RESUMO
Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.
Assuntos
Caenorhabditis elegans , Proteína Neuronal da Síndrome de Wiskott-Aldrich , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Animais , Humanos , Caenorhabditis elegans/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Sinapsinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
The biomechanical regulation of the cytoskeleton and cell adhesions underlies various essential cellular functions. Studying them requires visualizing their nanostructure and molecular dynamics with evermore precise spatio-temporal resolution. In this review we will focus on the recent advances in single molecule fluorescence imaging techniques and discuss how they improve our understanding of mechanically sensitive cellular structures such as adhesions and the cytoskeleton. We will also discuss future directions for research, emphasizing on the 3D nature of cellular structures and tissues, their mechanical regulation at the molecule level, as well as how super-resolution microscopy will enhance our knowledge on protein structure and conformational changes in the cellular context.
Assuntos
Citoesqueleto , Imagem Individual de Molécula , Humanos , Animais , Citoesqueleto/metabolismo , Fenômenos Biomecânicos , Adesão Celular , BiofísicaRESUMO
Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.
Assuntos
Corantes Fluorescentes , Neurônios , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Neurônios/metabolismo , Corantes Fluorescentes/metabolismo , Sinapses/metabolismoRESUMO
Neuronal transmission relies on the regulated secretion of neurotransmitters, which are packed in synaptic vesicles (SVs). Hundreds of SVs accumulate at synaptic boutons. Despite being held together, SVs are highly mobile, so that they can be recruited to the plasma membrane for their rapid release during neuronal activity. However, how such confinement of SVs corroborates with their motility remains unclear. To bridge this gap, we employ ultrafast single-molecule tracking (SMT) in the reconstituted system of native SVs and in living neurons. SVs and synapsin 1, the most highly abundant synaptic protein, form condensates with liquid-like properties. In these condensates, synapsin 1 movement is slowed in both at short (i.e., 60-nm) and long (i.e., several hundred-nm) ranges, suggesting that the SV-synapsin 1 interaction raises the overall packing of the condensate. Furthermore, two-color SMT and super-resolution imaging in living axons demonstrate that synapsin 1 drives the accumulation of SVs in boutons. Even the short intrinsically-disordered fragment of synapsin 1 was sufficient to restore the native SV motility pattern in synapsin triple knock-out animals. Thus, synapsin 1 condensation is sufficient to guarantee reliable confinement and motility of SVs, allowing for the formation of mesoscale domains of SVs at synapses in vivo.
Assuntos
Sinapsinas , Vesículas Sinápticas , Animais , Vesículas Sinápticas/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais Geneticamente ModificadosRESUMO
Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.
Assuntos
Citoesqueleto , Microtúbulos , Movimento Celular , Polímeros , Projetos de PesquisaRESUMO
Focal adhesions are composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Adhesion depends on the activation of integrins. Talin and kindlin proteins are intracellular activators of integrins that bind to ß-integrin cytoplasmic tails. Integrin activation and clustering through extracellular ligands guide the organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we used a biomimetic in vitro system, made of giant unilamellar vesicles, containing transmembrane integrins (herein αIIbß3), with purified talin (talin-1), kindlin (kindlin-2, also known as FERMT2) and actomyosin. Here, we show that talin and kindlin individually have the ability to cluster integrins. Talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin increases integrin-talin-actomyosin coupling. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion.
Assuntos
Integrinas , Talina , Integrinas/metabolismo , Talina/genética , Talina/metabolismo , Actomiosina , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Adesão CelularRESUMO
Detection and conversion of mechanical forces into biochemical signals is known as mechanotransduction. From cells to tissues, mechanotransduction regulates migration, proliferation, and differentiation in processes such as immune responses, development, and cancer progression. Mechanosensitive structures such as integrin adhesions, the actin cortex, ion channels, caveolae, and the nucleus sense and transmit forces. In vitro approaches showed that mechanosensing is based on force-dependent protein deformations and reorganizations. However, the mechanisms in cells remained unclear since cell imaging techniques lacked molecular resolution. Thanks to recent developments in super-resolution microscopy (SRM) and molecular force sensors, it is possible to obtain molecular insight of mechanosensing in live cells. We discuss how understanding of molecular mechanotransduction was revolutionized by these innovative approaches, focusing on integrin adhesions, actin structures, and the plasma membrane.
