Publications by authors named "Markus Moser"

120 Publications

Selective depletion of a CD64-expressing phagocyte subset mediates protection against toxic kidney injury and failure.

Proc Natl Acad Sci U S A 2021 09;118(39)

Walter-Brendel-Centre of Experimental Medicine, University Hospital, Ludwig-Maximilians-Universität in Munich, 82152 Planegg-Martinsried, Germany;

Dendritic cells (DC), macrophages, and monocytes, collectively known as mononuclear phagocytes (MPs), critically control tissue homeostasis and immune defense. However, there is a paucity of models allowing to selectively manipulate subsets of these cells in specific tissues. The steady-state adult kidney contains four MP subsets with Clec9a-expression history that include the main conventional DC1 (cDC1) and cDC2 subtypes as well as two subsets marked by CD64 but varying levels of F4/80. How each of these MP subsets contributes to the different phases of acute kidney injury and repair is unknown. We created a mouse model with a Cre-inducible lox-STOP-lox-diphtheria toxin receptor cassette under control of the endogenous CD64 locus that allows for diphtheria toxin-mediated depletion of CD64-expressing MPs without affecting cDC1, cDC2, or other leukocytes in the kidney. Combined with specific depletion of cDC1 and cDC2, we revisited the role of MPs in cisplatin-induced kidney injury. We found that the intrinsic potency reported for CD11c cells to limit cisplatin toxicity is specifically attributed to CD64 MPs, while cDC1 and cDC2 were dispensable. Thus, we report a mouse model allowing for selective depletion of a specific subset of renal MPs. Our findings in cisplatin-induced injury underscore the value of dissecting the functions of individual MP subsets in kidney disease, which may enable therapeutic targeting of specific immune components in the absence of general immunosuppression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2022311118DOI Listing
September 2021

Binding of Rap1 and Riam to Talin1 Fine-Tune β2 Integrin Activity During Leukocyte Trafficking.

Front Immunol 2021 19;12:702345. Epub 2021 Aug 19.

Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technische Universität München, Munich, Germany.

β2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent β2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the β2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that β2 integrin regulation primarily occurs these two pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2021.702345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417109PMC
August 2021

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts.

J Cell Sci 2021 Aug 23;134(16). Epub 2021 Aug 23.

Institute for Molecular and Cellular Anatomy, University of Regensburg, 93053 Regensburg, Germany.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.259013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8435292PMC
August 2021

Upregulation of VCAM-1 in lymphatic collectors supports dendritic cell entry and rapid migration to lymph nodes in inflammation.

J Exp Med 2021 07 14;218(7). Epub 2021 May 14.

Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.

Dendritic cell (DC) migration to draining lymph nodes (dLNs) is a slow process that is believed to begin with DCs approaching and entering into afferent lymphatic capillaries. From capillaries, DCs slowly crawl into lymphatic collectors, where lymph flow induced by collector contraction supports DC detachment and thereafter rapid, passive transport to dLNs. Performing a transcriptomics analysis of dermal endothelial cells, we found that inflammation induces the degradation of the basement membrane (BM) surrounding lymphatic collectors and preferential up-regulation of the DC trafficking molecule VCAM-1 in collectors. In crawl-in experiments performed in ear skin explants, DCs entered collectors in a CCR7- and β1 integrin-dependent manner. In vivo, loss of β1-integrins in DCs or of VCAM-1 in lymphatic collectors had the greatest impact on DC migration to dLNs at early time points when migration kinetics favor the accumulation of rapidly migrating collector DCs rather than slower capillary DCs. Taken together, our findings identify collector entry as a critical mechanism enabling rapid DC migration to dLNs in inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20201413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129804PMC
July 2021

The voltage-gated potassium channel KV1.3 regulates neutrophil recruitment during inflammation.

Cardiovasc Res 2021 Apr 21. Epub 2021 Apr 21.

Walter Brendel Centre of Experimental Medicine, Biomedical Center, Institute of Cardiovascular Physiology and Pathophysiology, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany.

Aims: Neutrophil trafficking within the vasculature strongly relies on intracellular calcium signaling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signaling.

Methods And Results: Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store operated Ca2+ entry (SOCE) through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signaling, thereby preventing cellular spreading, adhesion strengthening and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of E.coli particles by neutrophils in the absence of KV1.3.

Conclusion: We show that the voltage gated potassium channel KV1.3 is critical for Ca2+ signaling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signaling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.

