Tina Märker's research while affiliated with Heinrich-Heine-Universität Düsseldorf and other places

The human Usher syndrome (USH) is a complex ciliopathy with at least 12 chromosomal loci assigned to 3 clinical subtypes, USH1-3. The heterogeneous USH proteins are organized into protein networks. Here, we identified Magi2 (membrane-associated guanylate kinase inverted-2) as a new component of the USH protein interactome, binding to the multifunctional scaffold protein SANS (USH1G). We showed that the SANS-Magi2-complex assembly is regulated by the phosphorylation of an internal PDZ-binding motif in the SAM domain of SANS by the protein kinase CK2. We affirmed Magi2's role in receptor-mediated, clathrin-dependent endocytosis and showed that phosphorylated SANS tightly regulates Magi2-mediated endocytosis. Specific depletions by RNAi revealed that SANS and Magi2-mediated endocytosis regulates aspects of ciliogenesis. Furthermore, we demonstrated the localization of the SANS-Magi2 complex in the periciliary membrane complex facing the ciliary pocket of retinal photoreceptor cells in situ. Our data suggest that endocytotic processes may not only contribute to photoreceptor cell homeostasis but also counterbalance the periciliary membrane delivery accompanying the exocytosis processes for the cargo vesicle delivery. In USH1G patients, mutations in SANS eliminate Magi2 binding and thereby deregulate endocytosis, lead to defective ciliary transport modules and ultimately disrupt photoreceptor cell function inducing retinal degeneration.

Adipocytes release immune mediators that contribute to diabetes-associated inflammatory processes. As the stress protein heat shock protein 60 (Hsp60) induces proinflammatory adipocyte activities, we hypothesized that adipocytes of diabetes-predisposed mice exhibit an increased proinflammatory reactivity to Hsp60. Preadipocytes and mature adipocytes from nonobese diabetic (NOD), New Zealand obese (NZO), and C57BL/6J mice were analyzed for Hsp60 binding, Hsp60-activated signaling pathways, and Hsp60-induced release of the chemokine CXCL-1 (KC), interleukin 6 (IL-6), and macrophage chemoattractant protein-1 (MCP-1). Hsp60 showed specific binding to (pre-)adipocytes of NOD, NZO, and C57BL/6J mice. Hsp60 binding involved conserved binding structure(s) and Hsp60 epitopes and was strongest to NZO mouse-derived mature adipocytes. Hsp60 exposure induced KC, IL-6, and MCP-1 release from (pre-)adipocytes of all mouse strains with a pronounced increase of IL-6 release from NZO mouse-derived adipocytes. Compared to NOD and C57BL/6J mouse derived cells, Hsp60-induced formation of IL-6, KC, and MCP-1 from NZO mouse-derived (pre-)adipocytes strongly depended on NF κ B-activation. Increased Hsp60 binding and Hsp60-induced IL-6 release by mature adipocytes of NZO mice suggest that enhanced adipocyte reactivity to the stress signal Hsp60 contributes to inflammatory processes underlying diabetes associated with obesity and insulin resistance.

The human Usher syndrome (USH) is the most common form of combined deaf-blindness. The encoded molecules are integrated into protein networks by scaffolds including the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain). Previous studies indicated SANS´participationSANS´participation in vesicle transport and cargo handover at the periciliary region of photoreceptor cells. To decipher the precise cellular role of SANS, we searched for interacting partners. Therefore we adopted a yeast-2-hybrid screen of a retinal cDNA library using SANS´C SANS´C-terminus as bait. Amongst others we identified the MAGUK protein Magi2 (membrane-associated guanylate kinase inverted-2) as putative binding partner. Magi2 is known as scaffold protein involved in endocytosis at synapses. We confirmed Magi2 as direct interaction partner of SANS by complementary interaction assays. In addition, correlative light and electron microscopy showed partial co-localization of SANS and Magi2 in the periciliary region of photoreceptor cells. Furthermore, immunoelectron microscopy revealed the association of Magi2-SANS complex with membranous vesicles in this region. These vesicles may represent either vesicles related to ciliary transport or endocytotic vesicles. Preliminary data revealed endocytotic processes at the periciliary region of photoreceptor cells. In conclusion, we have identified Magi2 as component of the USH protein inter-actome. Present data indicate a role of SANS-Magi2 complex in endocytosis in the periciliary region of photo-receptor cells. These findings confirm the recently emphasized endocytotic function of the ciliary pocket in primary cilia. Furthermore, we provide first evidence for a molecular link of the USH protein interactome to the endocytosis machinery in photoreceptor cells.