Assuntos
Actinas , Mecanotransdução Celular , Humanos , Mecanotransdução Celular/fisiologia , Actinas/metabolismo , Citoesqueleto/metabolismo , Integrinas/metabolismo , Microscopia , Adesão Celular , Citoesqueleto de Actina/metabolismoRESUMO
The spatial organization of cell-surface receptors is fundamental for the coordination of biological responses to physical and biochemical cues of the extracellular matrix. How serine/threonine kinase receptors, ALK3-BMPRII, cooperate with integrins upon BMP2 to drive cell migration is unknown. Whether the dynamics between integrins and BMP receptors intertwine in space and time to guide adhesive processes is yet to be elucidated. We found that BMP2 stimulation controls the spatial organization of BMPRs by segregating ALK3 from BMPRII into ß3 integrin-containing focal adhesions. The selective recruitment of ALK3 to focal adhesions requires ß3 integrin engagement and ALK3 activation. BMP2 controls the partitioning of immobilized ALK3 within and outside focal adhesions according to single-protein tracking and super-resolution imaging. The spatial control of ALK3 in focal adhesions by optogenetics indicates that ALK3 acts as an adhesive receptor by eliciting cell spreading required for cell migration. ALK3 segregation from BMPRII in integrin-based adhesions is a key aspect of the spatio-temporal control of BMPR signaling.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Integrina beta3 , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Adesão Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Integrina beta3/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Intermediate filaments (IFs) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49-nanometer axial repeat both in cells and in vitro. As unit-length filaments (ULFs) were measured at ~59 nanometers, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical, and structural properties in cells.
RESUMO
Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration.
Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Movimento Celular , Fibroblastos/metabolismo , Mecanotransdução Celular , Pseudópodes/metabolismo , Citoesqueleto de Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Animais , Linhagem Celular Transformada , Camundongos , Microscopia de Fluorescência , Pinças Ópticas , Imagem Individual de Molécula , Estresse Mecânico , Fatores de TempoRESUMO
Giant unilamellar vesicles (GUV) are powerful tools to explore physics and biochemistry of the cell membrane in controlled conditions. For example, GUVs were extensively used to probe cell adhesion, but often using non-physiological linkers, due to the difficulty of incorporating transmembrane adhesion proteins into model membranes. Here we describe a new protocol for making GUVs incorporating the transmembrane protein integrin using gel-assisted swelling. We report an optimised protocol, enumerating the pitfalls encountered and precautions to be taken to maintain the robustness of the protocol. We characterise intermediate steps of small proteoliposome formation and the final formed GUVs. We show that the integrin molecules are successfully incorporated and are functional.
Assuntos
Géis/química , Integrinas/metabolismo , Lipossomas Unilamelares/química , Adesão Celular , Fluorescência , Humanos , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Tamanho da PartículaRESUMO
Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.
Assuntos
Membrana Celular/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Integrinas/genética , Proteínas de Membrana/genética , Camundongos Knockout , Movimento (Física) , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Proteínas de Neoplasias/genética , Ligação ProteicaRESUMO
Cell migration is a complex biophysical process which involves the coordination of molecular assemblies including integrin-dependent adhesions, signaling networks and force-generating cytoskeletal structures incorporating both actin polymerization and myosin activity. During the last decades, proteomic studies have generated impressive protein-protein interaction maps, although the subcellular location, duration, strength, sequence, and nature of these interactions are still concealed. In this chapter we describe how recent developments in superresolution microscopy (SRM) and single-protein tracking (SPT) start to unravel protein interactions and actions in subcellular molecular assemblies driving cell migration.