Translational Perspective: Neutrophils exert important immune functions during tissue injury or bacterial infection through leaving the vasculature and extravasate into affected tissues. Conversely, neutrophils trigger the pathogenesis of acute and chronic inflammatory disorders and are involved in the development and maintenance of various autoimmune diseases. Within this study, we show that the voltage-gated potassium channel KV1.3 is functionally expressed on neutrophils and affects calcium signaling thereby regulating neutrophil effector functions during immune responses. Hence, KV1.3 represents an interesting potential new target to treat unwanted excessive neutrophil invasion in various disorders ranging from autoinflammatory disorders to ischemic tissue injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvab133DOI Listing
April 2021

2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function.

Front Immunol 2020 18;11:619925. Epub 2021 Feb 18.

Sanquin Research and Landsteiner Laboratory, Department of Blood Cell Research, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, Netherlands.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of 2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of 2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two 2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting 2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.619925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930317PMC
July 2021

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.

Nat Commun 2020 11 3;11(1):5555. Epub 2020 Nov 3.

Hannover Medical School, Institute for Neurophysiology, 30625, Hannover, Germany.

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-19304-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641277PMC
November 2020

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation.

J Cell Biol 2020 12;219(12)

Department of Biochemistry and Molecular Biology, Universitat de València, Burjassot, Spain.

Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding- and fibrillogenesis through binding to α5β1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif-deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin-binding sites. Our results demonstrate that α5β1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5β1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.202004198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644020PMC
December 2020

The integrin-linked kinase is required for chemokine-triggered high-affinity conformation of the neutrophil β2-integrin LFA-1.

Blood 2020 11;136(19):2200-2205

Department of Anesthesiology, Intensive Care Medicine and Pain Therapy, University Hospital Muenster, Muenster, Germany.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the β2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of β2-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-α deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3-dependent conformational activation of LFA-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020004948DOI Listing
November 2020

The alternative cap-binding complex is required for antiviral defense in vivo.

PLoS Pathog 2019 12 19;15(12):e1008155. Epub 2019 Dec 19.

Institute of Virology, Technical University of Munich, School of Medicine, Munich, Germany.

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946169PMC
December 2019

Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation.

J Cell Sci 2019 11 1;132(21). Epub 2019 Nov 1.

Max-Planck-Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany

Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP bind talin proteins and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin F2 domain during talin membrane targeting and integrin activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1-binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and integrin activation similarly to the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP binding to spatiotemporally position and activate talins to regulate integrin activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.235531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857594PMC
November 2019

A kindlin-3-leupaxin-paxillin signaling pathway regulates podosome stability.

J Cell Biol 2019 10 19;218(10):3436-3454. Epub 2019 Sep 19.

Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany

Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling. How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we report that kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. We demonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation, requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes is kindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3-null cells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or any paxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes but also regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylation and downstream signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201903109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781449PMC
October 2019

Eosinophil-platelet interactions promote atherosclerosis and stabilize thrombosis with eosinophil extracellular traps.

Blood 2019 11;134(21):1859-1872

Medizinische Klinik und Poliklinik I, Ludwig-Maximilians-Universität, Munich, Germany.

Clinical observations implicate a role of eosinophils in cardiovascular diseases because markers of eosinophil activation are elevated in atherosclerosis and thrombosis. However, their contribution to atherosclerotic plaque formation and arterial thrombosis remains unclear. In these settings, we investigated how eosinophils are recruited and activated through an interplay with platelets. Here, we provide evidence for a central importance of eosinophil-platelet interactions in atherosclerosis and thrombosis. We show that eosinophils support atherosclerotic plaque formation involving enhanced von Willebrand factor exposure on endothelial cells and augmented platelet adhesion. During arterial thrombosis, eosinophils are quickly recruited in an integrin-dependent manner and engage in interactions with platelets leading to eosinophil activation as we show by intravital calcium imaging. These direct interactions induce the formation of eosinophil extracellular traps (EETs), which are present in human thrombi and constitute a substantial part of extracellular traps in murine thrombi. EETs are decorated with the granule protein major basic protein, which causes platelet activation by eosinophils. Consequently, targeting of EETs diminished thrombus formation in vivo, which identifies this approach as a novel antithrombotic concept. Finally, in our clinical analysis of coronary artery thrombi, we identified female patients with stent thrombosis as the population that might derive the greatest benefit from an eosinophil-inhibiting strategy. In summary, eosinophils contribute to atherosclerotic plaque formation and thrombosis through an interplay with platelets, resulting in mutual activation. Therefore, eosinophils are a promising new target in the prevention and therapy of atherosclerosis and thrombosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019000518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908806PMC
November 2019

A β2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation.

Front Immunol 2019 28;10:1138. Epub 2019 May 28.

Fagerholm Lab, MIBS, University of Helsinki, Helsinki, Finland.

