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  1. Article ; Online: Protocol for deriving proximity, affinity, and stoichiometry of protein interactions using image-based quantitative two-hybrid FRET

    Colin Feldmann / Michael Schänzler / Manu Ben-Johny / Christian Wahl-Schott

    STAR Protocols, Vol 4, Iss 3, Pp 102459- (2023)

    2023  

    Abstract: Summary: Two-hybrid Förster resonance energy transfer (FRET) provides proximity, affinity, and stoichiometry information in binding interactions. We present an image-based approach that surpasses traditional two-hybrid FRET assays in precision and ... ...

    Abstract Summary: Two-hybrid Förster resonance energy transfer (FRET) provides proximity, affinity, and stoichiometry information in binding interactions. We present an image-based approach that surpasses traditional two-hybrid FRET assays in precision and robustness. We outline instrument setup and image acquisition and further describe steps for image preprocessing and two-hybrid FRET analysis using provided software to simplify the workflow. This protocol is compatible with confocal microscopes for high-precision and imaging plate readers for high-throughput applications. A plasmid-based reference system supports fast establishment of the protocol.For complete details on the use and execution of this protocol, please refer to Rivas et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
    Keywords Microscopy ; Science (General) ; Q1-390
    Subject code 006 ; 004
    Language English
    Publishing date 2023-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

    Barbara Spix / Elisabeth S. Butz / Cheng-Chang Chen / Anna Scotto Rosato / Rachel Tang / Aicha Jeridi / Veronika Kudrina / Eva Plesch / Philipp Wartenberg / Elisabeth Arlt / Daria Briukhovetska / Meshal Ansari / Gizem Günes Günsel / Thomas M. Conlon / Amanda Wyatt / Sandra Wetzel / Daniel Teupser / Lesca M. Holdt / Fabien Ectors /
    Ingrid Boekhoff / Ulrich Boehm / Jaime García-Añoveros / Paul Saftig / Martin Giera / Sebastian Kobold / Herbert B. Schiller / Susanna Zierler / Thomas Gudermann / Christian Wahl-Schott / Franz Bracher / Ali Önder Yildirim / Martin Biel / Christian Grimm

    Nature Communications, Vol 13, Iss 1, Pp 1-

    2022  Volume 18

    Abstract: Excess macrophage elastase MMP-12 is a major driver of chronic obstructive pulmonary disease. Here the authors show that the endolysosomal ion channel TRPML3 is a regulator of the cellular reuptake of MMP-12, thus neutralizing harmful MMP-12 in the lung. ...

    Abstract Excess macrophage elastase MMP-12 is a major driver of chronic obstructive pulmonary disease. Here the authors show that the endolysosomal ion channel TRPML3 is a regulator of the cellular reuptake of MMP-12, thus neutralizing harmful MMP-12 in the lung.
    Keywords Science ; Q
    Language English
    Publishing date 2022-01-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann–Pick type C1, and Batten disease

    Anna Scotto Rosato / Einar K Krogsaeter / Dawid Jaślan / Carla Abrahamian / Sandro Montefusco / Chiara Soldati / Barbara Spix / Maria Teresa Pizzo / Giuseppina Grieco / Julia Böck / Amanda Wyatt / Daniela Wünkhaus / Marcel Passon / Marc Stieglitz / Marco Keller / Guido Hermey / Sandra Markmann / Doris Gruber‐Schoffnegger / Susan Cotman /
    Ludger Johannes / Dennis Crusius / Ulrich Boehm / Christian Wahl‐Schott / Martin Biel / Franz Bracher / Elvira De Leonibus / Elena Polishchuk / Diego L Medina / Dominik Paquet / Christian Grimm

    EMBO Molecular Medicine, Vol 14, Iss 9, Pp n/a-n/a (2022)

    2022  

    Abstract: Abstract Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. ...

    Abstract Abstract Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+‐permeable endolysosomal two‐pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann–Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in‐vivo.
    Keywords Batten ; MLIV ; NPC1 ; TPC2 ; TRPML ; Medicine (General) ; R5-920 ; Genetics ; QH426-470
    Subject code 572
    Language English
    Publishing date 2022-09-01T00:00:00Z
    Publisher Wiley
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells

    Stefanie Fenske / Konstantin Hennis / René D. Rötzer / Verena F. Brox / Elvir Becirovic / Andreas Scharr / Christian Gruner / Tilman Ziegler / Verena Mehlfeld / Jaclyn Brennan / Igor R. Efimov / Audrys G. Pauža / Markus Moser / Carsten T. Wotjak / Christian Kupatt / Rasmus Gönner / Rai Zhang / Henggui Zhang / Xiangang Zong /
    Martin Biel / Christian Wahl-Schott

    Nature Communications, Vol 11, Iss 1, Pp 1-

    2020  Volume 22

    Abstract: The involvement of cAMP-dependent regulation of HCN4 in the chronotropic heart rate response is a matter of debate. Here the authors use a knockin mouse model expressing cAMP-insensitive HCN4 channels to discover an inhibitory nonfiring cell pool in the ... ...

