently, the impact of a daily intraperitoneal injection of the miR-200a-3p mimic over a four-week duration was investigated, focusing on its influence on hippocampal dysregulation in ob/ob mice. Main findingsThe light-intensity exercise intervention increased gastrocnemius muscle-derived and plasma exosomal miR-200a-3p levels in ob/ob mice, concomitant with improved memory dysfunction. Intriguingly, the daily intraperitoneal injection of mmu-miR-200a-3p mimic also demonstrated an ameliorative effect on memory function in ob/ob mice. Notably, both the exercise intervention and miR-200a-3p mimic treatment induced downregulation in hippocampal Keap1 mRNA and upregulation in mRNA of Hsp90aa1 and Mct2 in ob/ob mice. Principal conclusionsThe current results imply that the augmentation of exosomal miR-200a-3p derived from the gastrocnemius muscle contributes to the amelioration of memory dysfunction in T2DM undergoing light-intensity exercise. Additionally, it is proposed that miR-200a-3p emulates the effects of light-intensity exercise, suggesting a potential therapeutic pathway for addressing hippocampal complications in the context of T2DM.

neuroscience↗

The Rubicon-WIPI axis regulates exosome biogenesis during aging

Cells release intraluminal vesicles (ILVs) in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNAi screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for ILV formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for the age-dependent changes in exosome quantity and quality.

cell biology↗

Heterogeneous local structures of the microtubule lattice revealed by cryo-ET and non-averaging analysis

Microtubule cytoskeletons play pivotal roles in various cellular processes, including cell division and locomotion, by dynamically changing their length and distribution in cells through tubulin polymerization/depolymerization. Recent structural studies have revealed the polymorphic lattice structure of microtubules closely correlate with the microtubule dynamics, but the studies were limited to averaged structures. To reveal the transient and localized structures, such as GTP-cap, we developed several non-averaging methods for cryogenic electron tomography to precisely measure the longitudinal spacing and helical twisting of individual microtubule lattices at the tubulin subunit level. Our analysis revealed that polymerizing and depolymerizing ends share a similar structure with regards to lattice spacing. The most distinctive property specific to the polymerizing plus end was left-handed twisting in the inter-dimer interface, suggesting that the twisting might accelerate fast polymerization at the plus ends. Our analysis uncovered the heterogeneity of native microtubules and will be indispensable for the study of microtubules dynamics under physiological contexts or during specific cellular events.

biophysics↗

Feeding a low-carbohydrate and high-protein diet diminishes working memory in healthy mice: possible involvement of miR-539-3p/Lrp6/Igf1r axis in the hippocampus

A low-carbohydrate and high-protein (LC-HP) diet demonstrates favorable impacts on metabolic parameters, albeit it leads to a decline in hippocampal function among healthy mice. The reduction in working memory induced by LC-HP diets is attributed to the decreased expression of hippocampal IGF-1 receptor (IGF-1R). However, the precise mechanisms underlying this phenomenon remain unexplored. Here, we investigated that by analyzing alterations in hippocampal miRNA profiles. C57BL/6 mice were divided into the LC-HP diet-fed group (25.1% carbohydrate, 57.2% protein, and 17.7% fat as percentages of calories) and the control diet-fed group (58.9% carbohydrate, 24.0% protein, and 17.1% fat as percentages of calories). After four weeks, all mice underwent the Y-maze test, followed by analyses of mRNA and miRNA expressions in the hippocampus. Our investigation revealed that feeding the LC-HP diet suppressed working memory function and hippocampal Igf1r mRNA levels in mice. Sequencing of miRNA demonstrated 17 upregulated and 27 downregulated miRNAs in the hippocampus of LC-HP diet-fed mice. Notably, upregulation of miR-539-3p, predicted to modulate Igf1r gene expression, was observed. Consequently, we found decreased hippocampal mRNA levels of low-density lipoprotein receptor-related protein 6 (Lrp6), a gene modulated by miR-539-3p, in mice fed the LC-HP diet. Furthermore, a significant positive correlation was observed between Lrp6 and Igf1r mRNA levels in the hippocampus. These findings suggest that LC-HP diets may suppress hippocampal function via the miR-539-3p/Lrp6/Igf1r axis, offering a potential target for nutritional strategies to preserve hippocampal health.