Human Usher syndrome (USH) is the most common form of combined deaf-blindness, characterized by profound congenital deafness, constant vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. The USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) is associated with microtubules and mediates a transport related periciliary protein network in photoreceptor cells. Here we aim to enlighten the involvement of SANS in ciliary transport of photoreceptor cells by identifying proteins associated with SANS in transport complexes. In Y2H screen of retinal cDNA library we identified the direct binding of SANS to dynactin-1 (p150Glued), a subunit of the dynactin complex and cargo linker to the cytoplasmic dynein motor. This interaction was validated in complementary interaction assays in vitro and in cell culture. In addition, we demonstrated the integration of SANS into the cytoplasmic dynein transport complex by GST-pull down of SANS with cytoplasmic dynein intermediate chain (cDIC74). Correlative immuno-fluorescence and-electron microscopy analyses of photore-ceptor inner segments revealed co-localization of essential dynein-dynactin components and SANS at microtubules, the known transport routes for opsin bearing vesicles. Co-immunoprecipitation revealed that SANS is part of an opsin containing protein complex together with transport regulating rab-GTPases. Finally, the analysis of SANS deficient mice revealed altered distribution of opsin in the inner segment, indicating a transport delay. In conclusion we identified SANS as a component of the cytoplasmic dynein motor complex which is crucial for opsin transport to photoreceptor connecting cilia. Defects in these transport mechanisms lead to retinal degeneration as characteristic for USH1G patients.

Purpose: : Human Usher syndrome (USH) is the most common form of combined deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. The USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) is associated with microtubules and mediates a transport related periciliary protein network in photoreceptor cells. The present work was designed to enlighten the function of SANS in intracellular transport in photoreceptor cells. Methods: : Identification and validation of protein-protein interactions via yeast-2-hybrid (Y2H) screen of retinal cDNA library, GST-pull down, co-transfection assays, microtubule spin-down and microtubule destabilization assays. Investigation of protein localization in mouse and human retina by indirect immunofluorescence and immunoelectron microscopy. Results: : In our Y2H screen we identified the interaction between SANS and dynactin-1 (p150Glued), a subunit of the dynactin complex and cargo linker to the cytoplasmic dynein motor. This interaction was validated in independent interaction assays in vitro and in cell culture. Furthermore, we demonstrated the integration of SANS into dynein transport complex by pull down of SANS with GST-fused cytoplasmic dynein intermediate chain (cDIC74). Correlative immunofluorescence and immunoelectron microscopy revealed co-localization of p150Glued, basic dynein components and SANS at microtubule tracks of photoreceptor inner segments. Conclusions: : The present data strengthen our hypothesis that SANS participates in cargo transport to its ciliary destination. Here we demonstrate the integration of SANS into cytoplasmic dynein-dynactin complexes, essential for microtubule based transport in photoreceptor cells. Defects in these transport mechanisms may lead to retinal degeneration as characteristic for USH1G patients.

The stress protein heat shock protein 60 (Hsp60) induces secretion of proinflammatory mediators from murine adipocytes. This study aimed to study Hsp60 as a mediator of adipose tissue inflammation and skeletal muscle cell (SkMC) insulin sensitivity and to quantify plasma Hsp60 concentrations in lean and obese individuals. Regulation of Hsp60 release and Hsp60-induced cytokine secretion and signaling was measured in human adipocytes and SkMCs. Adipocytes exhibited higher Hsp60 release than preadipocytes and SkMCs, which was further stimulated by cytokines and Toll-like receptor (TLR)-4 activation. Hsp60 activated extracellular signal-related kinase (ERK)-1/2, Jun NH(2)-terminal kinase (JNK), p38, nuclear factor (NF)-κB, and impaired insulin-stimulated Akt phosphorylation in adipocytes. Furthermore, Hsp60 stimulated adipocytes to secrete tumor necrosis factor-α, interleukin (IL)-6, and IL-8. In SkMCs, Hsp60 activated ERK1/2, JNK, and NF-κB and inhibits insulin signaling and insulin-stimulated glucose uptake. SkMCs released IL-6, IL-8, and monocyte chemoattractant protein-1 on Hsp60 stimulation. Plasma Hsp60 was higher in obese males than in lean males and correlated positively with BMI, blood pressure, leptin, and homeostasis model assessment-insulin resistance. In summary, Hsp60 is released by human adipocytes, increased in plasma of obese humans, and induces insulin resistance. This is accompanied by activation of proinflammatory signaling in human adipocytes and SkMCs. Thus, Hsp60 might be a factor underlying adipose tissue inflammation and obesity-associated metabolic disorders.