    Abstract The involvement of cAMP-dependent regulation of HCN4 in the chronotropic heart rate response is a matter of debate. Here the authors use a knockin mouse model expressing cAMP-insensitive HCN4 channels to discover an inhibitory nonfiring cell pool in the sinoatrial node and a tonic and mutual interaction between firing and nonfiring pacemaker cells that is controlled by cAMP-dependent regulation of HCN4, with implications in chronotropic heart rate responses.
    Keywords Science ; Q
    Language English
    Publishing date 2020-11-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function

    Susanne Gerndt / Cheng-Chang Chen / Yu-Kai Chao / Yu Yuan / Sandra Burgstaller / Anna Scotto Rosato / Einar Krogsaeter / Nicole Urban / Katharina Jacob / Ong Nam Phuong Nguyen / Meghan T Miller / Marco Keller / Angelika M Vollmar / Thomas Gudermann / Susanna Zierler / Johann Schredelseker / Michael Schaefer / Martin Biel / Roland Malli /
    Christian Wahl-Schott / Franz Bracher / Sandip Patel / Christian Grimm

    eLife, Vol

    2020  Volume 9

    Abstract: Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., ...

    Abstract Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.
    Keywords TPC2 ; two-pore channel 2 ; lysosome ; NAADP ; PI(3,5)P2 ; TPC ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 572
    Language English
    Publishing date 2020-03-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells

    Stefanie Fenske / Konstantin Hennis / René D. Rötzer / Verena F. Brox / Elvir Becirovic / Andreas Scharr / Christian Gruner / Tilman Ziegler / Verena Mehlfeld / Jaclyn Brennan / Igor R. Efimov / Audrys G. Pauža / Markus Moser / Carsten T. Wotjak / Christian Kupatt / Rasmus Gönner / Rai Zhang / Henggui Zhang / Xiangang Zong /
    Martin Biel / Christian Wahl-Schott

    Nature Communications, Vol 11, Iss 1, Pp 1-

    2020  Volume 22

    Abstract: The involvement of cAMP-dependent regulation of HCN4 in the chronotropic heart rate response is a matter of debate. Here the authors use a knockin mouse model expressing cAMP-insensitive HCN4 channels to discover an inhibitory nonfiring cell pool in the ... ...

    Abstract The involvement of cAMP-dependent regulation of HCN4 in the chronotropic heart rate response is a matter of debate. Here the authors use a knockin mouse model expressing cAMP-insensitive HCN4 channels to discover an inhibitory nonfiring cell pool in the sinoatrial node and a tonic and mutual interaction between firing and nonfiring pacemaker cells that is controlled by cAMP-dependent regulation of HCN4, with implications in chronotropic heart rate responses.
    Keywords Science ; Q
    Language English
    Publishing date 2020-11-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: The two-pore channel TPC1 is required for efficient protein processing through early and recycling endosomes

    Jan Castonguay / Joachim H. C. Orth / Thomas Müller / Faten Sleman / Christian Grimm / Christian Wahl-Schott / Martin Biel / Robert Theodor Mallmann / Wolfgang Bildl / Uwe Schulte / Norbert Klugbauer

    Scientific Reports, Vol 7, Iss 1, Pp 1-

    2017  Volume 15

    Abstract: Abstract Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and ... ...

    Abstract Abstract Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and pathogenicity of Ebola viruses. Here, we investigate the cellular function of TPC1 using protein toxins as model substrates for distinct endosomal processing routes. Toxin uptake and activation through early endosomes but not processing through other compartments were reduced in TPC1 knockout cells. Detailed co-localization studies with subcellular markers confirmed predominant localization of TPC1 to early and recycling endosomes. Proteomic analysis of native TPC1 channels finally identified direct interaction with a distinct set of syntaxins involved in fusion of intracellular vesicles. Together, our results demonstrate a general role of TPC1 for uptake and processing of proteins in early and recycling endosomes, likely by providing high local Ca2+ concentrations required for SNARE-mediated vesicle fusion.
    Keywords Medicine ; R ; Science ; Q
    Subject code 571
    Language English
    Publishing date 2017-08-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Recombinant tandem of pore-domains in a Weakly Inward rectifying K+ channel 2 (TWIK2) forms active lysosomal channels

    Nicole Bobak / Sylvain Feliciangeli / Cheng-Chang Chen / Ismail Ben Soussia / Stefan Bittner / Sophie Pagnotta / Tobias Ruck / Martin Biel / Christian Wahl-Schott / Christian Grimm / Sven G. Meuth / Florian Lesage

    Scientific Reports, Vol 7, Iss 1, Pp 1-

    2017  Volume 13

    Abstract: Abstract Recombinant TWIK2 channels produce weak basal background K+ currents. Current amplitudes depend on the animal species the channels have been isolated from and on the heterologous system used for their re-expression. Here we show that this ... ...

