Membrane-induced 2D phase separation of the focal adhesion protein talin.

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P2-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.

Molecular cartography within axons.

Recent advances in imaging methods begin to further illuminate the inner workings of neurons. Views of the axonal landscape through the lens of in situ cryo-electron tomography (cryo-ET) provide a high-resolution atlas of the macromolecular organization in near-native conditions, leading to our growing understanding of the vital roles of compositional and structural organization in maintaining neuronal homeostasis. In this review, we discuss the latest observations concerning the fundamental components found within neuronal compartments, with special emphasis on the axon, branch points, and growth cone. We describe the similarity and difference in organization of organelles and molecules in varying compartments. Finally, we highlight outstanding questions on the dynamics and localization of various components along the axon that may be answered using orthogonal approaches.

Cryo-electron microscopy in the fight against COVID-19-mechanism of virus entry.

Cryogenic electron microscopy (cryo-EM) and electron tomography (cryo-ET) have become a critical tool for studying viral particles. Cryo-EM has enhanced our understanding of viral assembly and replication processes at a molecular resolution. Meanwhile, in situ cryo-ET has been used to investigate how viruses attach to and invade host cells. These advances have significantly contributed to our knowledge of viral biology. Particularly, prompt elucidations of structures of the SARS-CoV-2 spike protein and its variants have directly impacted the development of vaccines and therapeutic measures. This review discusses the progress made by cryo-EM based technologies in comprehending the severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2), the virus responsible for the devastating global COVID-19 pandemic in 2020 with focus on the SARS-CoV-2 spike protein and the mechanisms of the virus entry and replication.

The prevalence of shoulders with a large glenoid defect and small bone fragment increases after several instability events during conservative treatment for traumatic anterior instability.

Background: Unstable shoulders with a large glenoid defect and small bone fragment are at higher risk for postoperative recurrence after arthroscopic Bankart repair. The purpose of the present study was to clarify the changes in the prevalence of such shoulders during conservative treatment for traumatic anterior instability. Methods: We retrospectively investigated 114 shoulders that underwent conservative treatment and computed tomography (CT) examination at least twice after an instability event in the period from July 2004 to December 2021. We investigated the changes in glenoid rim morphology, glenoid defect size, and bone fragment size from the first to the final CT. Results: At first CT, 51 shoulders showed no glenoid bone defect, 12 showed glenoid erosion, and 51 showed a glenoid bone fragment [33 small bone fragment (<7.5%) and 18 large bone fragment (≥7.5%); mean size: 4.9 ± 4.2% (0-17.9%)]. Among patients with glenoid defect (fragment and erosion), the mean glenoid defect was 5.4 ± 6.6% (0-26.6%); 49 were considered a small glenoid defect (<13.5%) and 14 were a large glenoid defect (≥13.5%). While all 14 shoulders with large glenoid defect had a bone fragment, small fragment was solely seen in 4 shoulders. At final CT, 23 of the 51 shoulders persisted without glenoid defect. The number of shoulders presenting glenoid erosion increased from 12 to 24, and the number of shoulders with bone fragment increased from 51 to 67 [36 small bone fragment and 31 large bone fragment; mean size: 5.1 ± 4.9% (0-21.1%)]. The prevalence of shoulders with no or a small bone fragment did not increase from first CT (71.4%) to final CT (65.9%; P = .488), and the bone fragment size did not decrease (P = .753). The number of shoulders with glenoid defect increased from 63 to 91 and the mean glenoid defect significantly increased to 9.9 ± 6.6% (0-28.4%) (P < .001). The number of shoulders with large glenoid defect increased from 14 to 42 (P < .001). Of these 42 shoulders, 19 had no or a small bone fragment. Accordingly, among a total of 114 shoulders, the increase from first to final CT in the prevalence of a large glenoid defect accompanied by no or a small bone fragment was significant [4 shoulders (3.5%) vs. 19 shoulders (16.7%); P = .002]. Conclusions: The prevalence of shoulders with a large glenoid defect and small bone fragment increases significantly after several instability events.

Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 spike protein.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging shows that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Cellular cryo-electron tomography reveals dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and find that S protein recognizes integrin αvß3. Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

Anterior glenoid rim erosion in the early stage after arthroscopic Bankart repair affects postoperative recurrence.