β2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of β-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the β2-integrin (TTT/AAA-β2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-β2-integrin KI dendritic cells, which leads to a failure of MRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of β2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of β2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.01138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546827PMC
August 2020

Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures.

JCI Insight 2019 05 2;4(9). Epub 2019 May 2.

Department of Pharmacy, Center for Drug Research, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Munich, Germany.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.126418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538325PMC
May 2019

Microenvironment-derived ADAM28 prevents cancer dissemination.

Oncotarget 2018 Dec 14;9(98):37185-37199. Epub 2018 Dec 14.

Laboratory of Tumor and Development Biology, GIGA-Cancer and GIGA-I3, GIGA-Research, University of Liege, Liege, Belgium.

Previous studies have linked cancer cell-associated ADAM28 expression with tumor progression and metastatic dissemination. However, the role of host-derived ADAM28 in cancer dissemination processes remains unclear. Genetically engineered-mice fully deficient for ADAM28 unexpectedly display increased lung colonization by pulmonary, melanoma or breast tumor cells. In experimental tumor cell dissemination models, host ADAM28 deficiency is further associated with a decreased lung infiltration by CD8 T lymphocytes. Notably, naive ADAM28-deficient mice already display a drastic reduction of CD8 T cells in spleen which is further observed in lungs. Interestingly, CD8 T cell characterization revealed that ADAM28-deficiency does not impact proliferation, migration nor activation of CD8 T cells. Our data highlight a functional role of ADAM28 in T cell mobilization and point to an unexpected protective role for host ADAM28 against metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.26449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324684PMC
December 2018

Direct Rap1/Talin1 interaction regulates platelet and neutrophil integrin activity in mice.

Blood 2018 12 15;132(26):2754-2762. Epub 2018 Nov 15.

Department Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany.

Targeting Talin1 to the plasma membrane is a crucial step in integrin activation, which in leukocytes is mediated by a Rap1/RIAM/Talin1 pathway, whereas in platelets, it is RIAM independent. Recent structural, biochemical, and cell biological studies have suggested direct Rap1/Talin1 interaction as an alternative mechanism to recruit Talin1 to the membrane and induce integrin activation. To test whether this pathway is of relevance in vivo, we generated Rap1 binding-deficient Talin1 knockin (Tln1) mice. Although Tln1 mice showed no obvious abnormalities, their platelets exhibited reduced integrin activation, aggregation, adhesion, and spreading, resulting in prolonged tail-bleeding times and delayed thrombus formation and vessel occlusion in vivo. Surprisingly, neutrophil adhesion to different integrin ligands and β2 integrin-dependent phagocytosis were also significantly impaired, which caused profound leukocyte adhesion and extravasation defects in Tln1 mice. In contrast, macrophages exhibited no defect in adhesion or spreading despite reduced integrin activation. Taken together, our findings suggest that direct Rap1/Talin1 interaction is of particular importance in regulating the activity of different integrin classes expressed on platelets and neutrophils, which both depend on fast and dynamic integrin-mediated responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2018-04-846766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307989PMC
December 2018

Differential requirement of kindlin-3 for T cell progenitor homing to the non-vascularized and vascularized thymus.

Elife 2018 09 6;7. Epub 2018 Sep 6.

Department Molecular Medicine, Max-Planck-Institute of Biochemistry, Martinsried, Germany.

The role of integrin-mediated adhesion during T cell progenitor homing to and differentiation within the thymus is ill-defined, mainly due to functional overlap. To circumvent compensation, we disrupted the hematopoietic integrin regulator kindlin-3 in mice and found a progressive thymus atrophy that is primarily caused by an impaired homing capacity of T cell progenitors to the vascularized thymus. Notably, the low shear flow conditions in the vascular system at midgestation allow kindlin-3-deficient fetal liver-derived T cell progenitors to extravasate via pharyngeal vessels and colonize the avascular thymus primordium. Once in the thymus, kindlin-3 promotes intrathymic T cell proliferation by facilitating the integrin-dependent crosstalk with thymic antigen presenting cells, while intrathymic T cell migration, maturation into single positive CD4 and CD8 T cells and release into the circulation proceed without kindlin-3. Thus, kindlin-3 is dispensable for integrin-mediated T cell progenitor adhesion and signalling at low and indispensable at high shear forces.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.35816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126919PMC
September 2018

Pathogenicity of human antibodies against myelin oligodendrocyte glycoprotein.

Ann Neurol 2018 08 9;84(2):315-328. Epub 2018 Sep 9.

Institute of Clinical Neuroimmunology, Biomedical Center and University Hospitals, Ludwig-Maximilians-Universität München, Munich, Germany.

