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1.
Cell ; 179(1): 120-131.e13, 2019 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-31539492

RESUMEN

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.


Asunto(s)
Adhesiones Focales/metabolismo , Dominios y Motivos de Interacción de Proteínas , Talina/química , Talina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Sitios de Unión , Adhesión Celular/fisiología , Microscopía por Crioelectrón , Citoesqueleto/metabolismo , Dimerización , Escherichia coli/metabolismo , Humanos , Integrinas/metabolismo , Modelos Moleculares , Unión Proteica , Transducción de Señal/fisiología , Vinculina/metabolismo
2.
Knee Surg Sports Traumatol Arthrosc ; 28(7): 2325-2333, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31667568

RESUMEN

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.


Asunto(s)
Artroplastia/métodos , Artroscopía/métodos , Lesiones de Bankart/cirugía , Trasplante Óseo , Luxación del Hombro/cirugía , Articulación del Hombro/cirugía , Adolescente , Adulto , Anciano , Amputación Quirúrgica , Atletas , Femenino , Humanos , Inestabilidad de la Articulación/cirugía , Masculino , Persona de Mediana Edad , Rango del Movimiento Articular , Escápula/cirugía , Hombro/cirugía , Deportes , Adulto Joven
3.
J Orthop Sci ; 25(6): 992-998, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32122745

RESUMEN

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.


Asunto(s)
Lesiones del Manguito de los Rotadores , Traumatismos de los Tendones , Artroscopía , Humanos , Manguito de los Rotadores , Lesiones del Manguito de los Rotadores/diagnóstico por imagen , Lesiones del Manguito de los Rotadores/cirugía , Traumatismos de los Tendones/diagnóstico por imagen , Traumatismos de los Tendones/cirugía , Tendones/cirugía , Resultado del Tratamiento
4.
Nature ; 483(7387): 53-8, 2012 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-22327295

RESUMEN

Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.


Asunto(s)
Proteínas Bacterianas/química , Hierro/metabolismo , Neisseria/metabolismo , Proteína A de Unión a Transferrina/química , Proteína A de Unión a Transferrina/metabolismo , Proteína B de Unión a Transferrina/química , Proteína B de Unión a Transferrina/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Transporte Biológico , Bovinos , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Neisseria/patogenicidad , Conformación Proteica , Dispersión del Ángulo Pequeño , Especificidad de la Especie , Relación Estructura-Actividad , Transferrina/química , Transferrina/metabolismo , Transferrina/ultraestructura , Proteína A de Unión a Transferrina/ultraestructura , Proteína B de Unión a Transferrina/ultraestructura , Difracción de Rayos X
5.
Proc Natl Acad Sci U S A ; 111(31): 11347-52, 2014 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-25059720

RESUMEN

p150(glued) belongs to a group of proteins accumulating at microtubule plus ends (+TIPs). It plays a key role in initiating retrograde transport by recruiting and tethering endosomes and dynein to microtubules. p150(glued) contains an N-terminal microtubule-binding cytoskeleton-associated protein glycine-rich (CAP-Gly) domain that accelerates tubulin polymerization. Although this copolymerization is well-studied using light microscopic techniques, structural consequences of this interaction are elusive. Here, using electron-microscopic and spectroscopic approaches, we provide a detailed structural view of p150(glued) CAP-Gly binding to microtubules and tubulin. Cryo-EM 3D reconstructions of p150(glued)-CAP-Gly complexed with microtubules revealed the recognition of the microtubule surface, including tubulin C-terminal tails by CAP-Gly. These binding surfaces differ from other retrograde initiation proteins like EB1 or dynein, which could facilitate the simultaneous attachment of all accessory components. Furthermore, the CAP-Gly domain, with its basic extensions, facilitates lateral and longitudinal interactions of tubulin molecules by covering the tubulin acidic tails. This shielding effect of CAP-Gly and its basic extensions may provide a molecular basis of the roles of p150(glued) in microtubule dynamics.


Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Frío , Microscopía por Crioelectrón , Complejo Dinactina , Dineínas/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólisis , Procesamiento de Imagen Asistido por Computador , Cinética , Microtúbulos/ultraestructura , Péptidos/química , Péptidos/metabolismo , Polimerizacion , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Eliminación de Secuencia , Relación Estructura-Actividad , Tubulina (Proteína)/ultraestructura
6.
Nucleic Acids Res ; 42(20): 12912-27, 2014 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-25348395

RESUMEN

The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork.


Asunto(s)
Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , ADN de Cadena Simple/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Nucleares/química , Proteína de Replicación A/química , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestructura , Proteínas de Unión al ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Modelos Moleculares , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestructura , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteína de Replicación A/metabolismo , Proteína de Replicación A/ultraestructura
7.
Proc Natl Acad Sci U S A ; 110(27): E2441-50, 2013 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-23776210

RESUMEN

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.


Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Bacteriófago mu/enzimología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Bacteriófago mu/genética , Sitios de Unión/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Imagenología Tridimensional , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Multimerización de Proteína/genética , Homología de Secuencia de Aminoácido , Transposasas/genética , Transposasas/metabolismo , Proteínas Virales/genética
8.
Int Orthop ; 40(1): 81-6, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26152243

RESUMEN

PURPOSE: We evaluated the pathologies of anterior capsular mechanism in older patients with recurrent anterior shoulder dislocation in the absence of full-thickness rotator cuff tears. METHODS: Three hundred and ninety-five shoulders with recurrent anterior shoulder dislocation were assessed. The patients were divided into three groups by the age at the first dislocation and the surgical treatment: group A (onset and treatment were at an age over 40 years), group B (onset was at an age under 40 years and treatment was at an age over 40 years) and group C (onset and treatment were at an age under 40 years). Groups A, B and C involved nine, 31 and 355 shoulders, respectively. RESULTS: The prevalence of an isolated Bankart lesion was 81.7 % in group C, 33.3 % in group A and 64.5 % in group B, and each of A and B was significantly lower than group C. The prevalence of an isolated capsular tear was 3.1 % in group C, while it was 33.3 % in group A, which was significantly higher. CONCLUSIONS: The prevalence of an isolated Bankart lesion was low and the prevalence of a capsular tear was high in older patients. We should keep in mind the existence of a capsular tear in older patients and examine the whole anterior capsular mechanism meticulously.


Asunto(s)
Cápsula Articular/patología , Luxación del Hombro/patología , Articulación del Hombro/patología , Adulto , Anciano , Artroscopía , Femenino , Humanos , Inestabilidad de la Articulación/cirugía , Laceraciones , Masculino , Persona de Mediana Edad , Prevalencia , Recurrencia , Luxación del Hombro/cirugía , Adulto Joven
9.
Biophys J ; 106(10): 2134-42, 2014 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-24853742

RESUMEN

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of ß-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-ß structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10-20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated ß-structure model for PolyQKd-33 and a model with two ß-solenoid protofibrils for PolyQKd-32. These data provide evidence for ß-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.


Asunto(s)
Sustitución de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos , Multimerización de Proteína , Concentración de Iones de Hidrógeno , Estructura Secundaria de Proteína
10.
J Biol Chem ; 288(24): 17620-30, 2013 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-23609437

RESUMEN

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.


