TNF-α influences the lateral dynamics of TNF receptor I in living cells

Heidbreder, Meike; Zander, Christin; Malkusch, Sebastian; Widera, Darius; Kaltschmidt, Barbara; Kaltschmidt, Christian; Nair, Deepak; Choquet, Daniel; Sibarita, Jean‐Baptiste; Heilemann, Mike

doi:10.1016/j.bbamcr.2012.06.026

Cited by 28 publications

(25 citation statements)

References 42 publications

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“…QD-labelled molecules were localized by a wavelet-based method performing well for data with low SNR described recently22 and tracked over time using a simulated annealing algorithm23 to quantify single-molecule dynamics242526. We also tested the Gaussian fitting method for QD localization2728 and the Vogel algorithm for tracking individual QDs29, which yielded similar QD localization accuracy and dynamics but required much longer calculation times (the wavelet-based method worked 20 times faster with similar localization accuracy22).…”

Section: Resultsmentioning

confidence: 99%

Imaging of molecular surface dynamics in brain slices using single-particle tracking

et al. 2014

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Organization of signalling molecules in biological membranes is crucial for cellular communication. Many receptors, ion channels and cell adhesion molecules are associated with proteins important for their trafficking, surface localization or function. These complexes are embedded in a lipid environment of varying composition. Binding affinities and stoichiometry of such complexes were so far experimentally accessible only in isolated systems or monolayers of cell culture. Visualization of molecular dynamics within signalling complexes and their correlation to specialized membrane compartments demand high temporal and spatial resolution and has been difficult to demonstrate in complex tissue like brain slices. Here we demonstrate the feasibility of single-particle tracking (SPT) in organotypic brain slices to measure molecular dynamics of lipids and transmembrane proteins in correlation to synaptic membrane compartments. This method will provide important information about the dynamics and organization of surface molecules in the complex environment of neuronal networks within brain slices.

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Section: Resultsmentioning

confidence: 99%

Imaging of molecular surface dynamics in brain slices using single-particle tracking

et al. 2014

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“…SPT and super-resolution imaging methods have been used extensively to study interactions of EGFR and ErbB receptors [61,132–136] and to a lesser extent TNFRs [137–139]. EGFR structure, dynamics and oligomerization/interactions have been widely studied using a variety of biophysical methods which also includes localization microscopy (discussed below), number and brightness [58], FRET and/or FLIM [120,133,135,140], fluorescence recovery after photobleaching (FRAP) [141–144], fluorescence correlation/cross-correlation spectroscopy (FCS, FCCS) [59,60,145], photon counting histogram (PCH) and fluorescence intensity distribution analysis (FIDA) [146], spatial intensity distribution analysis (SpIDA) [147], and single molecule photobleaching and single molecule localization and photobleaching [61,66–68,135].…”

Section: New Experimental Techniques Are Changing the Game: Singlementioning

confidence: 99%

“…Heidbreder et al demonstrated that the ensemble average mobility of TNFR1 in the presence of TNFα is indistinguishable from the resting receptor, however the distribution of mobilities of individual receptor molecules showed a small but significant shift toward faster moving receptors [137]. This shift in the distribution of receptor mobilities is accompanied by an overall decrease in the percentage of nearly-immobile receptors upon the addition of ligand, and the change is exacerbated when cells are pre-treated with MβCD [137]. This raises an interesting question: If membrane cholesterol facilitates interactions between TNFR TM helices, as described above [85], does this increase in TNFR1 mobility reflect a change in the oligomeric state of the receptor?…”

Section: New Experimental Techniques Are Changing the Game: Singlementioning

confidence: 99%

Piecing it together: Unraveling the elusive structure-function relationship in single-pass membrane receptors

Valley¹,

Lewis²,

Sachs³

2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The challenge of crystallizing single-pass plasma membrane receptors has remained an obstacle to understanding the structural mechanisms that connect extracellular ligand binding to cytosolic activation. For example, the complex interplay between receptor oligomerization and conformational dynamics has been, historically, only inferred from static structures of isolated receptor domains. A fundamental challenge in the field of membrane receptor biology, then, has been to integrate experimentally observable dynamics of full-length receptors (e.g. diffusion and conformational flexibility) into static structural models of the disparate domains. In certain receptor families, e.g. the ErbB receptors, structures have led somewhat linearly to a putative model of activation. In other families, e.g. the tumor necrosis factor (TNF) receptors, structures have produced divergent hypothetical mechanisms of activation and transduction. Here, we discuss in detail these and other related receptors, with the goal of illuminating the current challenges and opportunities in building comprehensive models of single-pass receptor activation. The deepening understanding of these receptors has recently been accelerated by new experimental and computational tools that offer orthogonal perspectives on both structure and dynamics. As such, this review aims to contextualize those technological developments as we highlight the elegant and complex conformational communication between receptor domains. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

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“…Profiting from the pool of labelled biomolecules in a sample, a large number of single-molecule trajectories are recorded. SPT with photoactivated-localization microscopy (SPT-PALM 27 ) allows longer observation times, provides better statistics, 28,29 and allows high-density mapping of molecular movements. 30 In order to study the dynamics of retrogradely transported NF-κB in neurons at the single-molecule level, we applied SPT-PALM 27,31 and used the fluorescent protein tandem-Eos-FP (tdEos) as a reporter.…”

Section: Introductionmentioning

confidence: 99%

“…30 In order to study the dynamics of retrogradely transported NF-κB in neurons at the single-molecule level, we applied SPT-PALM 27,31 and used the fluorescent protein tandem-Eos-FP (tdEos) as a reporter. 27,28 tdEos is photoconverted from a green-fluorescent to an orange-fluorescent species by irradiation with 405 nm light. 32 Following this procedure, a small stochastic subset of the tdEos is transferred into the active (orange-fluorescent) state and tracked as single molecules.…”

Section: Introductionmentioning

confidence: 99%

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

et al. 2016

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Abstract. Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/ dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (D mean ) in neurites from 0.12 AE 0.05 to 0.61 AE 0.03 μm 2 ∕s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 AE 1.9 to 15 AE 4.9 μm∕s, whereas a velocity increase from 9 AE 1.3 to 14 AE 3 μm∕s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.

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TNF-α influences the lateral dynamics of TNF receptor I in living cells

Cited by 28 publications

References 42 publications

Imaging of molecular surface dynamics in brain slices using single-particle tracking

Imaging of molecular surface dynamics in brain slices using single-particle tracking

Piecing it together: Unraveling the elusive structure-function relationship in single-pass membrane receptors

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

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