neuroscience↗

Augmented-reality-based exercise increases salivary oxytocin

Exercise is known to have positive effects on psychological well-being, with team sports often associated with superior mental health compared to individual sports. Augmented reality (AR) technology has the potential to convert solitary exercise into multi-person exercise. Given the role of oxytocin in mediating the psychological benefits of exercise and sports, this study aimed to investigate the impact of AR-based multi-person exercise on mood and salivary oxytocin levels. Fourteen participants underwent three distinct regimens: non-exercise (Rest), standard solitary cycling exercise (Ex), and AR-based multi-person cycling exercise (Ex+AR). In both exercise conditions (Ex and Ex+AR), participants engaged in cycling at a self-regulated pace to maintain a Rating of Perceived Exertion of 10. In the Ex+AR condition, participants avatars were projected onto a tablet screen, allowing them to cycle alongside ten other virtual avatars in an AR environment. Mood states (assessed using POMS2) and saliva samples were collected before and immediately after each 10-minute regimen. Subsequently, the levels of salivary oxytocin were measured. Participants exhibited higher cycling speeds during Ex+AR compared to Ex, despite comparable levels of self-reported fatigue between the two groups. Notably, only the Ex+AR condition significantly improved mood states associated with depression-dejection and exhibited a trend toward suppressing anger-hostility in participants. Moreover, the Ex+AR condition led to a significant elevation in salivary oxytocin levels, while the Ex condition showed a trend toward an increase. However, changes in salivary oxytocin did not show a significant correlation with changes in mood states. These findings suggest that Ex+AR enhances mood states and promotes oxytocin release. AR-based multi-person exercise may offer greater psychological benefits compared to standard solitary exercise, although the relationship between oxytocin and mood changes remains inconclusive. HighlightsO_LIExercise, especially team sports, has positive effects on mood state, and oxytocin partially supports these effects. C_LIO_LIAugmented reality (AR) technology can potentially convert standard solitary exercise into multi-person exercise. C_LIO_LIAR-based multi-person exercise (Ex+AR) significantly ameliorated depression-dejection and increased salivary oxytocin levels, but standard solitary exercise did not. C_LIO_LIEx+AR may provide more significant psychological benefits than standard solitary exercise. C_LI

physiology↗

Golgi membrane proteins YIPF3 and YIPF4 regulate turnover of the Golgi apparatus through autophagy

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the autophagosome. Using quantitative proteomic analysis and two Golgiphagy-reporter systems that we developed, we found that five-transmembrane Golgi-resident proteins, YIPF3 and YIPF4, constitute a Golgiphagy receptor. The YIPF3-YIPF4 complex interacts with LC3B, GABARAP, and GABARAPL1 via the LIR motif in YIPF3, whose stability is dependent on YIPF4. Phosphorylation of the LIR in YIPF3 seems to be required for YIPF3-ATG8 interaction. Moreover, expression of the YIPF3 LIR mutant caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays that we established will pave the way for future studies to obtain deeper insights into Golgiphagy.

cell biology↗

Preference of CAMSAP3 for microtubules with expanded lattice contributes to stabilization of the minus end

CAMSAPs are proteins that show microtubule minus-end-specific localization, decoration and stabilization. Although the mechanism for minus-end recognition via their C-terminal CKK domain has been well described in recent studies, it is unclear how CAMSAPs stabilize microtubules. Our several binding assays revealed that D2 region of CAMSAP3 specifically binds to microtubules with the expanded lattice. To investigate the relationship between this preference and the stabilization effect of CAMSAP3, we precisely measured individual microtubule lengths and found that D2-binding expanded the microtubule lattice by [~]3%. Consistent with the notion that the expanded lattice is a common feature of stable microtubules, the presence of D2 slowed the microtubule depolymerization rate to approximately 1/20, suggesting that the D2-triggered lattice expansion stabilizes microtubules. Combining these results, we propose that CAMSAP3 stabilizes microtubules by lattice expansion upon D2-binding, which further accelerates the recruitment of other CAMSAP3 molecules. Since only CAMSAP3 has D2 and the highest microtubule stabilizing effect among mammalian CAMSAPs, our model also explains the molecular basis for the functional diversity of CAMSAP family members. Summary blurbD2 region in CAMSAP3 preferentially bound to expanded microtubule lattices and also induced lattice expansion, explaining the molecular functions of CAMSAP3.

biophysics↗

N-terminal region of Drosophila melanogaster Argonaute2 forms amyloid-like aggregates

Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. Because the N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains (PrLD), the possibility of the N-terminal region functioning as a PrLD was tested. Our biochemical assays demonstrated that the N-terminal region can form aggregates that are not dissociated even in the presence of SDS. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting the self-propagating activity. Further, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region is a PrLD. Many other PrLDs have been reported to modulate the function of proteins through their aggregation. Therefore, our results raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.

biochemistry↗

Key residue on cytoplasmic dynein for asymmetric unbinding and unidirectional movement along microtubule

