RESUMO
Optimal single particle tracking experiments in live cells requires small and photostable probes, which do not modify the behavior of the molecule of interest. Current fluorescence-based microscopy of single molecules and nanoparticles is often limited by bleaching and blinking or by the probe size. As an alternative, we present in this chapter the synthesis of a small and highly specific gold nanoprobe whose detection is based on its absorption properties. We first present a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them with a nanobody, a single-domain antibody from camelid, targeting the widespread green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we describe how to detect and track these individual gold nanoparticles in live cell using photothermal imaging microscopy. The combination of a probe with small size, perfect photostability, high specificity, and versatility through the vast existing library of GFP-proteins, with a highly sensitive detection technique enables long-term tracking of proteins with minimal hindrance in confined and crowded environments such as intracellular space.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Imagem Molecular/métodos , Animais , Células COS , Sobrevivência Celular , Chlorocebus aethiops , Endocitose , Proteínas de Fluorescência Verde/metabolismoRESUMO
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
The Scar/Wave complex (SWC) generates lamellipodia through Arp2/3-dependent polymerisation of branched actin networks. In order to identify new SWC regulators, we conducted a screen in Drosophila cells combining proteomics with functional genomics. This screen identified Clathrin heavy chain (CHC) as a protein that binds to the SWC and whose depletion affects lamellipodium formation. This role of CHC in lamellipodium formation can be uncoupled from its role in membrane trafficking by several experimental approaches. Furthermore, CHC is detected in lamellipodia in the absence of the adaptor and accessory proteins of endocytosis. We found that CHC overexpression decreased membrane recruitment of the SWC, resulting in reduced velocity of protrusions and reduced cell migration. By contrast, when CHC was targeted to the membrane by fusion to a myristoylation sequence, we observed an increase in membrane recruitment of the SWC, protrusion velocity and cell migration. Together these data suggest that, in addition to its classical role in membrane trafficking, CHC brings the SWC to the plasma membrane, thereby controlling lamellipodium formation.
Assuntos
Clatrina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas dos Microfilamentos/metabolismo , Pseudópodes/metabolismo , Animais , Movimento Celular/genética , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/patologia , Clatrina/genética , Drosophila , Proteínas de Drosophila/genética , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Ligação Proteica/genética , Transporte Proteico/genética , Proteômica , Pseudópodes/patologia , Deleção de Sequência/genética , Transgenes/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
The Arp2/3 complex generates branched actin networks when activated by Nucleation Promoting Factors (NPFs). Recently, the WASH family of NPFs has been identified, but its cellular role is unclear. Here, we show that WASH generates an actin network on a restricted domain of sorting and recycling endosomes. We found that WASH belongs to a multiprotein complex containing seven subunits, including the heterodimer of capping protein (CP). In vitro, the purified WASH complex activates Arp2/3-mediated actin nucleation and binds directly to liposomes. WASH also interacts with dynamin. WASH depletion gives rise to long membrane tubules pulled out from endosomes along microtubules, as does dynamin inhibition. Accordingly, WASH is required for efficient transferrin recycling. Together, these data suggest that the WASH molecular machine, integrating CP with a NPF, controls the fission of endosomes through an interplay between the forces generated by microtubule motors and actin polymerization.