Asunto(s)
Lipoproteínas/química , Nanopartículas/química , alfa-Sinucleína/química , Colesterol/química , Cromatografía en Gel , Microscopía por Crioelectrón , Transferencia Resonante de Energía de Fluorescencia , Humanos , Lipoproteínas/aislamiento & purificación , Lipoproteínas/ultraestructura , Membranas Artificiales , Membranas Mitocondriales/química , Nanopartículas/análisis , Nanopartículas/ultraestructura , Tamaño de la Partícula , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Fosfatidilserinas/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , alfa-Sinucleína/aislamiento & purificación , alfa-Sinucleína/ultraestructura
11.
Proc Natl Acad Sci U S A ; 108(8): 3252-7, 2011 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-21300906

RESUMEN

HET-s is a prion protein of the fungus Podospora anserina which, in the prion state, is active in a self/nonself recognition process called heterokaryon incompatibility. Its prionogenic properties reside in the C-terminal "prion domain." The HET-s prion domain polymerizes in vitro into amyloid fibrils whose properties depend on the pH of assembly; above pH 3, infectious singlet fibrils are produced, and below pH 3, noninfectious triplet fibrils. To investigate the correlation between structure and infectivity, we performed cryo-EM analyses. Singlet fibrils have a helical pitch of approximately 410 Å and a left-handed twist. Triplet fibrils have three protofibrils whose lateral dimensions (36 × 25 Å) and axial packing (one subunit per 9.4 Å) match those of singlets but differ in their supercoiling. At 8.5-Å resolution, the cross-section of the singlet fibril reconstruction is largely consistent with that of a ß-solenoid model previously determined by solid-state NMR. Reconstructions of the triplet fibrils show three protofibrils coiling around a common axis and packed less tightly at pH 3 than at pH 2, eventually peeling off. Taken together with the earlier observation that fragmentation of triplet fibrils by sonication does not increase infectivity, these observations suggest a novel mechanism for self-propagation, whereby daughter fibrils nucleate on the lateral surface of singlet fibrils. In triplets, this surface is occluded, blocking nucleation and thereby explaining their lack of infectivity.


Asunto(s)
Amiloide/química , Microscopía por Crioelectrón , Priones/química , Proteínas Fúngicas/química , Concentración de Iones de Hidrógeno , Infecciones/etiología , Espectroscopía de Resonancia Magnética , Podospora/química
12.
J Shoulder Elbow Surg ; 23(11): 1691-9, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24835298

RESUMEN

BACKGROUND: The Latarjet procedure is effective in managing anterior glenohumeral instability in the short term, but there is concern for postoperative arthritis. The purpose of this study was to evaluate the long-term functional outcome after the Latarjet procedure and to assess the prevalence of and risk factors for glenohumeral arthritis after this procedure. MATERIALS AND METHODS: A retrospective review was conducted of 68 Latarjet procedures at a mean of 20 years postoperatively. The mean age at surgery was 29.4 years. Functional outcome was determined by the Rowe score, subjective shoulder value, and recurrence of instability. Preoperative arthritis and postoperative radiographs were reviewed to evaluate the development or progression of arthritis. RESULTS: The mean Rowe score increased from 37.9 preoperatively to 89.6 at final follow-up (P < .001). The mean subjective shoulder value was 90.9% at final follow-up. The postoperative rate of recurrence was 5.9%. Of the 60 shoulders without arthritis preoperatively, 12 (20%) had developed arthritis at final follow-up. Among the 8 shoulders with preoperative arthritis (all stage 1), 4 (50%) demonstrated progression of arthritis at final follow-up. Overall, postoperative arthritis was stage 1 in 14.7%, stage 2 in 5.9%, and stage 3 in 8.8% of cases; no stage 4 arthritis was observed. Risk factors for postoperative arthritis were older age, high-demand sports activity, and lateral overhang of coracoid bone graft. CONCLUSION: The Latarjet procedure provides excellent long-term outcomes in the treatment of recurrent anterior glenohumeral instability. Twenty years after the Latarjet procedure, arthritis may develop or progress in 23.5% of cases, but the majority of arthritis is mild.


Asunto(s)
Inestabilidad de la Articulación/cirugía , Procedimientos Ortopédicos , Luxación del Hombro/cirugía , Articulación del Hombro/cirugía , Adolescente , Adulto , Artritis/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Procedimientos Ortopédicos/efectos adversos , Recuperación de la Función , Recurrencia , Estudios Retrospectivos , Adulto Joven
13.
Curr Opin Cell Biol ; 88: 102358, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38608424

RESUMEN

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.