Background: Recent studies reported that anterior glenoid rim erosion can occur in the early period after arthroscopic Bankart repair (ABR) for traumatic anterior shoulder instability. However, it is unknown whether such erosion is a risk factor for postoperative recurrence. This study evaluated risk factors for postoperative recurrence after ABR, specifically aiming to elucidate whether reduction of postoperative glenoid width due to anterior glenoid rim erosion is one of such factors. Methods: A total of 220 shoulders that underwent ABR alone between 2013 and 2020 were retrospectively investigated. Patient age at surgery, whether the patient was a collision/contact athlete, anchor placement, preoperative glenoid bone defect (%), localization of the Hill-Sachs lesion, and change of glenoid width (%) in the 6 months after surgery were investigated for their statistical relation to recurrence by univariate and multiple logistic regression analysis. Results: Postoperative recurrence occurred in 32 of 220 shoulders (14.5%). In univariate analysis, being a collision/contact athlete was the only variable with a significant effect on recurrence (odds ratio [OR], 2.555; 95% confidence interval [CI], 1.123-5.814; P = .03). Change of glenoid width reduction was larger in those with recurrence than without recurrence, but the difference was not statistically significant (-7.0 ± 6.6% vs. -5.0 ± 9.3%; P = .14). However, in multivariate logistic analysis, preoperative glenoid bone defect (%) (adjusted unit OR, 1.076; 95% CI, 1.018-1.137; P = .010) and postoperative change of glenoid width (%) (adjusted unit OR, 0.946; 95% CI, 0.900-0.994; P = .028) had a significant influence on postoperative recurrence. Conclusion: Glenoid width reduction due to anterior glenoid rim erosion after ABR is a risk factor for recurrence.

Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 Spike protein.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin α v ß 3 . Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

Molecular neuroscience community shares perspectives.

In May, an interdisciplinary group gathered in Crete for the Molecular Neurobiology Workshop. Scientists shared data acquired by vastly diverse techniques to understand how the nervous system, with only a limited number of components, is assembled to respond to infinite stimuli. Ideas of molecular cues, timing, switching, and context emerged.

In situ cryo-electron tomography reveals local cellular machineries for axon branch development.

Neurons are highly polarized cells forming an intricate network of dendrites and axons. They are shaped by the dynamic reorganization of cytoskeleton components and cellular organelles. Axon branching allows the formation of new paths and increases circuit complexity. However, our understanding of branch formation is sparse due to the lack of direct in-depth observations. Using in situ cellular cryo-electron tomography on primary mouse neurons, we directly visualized the remodeling of organelles and cytoskeleton structures at axon branches. Strikingly, branched areas functioned as hotspots concentrating organelles to support dynamic activities. Unaligned actin filaments assembled at the base of premature branches accompanied by filopodia-like protrusions. Microtubules and ER comigrated into preformed branches to support outgrowth together with accumulating compact, ∼500-nm mitochondria and locally clustered ribosomes. We obtained a roadmap of events supporting the hypothesis of local protein synthesis selectively taking place at axon branches, allowing them to serve as unique control hubs for axon development and downstream neural network formation.

Bottom-up reconstitution of focal adhesion complexes.

Focal adhesions (FA) are large macromolecular assemblies relevant for various cellular and pathological events such as migration, polarization, and metastatic cancer formation. At FA sites at the migrating periphery of a cell, hundreds of players gather and form a network to respond to extra cellular stimuli transmitted by the integrin receptor, the most upstream component within a cell, initiating the FA signaling pathway. Numerous cellular experiments have been performed to understand the FA architecture and functions; however, their intricate network formation hampers unraveling the precise molecular actions of individual players. Here, in vitro bottom-up reconstitution presents an advantageous approach for elucidating the FA machinery and the hierarchical crosstalk of involved cellular players.

Tolerability of the first infusion of once-yearly zoledronic acid within one to two weeks after hip fracture surgery.