Objective: Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the central nervous system (CNS). We analyzed their pathogenic activity by affinity-purifying these antibodies (Abs) from patients and transferring them to experimental animals.

Methods: Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and the recognized MOG epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in 2 models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically.

Results: We identified 17 patients with MOG Abs from our outpatient clinic and selected 2 with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs from the 2 patients recognized different epitopes on MOG, the CC' and the FG loop. In both patients, these Abs persisted during our observation period of 2 to 3 years. The anti-MOG Abs from both patients were pathogenic upon intrathecal injection in 2 different rat models. Together with cognate MOG-specific T cells, these Abs enhanced T-cell infiltration; together with myelin basic protein-specific T cells, they induced demyelination associated with deposition of C9neo, resembling a multiple sclerosis type II pathology.

Interpretation: MOG-specific Abs affinity purified from patients with inflammatory demyelinating disease induce pathological changes in vivo upon cotransfer with myelin-reactive T cells, suggesting that these Abs are similarly pathogenic in patients. Ann Neurol 2018;84:315-328.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ana.25291DOI Listing
August 2018

AP-2ε Expression in Developing Retina: Contributing to the Molecular Diversity of Amacrine Cells.

Sci Rep 2018 02 21;8(1):3386. Epub 2018 Feb 21.

Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta, Canada.

AP-2 transcription factors play important roles in the regulation of gene expression during development. Four of the five members of the AP-2 family (AP-2α, AP-2β, AP-2γ and AP-2δ) have previously been shown to be expressed in developing retina. Mouse knockouts have revealed roles for AP-2α, AP-2β and AP-2δ in retinal cell specification and function. Here, we show that the fifth member of the AP-2 family, AP-2ε, is also expressed in amacrine cells in developing mammalian and chicken retina. Our data indicate that there are considerably fewer AP-2ε-positive cells in the developing mouse retina compared to AP-2α, AP-2β and AP-2γ-positive cells, suggesting a specialized role for AP-2ε in a subset of amacrine cells. AP-2ε, which is restricted to the GABAergic amacrine lineage, is most commonly co-expressed with AP-2α and AP-2β, especially at early stages of retinal development. Co-expression of AP-2ε and AP-2γ increases with differentiation. Analysis of previously published Drop-seq data from single retinal cells supports co-expression of multiple AP-2s in the same cell. Since AP-2s bind to their target sequences as either homodimers or heterodimers, our work suggests spatially- and temporally-coordinated roles for combinations of AP-2 transcription factors in amacrine cells during retinal development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-21822-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821864PMC
February 2018

Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation.

Nat Commun 2017 11 23;8(1):1744. Epub 2017 Nov 23.

Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH, 44195, USA.

Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-01822-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701058PMC
November 2017

E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning.

Nat Commun 2017 11 1;8(1):1250. Epub 2017 Nov 1.

Department of Dermatology, University of Cologne, Cologne, 50931, Germany.

Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy and atomic force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of tension-bearing adherens junction (AJ) and F-actin organization to allow formation of an apical TJ network only in the uppermost viable layer. Molecularly, E-cadherin localizes and tunes EGFR activity and junctional tension to inhibit premature TJ complex formation in lower layers while promoting increased tension and TJ stability in the granular layer 2. In conclusion, our data identify an E-cadherin-dependent mechanical circuit that integrates adhesion, contractile forces and biochemical signaling to drive the polarized organization of junctional tension necessary to build an in vivo epithelial barrier.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-01170-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665913PMC
November 2017

Maturation of Platelet Function During Murine Fetal Development In Vivo.

Arterioscler Thromb Vasc Biol 2017 06 20;37(6):1076-1086. Epub 2017 Apr 20.

From the Walter Brendel Centre of Experimental Medicine, Munich, Germany (A.M., C.N., I.R., S.K., A.R.M.K., A.F., J.P., M.P., R.I., S.D., M.S.); Division of Neonatology, Hauner Children's University Hospital and Perinatal Centre, Ludwig Maximilians University, Munich, Germany (C.N., A.F., V.W., A.W.F.); Medizinische Klinik und Poliklinik I, Klinikum der Ludwig Maximilians Universität, Munich, Germany (J.P.); Max Planck Institute for Molecular Biomedicine, Münster, Germany (L.K., F.K.); Max PIanck Institute of Biochemistry, Department of Molecular Medicine, Martinsried, Germany (M.M.); Roche Inc, New York, NY (E.Q.); and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA (U.H.v.A.).

Objective: Platelet function has been intensively studied in the adult organism. However, little is known about the function and hemostatic capacity of platelets in the developing fetus as suitable in vivo models are lacking.

