A multi‐emitter fitting algorithm for potential live cell super‐resolution imaging over a wide range of molecular densities

AbstractThe collagen receptor DDR1 is a receptor tyrosine kinase that promotes progression of a wide range of human disorders. Little is known about how ligand binding triggers DDR1 kinase activity. We previously reported that collagen induces DDR1 activation through lateral dimer association and phosphorylation between dimers, a process that requires specific transmembrane association. Here we demonstrate ligand-induced DDR1 clustering by widefield and super-resolution imaging and provide evidence for a mechanism whereby DDR1 kinase activity is determined by its molecular density. Ligand binding resulted in initial DDR1 reorganisation into morphologically distinct clusters with unphosphorylated DDR1. Further compaction over time led to clusters with highly aggregated and phosphorylated DDR1. Ligand-induced DDR1 clustering was abolished by transmembrane mutations but did not require kinase activity. Our results significantly advance our understanding of the molecular events underpinning ligand-induced DDR1 kinase activity and provide an explanation for the unusually slow DDR1 activation kinetics.

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Cell-permeable organic fluorescent probes for live-cell long-term super-resolution imaging reveal lysosome-mitochondrion interactions

Nature Communications ◽

10.1038/s41467-017-01503-6 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 67

Author(s):

Yubing Han ◽

Meihua Li ◽

Fengwu Qiu ◽

Meng Zhang ◽

Yu-Hui Zhang

Keyword(s):

Fluorescent Probes ◽

Super Resolution ◽

Live Cell ◽

Resolution Imaging

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FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

10.1101/2020.02.06.937045 ◽

2020 ◽

Cited By ~ 3

Author(s):

Matthieu Lagardère ◽

Ingrid Chamma ◽

Emmanuel Bouilhol ◽

Macha Nikolski ◽

Olivier Thoumine

Keyword(s):

Real Time ◽

Protein Dynamics ◽

Single Molecule ◽

Imaging Techniques ◽

Simulated Data ◽

Super Resolution ◽

Molecular Complexes ◽

Live Cell ◽

Single Molecule Level ◽

Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

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A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo

Scientific Reports ◽

10.1038/s41598-021-04544-6 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Sara Hernández-Pérez ◽

Pieta K. Mattila

Keyword(s):

Super Resolution ◽

Cell Receptor ◽

Live Cell ◽

Receptor Ligand ◽

Vesicle Traffic ◽

Specific Hybridization ◽

Multiple Imaging ◽

Resolution Imaging ◽

Membrane Bound ◽

Quenching Probe

AbstractFacilitated by the advancements in microscopy, our understanding of the complexity of intracellular vesicle traffic has dramatically increased in recent years. However, distinguishing between plasma membrane-bound or internalised ligands remains a major challenge for the studies of cargo sorting to endosomal compartments, especially in small and round cells such as lymphocytes. The specific hybridization internalisation probe (SHIP) assay, developed for flow cytometry studies, employs a ssDNA fluorescence internalisation probe and a complementary ssDNA quenching probe to unambiguously detect the internalized receptors/cargo. Here, we adopted the SHIP assay to study the trafficking of receptor/ligand complexes using B lymphocytes and B cell receptor-mediated antigen internalization as a model system. Our study demonstrates the potential of the SHIP assay for improving the imaging of internalized receptor/ligand complexes and establishes the compatibility of this assay with multiple imaging modalities, including live-cell imaging and super-resolution microscopy.

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Quencher based chemical tag technology using exchangeable probes for live-cell super-resolution imaging

Proceedings for Annual Meeting of The Japanese Pharmacological Society ◽

10.1254/jpssuppl.94.0_2-o-c3-1 ◽

2021 ◽

Vol 94 (0) ◽

pp. 2-O-C3-1

Author(s):

Daisuke Asanuma ◽

Shigeyuki Namiki ◽

Kenzo Hirose

Keyword(s):

Super Resolution ◽

Live Cell ◽

Chemical Tag ◽

Resolution Imaging

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