OBJECTIVE: Once-yearly infusions of zoledronic acid (ZA) 5 mg may be optimal for secondary fracture prevention after hip fracture (HF), but there are crucial side effects of ZA. This study assessed the tolerability of the first infusion of once-yearly ZA within one to two weeks after HF surgery and to identify risk factors for acute-phase reactions (APRs) and the decrease in serum calcium (Ca) concentration. METHODS: We analyzed 84 patients (average age: 83 years, 18 men and 66 women) who met the inclusion criteria. The patients underwent the first infusion of ZA one to two weeks after HF surgery and received antipyretic analgesics and active vitamin D analog. RESULTS: APRs occurred in ten patients (11.9%) and all these patients had pyrexia (>37.5 °C) and/or other symptoms. The asymptomatic hypocalcemia (serum Ca < 8.3 mg/dL) incidence was 6.0% at 7 days after ZA infusion. Compared with female patients without APRs, female patients with APRs had significantly higher levels of serum 25-dihydroxyvitamin D at baseline and serum C-reactive protein on the day ZA was administered (day 0). Multiple linear regression analyses showed that serum level of tartrate-resistant acid phosphatase-5b were significantly associated with an absolute decrease in serum corrected Ca from day 0 to day 7. CONCLUSIONS: The first infusion of ZA within one to two weeks after HF surgery was well tolerated with the combined use of antipyretic analgesics and active vitamin D analog. Higher inflammatory condition after surgery which is more likely sensitized by ZA administration may increase the risk of APRs, and high bone turnover may increase hypocalcemia risk.

Cytoskeleton and Membrane Organization at Axon Branches.

Axon branching is a critical process ensuring a high degree of interconnectivity for neural network formation. As branching occurs at sites distant from the soma, it is necessary that axons have a local system to dynamically control and regulate axonal growth. This machinery depends on the orchestration of cellular functions such as cytoskeleton, subcellular transport, energy production, protein- and membrane synthesis that are adapted for branch formation. Compared to the axon shaft, branching sites show a distinct and dynamic arrangement of cytoskeleton components, endoplasmic reticulum and mitochondria. This review discusses the regulation of axon branching in the context of cytoskeleton and membrane remodeling.

Structural insights into integrin α5ß1 opening by fibronectin ligand.

Integrin α5ß1 is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and α5ß1 undergo conformational changes. While this is key for cell-tissue connections, its mechanism is unknown. Here, we report cryo-electron microscopy structures of native human α5ß1 with fibronectin to 3.1-angstrom resolution, and in its resting state to 4.6-angstrom resolution. The α5ß1-fibronectin complex revealed simultaneous interactions at the arginine-glycine-aspartate loop, the synergy site, and a newly identified binding site proximal to adjacent to metal ion-dependent adhesion site, inducing the translocation of helix α1 to secure integrin opening. Resting α5ß1 adopts an incompletely bent conformation, challenging the model of integrin sharp bending inhibiting ligand binding. Our biochemical and structural analyses showed that affinity of α5ß1 for fibronectin is increased with manganese ions (Mn2+) while adopting the half-bent conformation, indicating that ligand-binding affinity does not depend on conformation, and α5ß1 opening is induced by ligand-binding.

Reconstitution of contractile actomyosin rings in vesicles.

One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.

Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin.

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

Mitochondrial dysfunction generates aggregates that resist lysosomal degradation in human breast cancer cells.

Disrupting functional protein homeostasis is an established therapeutic strategy for certain tumors. Ongoing studies are evaluating autophagy inhibition for overcoming chemotherapeutic resistance to such therapies by neutralizing lysosomal pH. New and sensitive methods to monitor autophagy in patients are needed to improve trial design and interpretation. We report that mitochondrial-damaged breast cancer cells and rat breast tumors accumulate p53-positive protein aggregates that resist lysosomal degradation. These aggregates were localized to enzymatically-active autolysosomes that were degrading autophagosomes and the autophagic receptor proteins TAX1BP1 and NDP52. NDP52 was identified to associate with aggregated proteins and knocking down NDP52 led to the accumulation of protein aggregates. TAX1BP1 was identified to partly localize with aggregates, and knocking down TAX1BP1 enhanced aggregate formation, suppressed autophagy, impaired NDP52 autophagic degradation and induced cell death. We propose that quantifying aggregates and autophagic receptors are two potential methods to evaluate autophagy and lysosomal degradation, as confirmed using primary human tumor samples. Collectively, this report establishes protein aggregates and autophagy receptors, TAX1BP1 and NDP52, as potential endpoints for monitoring autophagy during drug development and clinical studies.