The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically heterogeneous with at least 11 chromosomal loci assigned to 3 clinical types, USH1-3. We have previously demonstrated that all USH1 and 2 proteins in the eye and the inner ear are organized into protein networks by scaffold proteins. This has contributed essentially to our current understanding of the function of USH proteins and explains why defects in proteins of different families cause very similar phenotypes. We have previously shown that the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) contributes to the periciliary protein network in retinal photoreceptor cells. This study aimed to further elucidate the role of SANS by identifying novel interaction partners. In yeast two-hybrid screens of retinal cDNA libraries we identified 30 novel putative interacting proteins binding to the central domain of SANS (CENT). We confirmed the direct binding of the phosphodiesterase 4D interacting protein (PDE4DIP), a Golgi associated protein synonymously named myomegalin, to the CENT domain of SANS by independent assays. Correlative immunohistochemical and electron microscopic analyses showed a co-localization of SANS and myomegalin in mammalian photoreceptor cells in close association with microtubules. Based on the present results we propose a role of the SANS-myomegalin complex in microtubule-dependent inner segment cargo transport towards the ciliary base of photoreceptor cells.

Fragestellung: Adipositas ist ein wesentlicher Risikofaktor fur die Entstehung von Insulinresistenz und Typ 2 Diabetes. Im Verlauf der Adipositas werden vermehrt Mediatoren aus dem Fettgewebe freigesetzt, die zur Fettgewebsentzundung fuhren. Diese Entzundungsmediatoren konnen lokal (Adipozyten) sowie systemisch (Skelettmuskelzellen) wirken und zur Entwicklung von Insulinresistenz beitragen. Die Signale, die eine Fettgewebsentzundung initiieren sind bislang unbekannt. Aktuelle Untersuchungen zeigen, dass dem Stress- und Hitzeschockprotein 60 (Hsp60) neben seiner Rolle als Chaperon auch regulatorische Eigenschaften zukommen. Daher war das Ziel, die Wirkung von autologem Hsp60 auf humane Adipozyten und Skelettmuskelzellen (SkMC) sowie die Bedeutung von Hsp60 in der Entstehung der Fettgewebsentzundung und der Insulinresistenz aufzuklaren. Methodik: Primare humane subkutane Praadipozyten wurden zu Adipozyten differenziert. Die Hsp60-Freisetzung aus unbehandelten und Zytokin-behandelten (je 1000 U/ml TNF-α, IFNγ, IL-β) Adipozyten sowie die Hsp60-Plasmaspiegel 18 normalgewichtiger (BMI: 23,4±5,6kg/m2) und 23 adiposer Individuen (BMI: 44,5±5,6kg/m2) wurden mittels ELISA quantifiziert. Die Hsp60-Bindung wurde mittels FACS quantifiziert. Die Hsp60-induzierte Freisetzung von Entzundungsmediatoren wurde mithilfe des Multiplex-Beads-Verfahrens untersucht. Adipozyten und SkMC wurden mit Hsp60 (5–20µg/ml) inkubiert und die Aktivierung spezifischer Signalproteine analysiert. Ergebnisse: Humane Adipozyten exprimieren und setzen Hsp60 frei. Nach Simulation einer Entzundung durch Inkubation mit pro-inflammatorischen Zytokinen wurden in den Zellkulturuberstanden der Adipozyten hohere Hsp60-Spiegel im Vergleich zu unbehandelten Zellen gemessen (0,9±0,3ng/ml vs. 0,4±0,3ng/ml; p<0,01). Adipose Individuen wiesen im Vergleich zu schlanken Probanden hohere Hsp60-Plasmaspiegel auf (20,3±1,0ng/ml vs. 12,6±11,0ng/ml; p<0,05). Ferner bindet Hsp60 spezifisch an humane Adipozyten und SkMC. In Adipozyten induzierte Hsp60 konzentrationsabhangig die Freisetzung von TNF-α, MIP1α, RANTES, MCP-1, IL-6 und IL-8. Erstmalig konnte auch eine Hsp60-induzierte Sekretion der Myokine MCP-1 (1,2±0,6ng/ml; p=0,001), IL-6 (0,4±0,3ng/ml; p<0,001) und IL-8 (1,7±0,9ng/ml; p<0,01) gezeigt werden. Hsp60 aktivierte sowohl in Adipozyten als auch in SkMC pro-inflammatorische Signalwege (JNK, ERK1/2 und NFκB) und reduzierte die Insulin-stimulierte Phosphorylierung der Akt auf 55,0±28,2% (p<0,05) bzw. 50,3±8,0% (p<0,01). Schlussfolgerung: Hsp60 wird von humanen Adipozyten freigesetzt und ist im Plasma adiposer Menschen erhoht. Hsp60 induziert in vitro Insulinresistenz in humanen Adipozyten und SkMC, die durch Aktivierung pro-inflammatorischer Signalwege und erhohte Sekretion von Entzundungsmediatoren erklarbar ist. Hsp60 konnte somit eine wichtige Rolle in der Entwicklung der Fettgewebsentzundung sowie der Insulinresistenz zukommen.