Approach And Results: To examine fetal platelet function in vivo, we generated a fetal thrombosis model and investigated light/dye-induced thrombus formation by intravital microscopy throughout gestation. We observed that significantly less and unstable thrombi were formed at embryonic day (E) 13.5 compared with E17.5. Flow cytometry revealed significantly lower platelet counts in E13.5 versus E17.5 fetuses versus adult controls. In addition, fetal platelets demonstrated changed activation responses of surface adhesion molecules and reduced P-selectin content and mobilization. Interestingly, we also measured reduced levels of the integrin-activating proteins Kindlin-3, Talin-1, and Rap1 during fetal development. Consistently, fetal platelets demonstrated diminished spreading capacity compared with adults. Transfusion of adult platelets into the fetal circulation led to rapid platelet aggregate formation even in young fetuses. Yet, retrospective data analysis of a neonatal cohort demonstrated no correlation of platelet transfusion with closure of a persistent ductus arteriosus, a process reported to be platelet dependent.

Conclusions: Taken together, we demonstrate an ontogenetic regulation of platelet function in vivo with physiologically low platelet numbers and hyporeactivity early during fetal development shedding new light on hemostatic function during fetal life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.116.308464DOI Listing
June 2017

Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins.

J Biol Chem 2017 05 21;292(19):7745-7760. Epub 2017 Mar 21.

From the Max-Planck Institute of Biochemistry, 82152 Martinsried and

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.739987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427257PMC
May 2017

MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane.

J Clin Invest 2016 11 4;126(11):4125-4139. Epub 2016 Oct 4.

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI87043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096904PMC
November 2016

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.

Nat Cell Biol 2016 Nov 24;18(11):1253-1259. Epub 2016 Oct 24.

Institute of Science and Technology Austria (IST Austria), am Campus 1, 3400 Klosterneuburg, Austria.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb3426DOI Listing
November 2016

An O-Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through α4β1 Integrin.

J Bone Miner Res 2017 01 3;32(1):70-81. Epub 2016 Aug 3.

Max-Planck Institute of Biochemistry, Martinsried, Germany.

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O-deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O-glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4β1 or α4β7 integrins, these integrins were evaluated. We show that integrin α4β1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4β1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O-glycosylation in the variable region that results in decreased integrin-mediated signaling. This deleterious effect can be thwarted by binding α4β1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model. © 2016 American Society for Bone and Mineral Research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.2916DOI Listing
January 2017

Loss of AP-2delta reduces retinal ganglion cell numbers and axonal projections to the superior colliculus.

Mol Brain 2016 06 4;9(1):62. Epub 2016 Jun 4.

Department of Oncology, Cross Cancer Institute, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada.

Background: AP-2δ is the most divergent member of the Activating Protein-2 (TFAP2) family of transcription factors. AP-2δ is restricted to specific regions of the CNS, including a subset of ganglion cells in the retina. Retinal ganglion cells (RGCs), the only output neurons of the retina, are responsible for transmitting the visual signal to the brain.

Results: AP-2δ knockout results in loss of Brn3c (Pou4f3) expression in AP-2δ -positive RGCs. While AP-2δ-/- mice have morphologically normal retinas at birth, there is a significant reduction in retinal ganglion cell numbers by P21, after eye opening. Chromatin immunoprecipitation indicates that Brn3c is a target of AP-2δ in the retina. Using fluorochrome-conjugated cholera toxin subunit B to trace ganglion cell axons from the eye to the major visual pathways in the brain, we found 87 % and 32 % decreases in ipsilateral and contralateral projections, respectively, to the superior colliculus in AP-2δ-/- mice. In agreement with anatomical data, visually evoked responses recorded from the brain confirmed that retinal outputs to the brain are compromised.

Conclusions: AP-2δ is important for the maintenance of ganglion cell numbers in the retina. Loss of AP-2δ alters retinal axonal projections to visual centers of the brain, with ipsilaterial projections to the superior colliculus being the most dramatically affected. Our results have important implications for integration of the visual signal at the superior colliculus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13041-016-0244-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893287PMC
June 2016

Minimal amounts of kindlin-3 suffice for basal platelet and leukocyte functions in mice.

Blood 2015 Dec 5;126(24):2592-600. Epub 2015 Oct 5.

Max-Planck-Institute of Biochemistry, Department of Molecular Medicine, Martinsried, Germany;

Hematopoietic cells depend on integrin-mediated adhesion and signaling, which is induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or 5% of normal kindlin-3 levels. We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearance were diminished. Quantification of protein copy numbers revealed stoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2015-04-639310DOI Listing
December 2015
-->