Varying label density allows artifact-free analysis of membrane-protein nanoclusters

Baumgart, Florian; Arnold, A.; Leskovar, Konrad; Staszek, Kaj; Fölser, Martin; Weghuber, Julian; Stockinger, Hannes; Schütz, Gerhard J.

doi:10.1038/nmeth.3897

Cited by 132 publications

(145 citation statements)

References 25 publications

(31 reference statements)

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“…In order to study the detailed assembly and regulation mechanisms of MCU complex structures in the mitochondrion, we employed Photo-activated localization microscopy (PALM). HeLa cells were transfected with MCU and MCU C97A tagged with photo switchable protein mEos3.2 (Baumgart et al, 2016). Similar to the above described imaging data, PALM acquisition images showed random distribution of MCU whereas MCU C97A appeared to assemble as clusters in the globular like inner mitochondrial membrane (Figure 5H).…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity

et al. 2017

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SUMMARY Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU has not been established. As an approach towards understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher-order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

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Section: Resultsmentioning

confidence: 99%

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity

et al. 2017

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“…The examinations touch the receptor clustering as a challenge for microscopic density recording dynamics, which is addressed by a receptor titration approach [92]. Approaches based on photoactivated light microscopy (PALM) as well as stochastic optical reconstruction microscopy (STORM) related techniques focus on the clustering dynamics of membrane receptors in cells of the immune system upon stimulation [93,94,95].…”

Section: Resultsmentioning

confidence: 99%

Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1β, and Trastuzumab Application

Pilarczyk

Nesnidal

Gunkel

et al. 2017

IJMS

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In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin-1sans-serifβ application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ-irradiation or alternatively neuregulin-1sans-serifβ application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation.

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“…Its drawback is that undercounting or overcounting of the fluorescent labels can alter the details of the reconstructed structures 173 . Micropipette assays.…”

Section: Structured Illumination Microscopy (Sim) This Technique Impmentioning

confidence: 99%

Cytoskeletal control of B cell responses to antigens

Tolar

2017

Nat Rev Immunol

123

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The actin cytoskeleton is essential for cell mechanics and has increasingly been implicated in the regulation of cell signalling. In B cells, the actin cytoskeleton couples extensively to B cell receptor (BCR) signalling pathways and its defects can either enhance or suppress B cell activation. Recent insights from single-cell imaging and biophysical techniques suggest that actin orchestrates BCR signalling at the plasma membrane through effects on protein diffusion, and that it regulates antigen discrimination through the biomechanics of immune synapses. These mechanical functions also play a role in the adaptation of B cell subsets to specialized tasks during antibody responses.3

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Varying label density allows artifact-free analysis of membrane-protein nanoclusters

Cited by 132 publications

References 25 publications

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity

Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1β, and Trastuzumab Application

Cytoskeletal control of B cell responses to antigens

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