RESUMO
Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Espinhas Dendríticas/fisiologia , Modelos Biológicos , Densidade Pós-Sináptica/metabolismo , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Forminas , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase , Proteínas , Ratos , Ratos Sprague-Dawley , Estatísticas não ParamétricasRESUMO
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
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
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
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
To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.
Assuntos
Movimento Celular/fisiologia , Transporte Proteico/fisiologia , Animais , Linhagem Celular , Humanos , Camundongos , Modelos BiológicosRESUMO
Expression of aversive memories is key for survival, but the underlying brain mechanisms are not fully understood. Medial habenular (MHb) axons corelease glutamate and acetylcholine onto target postsynaptic interpeduncular (IPN) neurons, but their role in aversive memories has not been addressed so far. We found that cannabinoid type 1 receptors (CB1R), key regulators of aversive responses, are present at presynaptic terminals of MHb neurons in the IPN. Conditional deletion of CB1R from MHb neurons reduces fear-conditioned freezing and abolishes conditioned odor aversion in mice, without affecting neutral or appetitively motivated memories. Interestingly, local inhibition of nicotinic, but not glutamatergic receptors in the target region IPN before retrieval, rescues these phenotypes. Finally, optogenetic electrophysiological recordings of MHb-to-IPN circuitry revealed that blockade of CB1R specifically enhances cholinergic, but not glutamatergic, neurotransmission. Thus, presynaptic CB1R control expression of aversive memories by selectively modulating cholinergic transmission at MHb synapses in the IPN.
Assuntos
Aprendizagem da Esquiva/fisiologia , Medo/fisiologia , Habenula/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Animais , Medo/psicologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor CB1 de Canabinoide/deficiênciaRESUMO
It was recently shown that perisomatic GABAergic inhibitory postsynaptic potentials (IPSPs) originating from basket and chandelier cells can be recorded as population IPSPs from the hippocampal pyramidal layer using extracellular electrodes (eIPSPs). Taking advantage of this approach, we have investigated the recruitment of perisomatic inhibition during spontaneous hippocampal activity in vitro. Combining intracellular and extracellular recordings from pyramidal cells and interneurons, we confirm that inhibitory signals generated by basket cells can be recorded extracellularly, but our results suggest that, during spontaneous activity, eIPSPs are mostly confined to the CA3 rather than CA1 region. CA3 eIPSPs produced the powerful time-locked inhibition of multi-unit activity expected from perisomatic inhibition. Analysis of the temporal dynamics of spike discharges relative to eIPSPs suggests significant but moderate recruitment of excitatory and inhibitory neurons within the CA3 network on a 10 ms time scale, within which neurons recruit each other through recurrent collaterals and trigger powerful feedback inhibition. Such quantified parameters of neuronal interactions in the hippocampal network may serve as a basis for future characterisation of pathological conditions potentially affecting the interactions between excitation and inhibition in this circuit.