Insufficient integrity of partial articular surface tendon avulsion lesions after arthroscopic trans-tendon repair: Comparison with integrity after arthroscopic repair after conversion to a full thickness tear.

BACKGROUND: For the surgical treatment of severe partial articular surface tendon avulsion (PASTA) lesions, 2 repair techniques, i.e., arthroscopic trans-tendon repair and arthroscopic repair after conversion to a full thickness tear, are widely used, and a satisfactory clinical outcome with both has been reported. The purpose of this study was to investigate integrity of the repaired rotator cuff based on second-look arthroscopy and to compare the above two repair techniques. METHODS: Thirty-seven shoulders underwent PASTA lesion repair arthroscopically, with 20 shoulders receiving second-look arthroscopy. According to the repair technique, the shoulders were divided into 2 groups, which were 10 shoulders with trans-tendon repair (group P) and 10 shoulders with repair after conversion to a full thickness tear (group C). Second-look arthroscopy was done at a minimum of 3 months after initial surgery, with the mean interval until second-look arthroscopy being 5.6 months (3-13) in group P and 5.1 months (3-9) in group C. The reasons for second-look arthroscopy were pain with contracture in 15 patients, as well as pain in 4 patients and muscle weakness at abduction in one patient. RESULTS: In group P, there was complete rotator cuff continuity in 3 shoulders, partial continuity in 4, and failure in 3, while group C had complete continuity in 8 shoulders, partial continuity in 1, and failure in 1. Adhesions of the subacromial bursa were seen in all shoulders, while contracture of the posterior capsule was noted in 5 shoulders from group P and 9 shoulders from group C, and contracture of the rotator interval was identified in 7 shoulders from group P and 9 shoulders from group C. CONCLUSIONS: Second-look arthroscopy revealed that the integrity of the rotator cuff after trans-tendon repair of severe PASTA lesions was often unsatisfactory in patients with symptomatic shoulder. On the other hand, complete continuity was seen in most shoulders underwent repair after conversion to a full thickness tear.

Benefits of bone graft augmentation to arthroscopic Bankart repair for recurrent anterior shoulder instability with glenoid bone loss.

PURPOSE: Glenoid bone loss contributes to recurrent instability after arthroscopic Bankart repair alone. With significant glenoid bone loss, better results have been reported after arthroscopic Bankart repair with glenoid arc reconstruction. However, no reports compare augmentation using bone graft with non-augmentation for glenoid bone loss. The purpose of this study was to assess clinical results of an arthroscopic Bankart repair with or without arthroscopic bone graft augmentation. It was hypothesized that such bone graft augmentation would restore shoulder stability, and lead to excellent outcomes. METHODS: Of 552 patients treated for anterior glenohumeral instability with arthroscopic Bankart repair, 68 met this study's inclusion criteria of glenoid bone loss over 20% and follow-up of at least 2 years. Patients were divided into 2 groups based on whether with bone graft augmentation for glenoid bone loss [Group A: n = 35, median age; 21 years (range 13-72 years)], or not (Group B: n = 33, median age; 21 years (range 13-50 years)]. For grafting, either autologous iliac bone or artificial bone made of hydroxyapatite was used. Rowe score, recurrence rate, and return to sport were used to assess the results. RESULTS: Mean Rowe score was 95.0 (SD 10.6) in Group A and 69.7 (SD 27.2) in Group B (p < 0.05). The recurrence rate was 2.9% (1/36) in Group A and 48.5% (16/33) in Group B (p < 0.05). Regarding contact/collision athletes, 24 were contained in Group A and 22 in Group B. Of the patients with recurrence in Group B, 13 (59.1%) were contact/collision athletes. Finally, 50% of the contact/collision sports athletes for both groups returned to their sports at the same as pre-injury level. Of the 11 patients who returned to the same level of contact/collision sports in Group B, seven returned with residual instability. Nine athletes in Group A and 3 in Group B quit their sports for personal or social reasons. CONCLUSIONS: Bone graft augmentation was beneficial when used with Arthroscopic Bankart repair for recurrent anterior shoulder instability with glenoid bone loss. Especially, for recurrent anterior shoulder instability with glenoid bone loss in contact/collision sports athletes, bone graft augmentation should be strongly considered as beneficial. LEVEL OF EVIDENCE: Level IV.

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.