Assuntos
Movimento Celular , Integrinas/metabolismo , Microscopia/métodos , Optogenética/métodos , Mapeamento de Interação de Proteínas/métodos , Imagem Individual de Molécula/métodos , Actinas/genética , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular Transformada , Criptocromos/genética , Criptocromos/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Expressão Gênica , Integrinas/genética , Camundongos , Microscopia/instrumentação , Miosinas/genética , Miosinas/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Detection and conversion of mechanical forces into biochemical signals controls cell functions during physiological and pathological processes. Mechanosensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell-stretching device compatible with super-resolution microscopy and single-protein tracking, we explored the nanoscale deformations and reorganizations of individual proteins inside mechanosensitive structures. We achieved super-resolution microscopy after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous single-protein tracking and stretching showed that while integrins followed the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodelling and talin deformations. Capturing acute reorganizations of single molecules during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements, enabling protein deformation and recruitment in mechanosensitive structures.
Assuntos
Actinas/fisiologia , Forma Celular , Animais , Fenômenos Biomecânicos , Células Cultivadas , Técnicas Citológicas , Humanos , Integrinas/metabolismo , Camundongos , Microscopia/métodos , Nanoestruturas , Dobramento de Proteína , Transporte Proteico , Talina/metabolismo , Vinculina/metabolismoRESUMO
Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Cell migration is an essential process, both in unicellular organisms such as amoeba and as individual or collective motility in highly developed multicellular organisms like mammals. It is controlled by a variety of activities combining protrusive and contractile forces, normally generated by actin filaments. Here, we summarize actin filament assembly and turnover processes, and how respective biochemical activities translate into different protrusion types engaged in migration. These actin-based plasma membrane protrusions include actin-related protein 2/3 complex-dependent structures such as lamellipodia and membrane ruffles, filopodia as well as plasma membrane blebs. We also address observed antagonisms between these protrusion types, and propose a model - also inspired by previous literature - in which a complex balance between specific Rho GTPase signaling pathways dictates the protrusion mechanism employed by cells. Furthermore, we revisit published work regarding the fascinating antagonism between Rac and Rho GTPases, and how this intricate signaling network can define cell behavior and modes of migration. Finally, we discuss how the assembly of actin filament networks can feed back onto their regulators, as exemplified for the lamellipodial factor WAVE regulatory complex, tightly controlling accumulation of this complex at specific subcellular locations as well as its turnover.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Polaridade Celular/fisiologia , Polimerização , Pseudópodes/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.
Assuntos
Movimento Celular , Extensões da Superfície Celular/metabolismo , Fibroblastos/metabolismo , Neuropeptídeos/metabolismo , Pseudópodes/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Camundongos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Multicolor single-molecule localization microscopy (λSMLM) is a powerful technique to reveal the relative nanoscale organization and potential colocalization between different molecular species. While several standard analysis methods exist for pixel-based images, λSMLM still lacks such a standard. Moreover, existing methods only work on 2D data and are usually sensitive to the relative molecular organization, a very important parameter to consider in quantitative SMLM. Here, we present an efficient, parameter-free colocalization analysis method for 2D and 3D λSMLM using tessellation analysis. We demonstrate that our method allows for the efficient computation of several popular colocalization estimators directly from molecular coordinates and illustrate its capability to analyze multicolor SMLM data in a robust and efficient manner.
RESUMO
Cells are mechanical living machines that remodel their microenvironment by adhering and generating forces on the extracellular matrix (ECM) using integrin-dependent adhesion sites (IAS). In return, the biochemical and physical nature of the ECM determines cellular behavior and morphology during proliferation, differentiation and migration. IAS come in different shapes and forms. They have specific compositions, morphologies, mechanical and biochemical signaling activities, which serve different cellular functions. Proteomic studies showed that IAS are composed of a large repertoire of proteins that could be linked to different functional activities, including signaling, force-transmission and force-sensing. Thanks to recent technological advances in microscopy and protein engineering, it is now possible to localize single proteins in three dimensions inside IAS, determine their diffusive behaviors, orientations, and how much mechanical force is transmitted across individual components. Here, we review how researchers have used those tools to investigate how IAS components assemble and dynamically interact to produce diverse functions of adhesive structures.