Cytoplasmic dynein 1 is almost exclusively responsible for intracellular transport toward the minus-end of microtubules in animal cells. One of the key factors for the unidirectional movement of dynein is the asymmetry of the unbinding of the motor from the microtubule when an external load is applied; it dissociates more easily from microtubules with minus-end directed loading than with plus-end directed loading. To elucidate the molecular basis for this property, we performed molecular dynamics simulations to identify the key residues responsible for asymmetry, which were then examined experimentally. First, we reproduced asymmetry in the unbinding behavior of dynein using coarse-grained simulations. Then, data analysis together with mutational analysis in silico predicted the specific residues that may be responsible for the asymmetry in unbinding. To examine this prediction, we expressed and purified recombinant dynein with mutations in either of the identified key residues. Consistent with the simulations, one of the mutants did not exhibit asymmetry in the in vitro unbinding assay. Moreover, the mutant dynein was able to bind and move diffusely along a microtubule but was unable to restrict its movement to the minus-end direction. Our results demonstrate both experimentally and theoretically how the key residue on the microtubule-binding domain generates asymmetry in unbinding, which is a critical mechanism for the unidirectional movement of dynein along a microtubule track. Significance StatementCytoplasmic dynein moves to the minus end of microtubules. This unidirectional dynein motility provides the driving force for various cellular activities including vesicle transport, organelle positioning and cell division. One of the key factors for dynein to exhibit unidirectional movement is the asymmetry of unbinding of dynein from the microtubule depending on the direction of external load. By combining computational simulations and in vitro experiments, we identified a residue responsible for the asymmetry. A point mutation at the residue indeed abolished unidirectional motility, highlighting the importance of the asymmetric unbinding property in dyneins unidirectional movement.

biophysics↗

Accumulation of TERT in Mitochondria Shows Two Opposing Effects on Apoptosis

Telomerase reverse transcriptase (TERT) is a protein that catalyzes the reverse transcription of telomere elongation. TERT is also expected to play a noncanonical role beyond telomere lengthening since it localizes not only in the nucleus but also in mitochondria, where telomeres do not exist. Several studies have reported that mitochondrial TERT regulates apoptosis induced by oxidative stress. However, there remains controversy about whether mitochondrial TERT promotes or inhibits apoptosis, mainly due to the lack of information on changes in the TERT distribution in individual cells over time. Here we simultaneously detected apoptosis and TERT localization after oxidative stress in individual HeLa cells by live-cell tracking. This tracking revealed that the stress-induced accumulation of TERT in mitochondria caused apoptosis but that the accumulation positively correlated with the time until cell death. The results suggest a new model in which mitochondrial TERT has two opposing effects at different stages of apoptosis: it predetermines apoptosis at the first stage of cell-fate determination but also delays apoptosis at the second stage. Because these distinct effects respectively support both sides of the controversy regarding the role of mitochondrial TERT in apoptosis, our model integrates two opposing hypotheses. Furthermore, detailed statistical analysis of TERT mutations, which have been predicted to inhibit TERT transport to mitochondria, revealed that these mutations suppress apoptosis independent of the mitochondrial localization of TERT. Together, these results indicate that the non-canonical functions of TERT affect a wide range of pathways.

cell biology↗

Aerobic glycolysis in cancer cells supplies ATP while preventing excess metabolic thermogenesis

Adenosine triphosphate (ATP) regeneration by substrate-level phosphorylation is a general feature of cancer metabolism, even under normoxic conditions (aerobic glycolysis). However, it is unclear why cancer cells prefer inefficient aerobic glycolysis over the highly efficient process of oxidative phosphorylation for ATP regeneration. To investigate the metabolic principles underlying aerobic glycolysis, we performed 13C-metabolic flux analysis of 12 cultured cancer cell lines and explored the metabolic constraints required to reproduce the results using in silico metabolic simulations. We found that the measured flux distribution can be reproduced by maximizing the ATP consumption in the flux balance analysis considering a limitation of metabolic heat dissipation (enthalpy change). It suggests that aerobic glycolysis may be preferable because metabolic heat production during one mol of ATP regeneration by aerobic glycolysis was less than that produced by oxidative phosphorylation (OXPHOS). Consistent with the simulation, OXPHOS inhibition induced metabolic redirection to aerobic glycolysis while maintaining the intracellular temperature. Furthermore, the dependency on aerobic glycolysis was partly alleviated upon culturing at low temperatures. Our data suggest that metabolic thermogenesis is an important factor in understanding aerobic glycolysis in cancer cells and that an advantage of aerobic glycolysis is the reduction in metabolic heat generation during ATP regeneration.

cancer biology↗

A fast and objective hidden Markov modeling for accurate analysis of biophysical data with numerous states