    Abstract Abstract Recombinant TWIK2 channels produce weak basal background K+ currents. Current amplitudes depend on the animal species the channels have been isolated from and on the heterologous system used for their re-expression. Here we show that this variability is due to a unique cellular trafficking. We identified three different sequence signals responsible for the preferential expression of TWIK2 in the Lamp1-positive lysosomal compartment. Sequential inactivation of tyrosine-based (Y308ASIP) and di-leucine-like (E266LILL and D282EDDQVDIL) trafficking motifs progressively abolishes the targeting of TWIK2 to lysosomes, and promotes its functional relocation at the plasma membrane. In addition, TWIK2 contains two N-glycosylation sites (N79AS and N85AS) on its luminal side, and glycosylation is necessary for expression in lysosomes. As shown by electrophysiology and electron microscopy, TWIK2 produces functional background K+ currents in the endolysosomes, and its expression affects the number and mean size of the lysosomes. These results show that TWIK2 is expressed in lysosomes, further expanding the registry of ion channels expressed in these organelles.
    Keywords Medicine ; R ; Science ; Q
    Subject code 572
    Language English
    Publishing date 2017-04-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting of retinitis pigmentosa-linked peripherin-2 mutants

    Sybille Böhm / Lisa M. Riedmayr / O. N. Phuong Nguyen / Andreas Gießl / Toni Liebscher / Elisabeth S. Butz / Christian Schön / Stylianos Michalakis / Christian Wahl-Schott / Martin Biel / Elvir Becirovic

    Scientific Reports, Vol 7, Iss 1, Pp 1-

    2017  Volume 13

    Abstract: Abstract Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous ... ...

    Abstract Abstract Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (perWT), mutant peripherin-2 (perMT), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to perWT and Rom-1. Furthermore, both mutants are preferentially forming non-covalent perMT-perMT, perWT-perMT, and Rom-1-perMT dimers. However, only perWT-perMT, but not perMT-perMT or Rom-1-perMT complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent perWT-perMT dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of perWT and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals.
    Keywords Medicine ; R ; Science ; Q
    Subject code 300
    Language English
    Publishing date 2017-05-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article ; Online: Selective agonist of TRPML2 reveals direct role in chemokine release from innate immune cells

    Eva Plesch / Cheng-Chang Chen / Elisabeth Butz / Anna Scotto Rosato / Einar K Krogsaeter / Hua Yinan / Karin Bartel / Marco Keller / Dina Robaa / Daniel Teupser / Lesca M Holdt / Angelika M Vollmar / Wolfgang Sippl / Rosa Puertollano / Diego Medina / Martin Biel / Christian Wahl-Schott / Franz Bracher / Christian Grimm

    eLife, Vol

    2018  Volume 7

    Abstract: Cytokines and chemokines are produced and secreted by a broad range of immune cells including macrophages. Remarkably, little is known about how these inflammatory mediators are released from the various immune cells. Here, the endolysosomal cation ... ...

    Abstract Cytokines and chemokines are produced and secreted by a broad range of immune cells including macrophages. Remarkably, little is known about how these inflammatory mediators are released from the various immune cells. Here, the endolysosomal cation channel TRPML2 is shown to play a direct role in chemokine trafficking and secretion from murine macrophages. To demonstrate acute and direct involvement of TRPML2 in these processes, the first isoform-selective TRPML2 channel agonist was generated, ML2-SA1. ML2-SA1 was not only found to directly stimulate release of the chemokine CCL2 from macrophages but also to stimulate macrophage migration, thus mimicking CCL2 function. Endogenous TRPML2 is expressed in early/recycling endosomes as demonstrated by endolysosomal patch-clamp experimentation and ML2-SA1 promotes trafficking through early/recycling endosomes, suggesting CCL2 being transported and secreted via this pathway. These data provide a direct link between TRPML2 activation, CCL2 release and stimulation of macrophage migration in the innate immune response.
    Keywords TRPML2 ; TRPML ; lysosome ; CCL2 ; macrophage ; Mcoln2 ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 616
    Language English
    Publishing date 2018-11-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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