RESUMO
Real-time optical imaging combined with single-molecule manipulation broadens the horizons for acquiring information about the spatiotemporal localization and the mechanical details of target molecules. To obtain an optical signal outside the focal plane without unintended interruption of the force signal in single-molecule optical imaging-force spectroscopy, we developed an optical method to extend the depth of field in a high numerical aperture objective (≥ 1.2), required to visualize a single fluorophore. By axial scanning, using an electrically tunable lens with a fixed sample, we were successfully able to visualize the epidermal growth factor receptor (EGFR) moving along the three-dimensionally elongated filamentous actin bundles connecting cells (intercellular nanotube), while another EGFR on the intercellular nanotube was trapped by optical tweezers in living cells. Our approach is simple, fast and inexpensive, but it is powerful for imaging target molecules axially in single-molecule optical imaging-force spectroscopy.
Assuntos
Citoesqueleto de Actina/química , Receptores ErbB/análise , Lentes , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Pinças Ópticas , Análise Espectral/métodos , Células HeLa , Humanos , Microscopia de Fluorescência/instrumentação , NanotubosRESUMO
Dendritic protrusions, mainly spines and filopodia, correlate with excitatory synapses in pyramidal neurons (PyNs), but this relationship may not apply universally. We found that ectopic H-Ras expression increased protrusions across various cortical cell types, including layer 2/3 PyNs, parvalbumin (PV)-, and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex. The probability of detecting protrusions correlated with local H-Ras activity, indicating its role in protrusion formation. H-Ras overexpression led to high turnover rates by adding protrusions. Two-photon photolysis of glutamate induced de novo spine formation in mature H-Ras expressing neurons, suggesting H-Ras's effect is not limited to early development. In PyNs and PV-INs, but not VIP-INs, spine neck lengths shifted to filopodia-like phenotypes. H-Ras primarily induced filopodia in PyNs and spines in PV- and VIP-INs. Increased protrusions in H-Ras-transfected PyNs lacked key excitatory synaptic proteins and did not affect miniature excitatory postsynaptic currents (mEPSCs), suggesting multifaceted roles beyond excitatory synapses.
RESUMO
Dendritic spines are structural correlates of excitatory synapses maintaining stable synaptic communications. However, this strong spine-synapse relationship was mainly characterized in excitatory pyramidal neurons (PyNs), raising a possibility that inferring synaptic density from dendritic spine number may not be universally applied to all neuronal types. Here we found that the ectopic expression of H-Ras increased dendritic spine numbers regardless of cortical cell types such as layer 2/3 pyramidal neurons (PyNs), parvalbumin (PV)- and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex (M1). The probability of detecting dendritic spines was positively correlated with the magnitude of H-Ras activity, suggesting elevated local H-Ras activity is involved in the process of dendritic spine formation. H-Ras overexpression caused high spine turnover rate via adding more spines rather than eliminating them. Two-photon photolysis of glutamate triggered de novo dendritic spine formation in mature neurons, suggesting H-Ras induced spine formation is not restricted to the early development. In PyNs and PV-INs, but not VIP-INs, we observed a shift in average spine neck length towards longer filopodia-like phenotypes. The portion of dendritic spines lacking key excitatory synaptic proteins were significantly increased in H-Ras transfected neurons, suggesting that these increased spines have other distinct functions. High spine density caused by H-Ras did not result in change in the frequency or the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Thus, our results propose that dendritic spines possess more multifaceted functions beyond the morphological proxy of excitatory synapse.
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Genetically defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We found that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, with tuning refined through suppression of irrelevant neuronal activity. ChCs contribute to learning-dependent refinements by providing selective inhibitory control over individual pyramidal neurons rather than global suppression. Quantitative analysis of structural plasticity across axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a targeted cell-specific manner.
Assuntos
Axônios , Controle Comportamental , Axônios/fisiologia , Neurônios/fisiologia , Células Piramidais/fisiologia , Sinapses/fisiologia , Interneurônios/fisiologiaRESUMO
Neural circuits are reorganized with specificity during learning. Genetically-defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions to learning. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We find that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, whose tuning is refined through suppression of irrelevant neuronal activity. ChCs are required for learning-dependent refinements via providing selective inhibitory control over pyramidal neurons rather than global suppression. Quantitative analysis on structural plasticity of axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a target cell specific manner, which highlights the significance of interneuron diversity.
RESUMO
Membrane nanotubes or tunneling nanotubes (TNTs) that connect cells have been recognized as a previously unidentified pathway for intercellular transport between distant cells. However, it is unknown how this delicate structure, which extends over tens of micrometers and remains robust for hours, is formed. Here, we found that a TNT develops from a double filopodial bridge (DFB) created by the physical contact of two filopodia through helical deformation of the DFB. The transition of a DFB to a close-ended TNT is most likely triggered by disruption of the adhesion of two filopodia by mechanical energy accumulated in a twisted DFB when one of the DFB ends is firmly attached through intercellular cadherin-cadherin interactions. These studies pinpoint the mechanistic questions about TNTs and elucidate a formation mechanism.
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We present a single-molecule imaging platform that quantitatively explores the spatiotemporal dynamics of individual insulin receptors in living cells. Modified DNA aptamers that specifically recognize insulin receptors (IRs) with a high affinity were selected through the SELEX process. Using quantum dot-labeled aptamers, we successfully imaged and analyzed the diffusive motions of individual IRs in the plasma membranes of a variety of cell lines (HIR, HEK293, HepG2). We further explored the cholesterol-dependent movement of IRs to address whether cholesterol depletion interferes with IRs and found that cholesterol depletion of the plasma membrane by methyl-ß-cyclodextrin reduces the mobility of IRs. The aptamer-based single-molecule imaging of IRs will provide better understanding of insulin signal transduction through the dynamics study of IRs in the plasma membrane.
Assuntos
Aptâmeros de Nucleotídeos/química , Imagem Molecular/métodos , Receptor de Insulina/análise , Receptor de Insulina/química , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular Tumoral , Colesterol/química , Colesterol/isolamento & purificação , Difusão , Humanos , Pontos Quânticos , Receptor de Insulina/metabolismo , Técnica de Seleção de AptâmerosRESUMO
The mismatch repair (MMR) initiation protein MutS forms at least two types of sliding clamps on DNA: a transient mismatch searching clamp (â¼1 s) and an unusually stable (â¼600 s) ATP-bound clamp that recruits downstream MMR components. Remarkably, direct visualization of single MutS particles on mismatched DNA has not been reported. We have combined real-time particle tracking with fluorescence resonance energy transfer (FRET) to image MutS diffusion dynamics on DNA containing a single mismatch. We show searching MutS rotates during diffusion independent of ionic strength or flow rate, suggesting continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps that are visually and successively released from the mismatch spin freely around the DNA, and their diffusion is affected by ionic strength and flow rate. These observations show that ATP binding alters the MutS diffusion mechanics on DNA, which has a number of implications for the mechanism of MMR.