The hidden Markov model (HMM) is widely used to analyze biophysical chronological data with discrete states, such as binding/detachment of biomolecules, protein/nucleotide conformational changes and step-like movement of single proteins. Despite its usefulness, classical HMM fitting has practical drawbacks that it requires the determination of the number of hidden states and fine initialization of many parameters before fitting. To overcome these drawbacks, several HMM pre-analyses have been reported, but do not provide enough accuracy when data have unknown kinetics and/or low signal-to-noise ratio. Therefore, in many cases, HMM fitting needs trial-and-error manual process that can impair the objectivity of the analysis. Moreover, for data composed of numerous hidden states, such as stepping data of cytoskeletal motors, there has been difficulty in HMM analysis because the large number of parameters were hardly properly initialized. Here, by combining a statistical step-finding method and the Gaussian mixture model clustering, we developed a new algorithm for more objective HMM analysis. Our algorithm can execute accurate state number estimation and parameter optimization with fully automated way. Simulation analysis demonstrated that our algorithm accurately fit both fast- and slow-transition trajectories. Compared with the previous method, the speed of our algorithm was 10-20 times faster for standard size data. Our algorithm also showed the accurate fit of the simulated motor-stepping data with more than 10 transition states, suggesting the applicability of the method to the data with numerous states. Furthermore, the algorithm is flexible enough to cope with cases where some kinetics are known in advance. Some available prior information, such as the dwell time of each state, can be integrated into the algorithm via two user-tunable parameters. In summary, our method enables fast, accurate and objective HMM analysis, and broadens the application range of HMM fitting that can provide more accurate interpretation of a wide variety of biophysical data.

biophysics↗

IN VITRO RECONSTITUTION OF DYNEIN FORCE EXERTION IN BULK CYTOPLASM

The forces generated by Microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [1, 2]. To date, most studies have focused on force exertion from motors anchored on a static surface, such as the cell cortex in vivo or glass surfaces in vitro [2-4]. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization and orientation [5-14], but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo size and medium viscosities, shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations, or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study places endomembrane transport as a significant mode of MT force exertion with far-reaching consequences for cellular organization.

biophysics↗

Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9

SHORT ABSTRACTThis paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRET LONG ABSTRACTThe CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.

biophysics↗

Diverse stepping motions of cytoplasmic dynein revealed by kinetic modeling

Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of microtubule (MT) using ATP hydrolysis free energy. By employing its two heads (motor domains), cytoplasmic dynein shows various bipedal stepping motions; the inchworm and hand-over-hand motions, as well as non-alternate steps of one head. However, the molecular basis to achieve such diverse stepping manners remains obscure. Here, we propose a kinetic model for bipedal motions of cytoplasmic dynein and performed Gillespie Monte Carlo simulations that reproduces most experimental data obtained to date. The model represents status of each motor domain as five states according to conformations, nucleotide- and MT-binding conditions of the domain. Also, the relative positions of the two domains were approximated by three discrete states. Accompanied by ATP hydrolysis cycles, the model dynein stochastically and processively moved forward in multiple steps via diverse pathways, including inchworm and hand-over-hand motions, same as experimental data. The model reproduced key experimental motility-related parameters including velocity and run-length as functions of ATP concentration and external force. Our model reveals that, in a typical inchworm motion, the leading domain moves via the ATP-dependent power-stroke of the linker coupled with a small change in the stalk angle, whereas the lagging domain moves via diffusion dragged by the leading domain. Moreover, the hand-over-hand motion in the model dynein clearly differs from that of kinesin by the usage of the power-stroke.\n\nAuthor SummaryCytoplasmic dynein is a two-headed molecular motor, which moves linearly and transports intra-cellar organelles along microtubules driven by ATP hydrolysis free energy. In contrast to other better-known molecular motors, such as kinesin, dynein is known to take various stepping motions including motions akin to human walking and inchworm-like motions. However, molecular mechanisms underpinning the diverse stepping motions are unclear. Here, based on recent high-resolution structure information and single-molecule motility assay data, we designed a kinetic model that explicitly include two heads, each of which makes ATP hydrolysis cycles and moves along the microtubules. Using the model, we performed Monte Carlo simulations. The simulation reproduced most of currently available experimental results. More importantly, the simulation suggested molecular mechanisms of various stepping motions. While stepping motions apparently resemble to those proposed before, once looking into details, we found the resulting mechanisms distinct from previously proposed ones in the usage of ATP and protein conformation changes coupled with stepping motions.

biophysics↗

Second harmonic generation polarization microscopy as a tool for protein structure analysis

Second-harmonic generation (SHG) is a nonlinear coherent scattering process that is sensitive to molecular structures in illuminated materials. We report SHG polarization measurement for the detection of protein conformational changes in solutions of macromolecular protein assemblies such as microtubules and protein crystals. The results illustrate the potential of this method for protein structural analysis in physiological solutions at room temperature without labelling.

biophysics↗

Real-time observation of flexible domain movements in Cas9

The CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

biophysics↗