Asunto(s)
Axones , Axones/metabolismo , Animales , Humanos , Microscopía por Crioelectrón , Conos de Crecimiento/metabolismo
14.
Nat Commun ; 15(1): 4986, 2024 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-38862544

RESUMEN

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.


Asunto(s)
Membrana Celular , Adhesiones Focales , Talina , Vinculina , Talina/metabolismo , Talina/química , Adhesiones Focales/metabolismo , Membrana Celular/metabolismo , Vinculina/metabolismo , Vinculina/química , Humanos , Animales , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Integrinas/metabolismo , Integrinas/química , Citoplasma/metabolismo , Unión Proteica , Separación de Fases
15.
J Biol Chem ; 287(35): 29301-11, 2012 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-22767608

RESUMEN

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.


Asunto(s)
Membrana Dobles de Lípidos/química , Micelas , Fosfatidilgliceroles/química , Pliegue de Proteína , alfa-Sinucleína/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Membrana Dobles de Lípidos/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosfatidilgliceroles/metabolismo , Estructura Secundaria de Proteína , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
16.
Knee Surg Sports Traumatol Arthrosc ; 21(7): 1598-602, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22729806

RESUMEN

PURPOSE: The role of posterior capsular tightness in throwing shoulder injury has not yet been clarified. Accordingly, the influence of posterior capsular tightness on the occurrence of throwing shoulder injury was investigated. METHODS: Sixty-one shoulders with throwing injury were retrospectively reviewed, including 50 tight shoulders and 11 non-tight shoulders. Occurrence of long head of biceps (LHB) lesions, superior glenohumeral ligament (SGHL) and middle glenohumeral ligament (MGHL) injuries, type 2 SLAP lesions, and supraspinatus and subscapularis tendon injuries was compared between the tight and non-tight groups. RESULTS: There were LHB lesions in 8 tight shoulders and 6 non-tight shoulders, SGHL injury in 14 and 8 shoulders, and subscapularis tendon injury in 6 and 5 shoulders, respectively, showing significant differences between tight and non-tight shoulders. In contrast, MGHL injury, type 2 SLAP lesions, and supraspinatus tendon injury showed no significant differences. The SLAP lesion was located anteriorly in 6 tight shoulders, posteriorly in 5, and combined in 4 versus 0, 3, and 0 for the non-tight shoulders, respectively, so anterior SLAP lesions only occurred in tight shoulders. Similarly, anterior supraspinatus tendon injuries had a higher incidence in tight shoulders than in non-tight shoulders (19 vs 3). CONCLUSIONS: Rotator interval lesions were frequent in non-tight shoulders, while anterior SLAP lesions and anterior supraspinatus tendon injuries were predominant in tight shoulders. The significance of posterior capsular tightness should be reconsidered. LEVEL OF EVIDENCE: Retrospective, Level IV.


Asunto(s)
Artroscopía , Traumatismos en Atletas/cirugía , Béisbol/lesiones , Cápsula Articular/fisiopatología , Ligamentos Articulares/cirugía , Lesiones del Hombro , Articulación del Hombro/fisiopatología , Articulación del Hombro/cirugía , Traumatismos de los Tendones/cirugía , Adulto , Traumatismos en Atletas/fisiopatología , Humanos , Ligamentos Articulares/lesiones , Masculino , Estudios Retrospectivos , Traumatismos de los Tendones/fisiopatología
17.
Front Mol Biosci ; 10: 1252529, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37867557

RESUMEN

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.

18.
JSES Int ; 7(1): 121-125, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36820429

RESUMEN

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.

19.
JSES Int ; 7(4): 538-543, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37426910

RESUMEN

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.

20.
Nat Commun ; 14(1): 620, 2023 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-36739444

RESUMEN

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.


Asunto(s)
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Plaquetas/metabolismo , Pandemias
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