Biology subjects

Linnert, J.

Publications and source records attributed to Linnert, J..

6 recordsLinked to original sources

bioRxiv2024

The BBS/CCT chaperonin complex ensures the localization of the adhesion G protein-coupled receptor ADGRV1 to primary cilia

Primary cilia are antenna-like sensory organelles present on almost all eukaryotic cells. Their sensory capacity relies on receptors, in particular G-protein-coupled receptors (GPCRs) which localize to the ciliary membrane. Here we show that ADGRV1, a member of the GPCR subfamily of adhesion GPCRs, is part of a large protein network, interacting with numerous proteins of a comprehensive ciliary proteome. ADGRV1 is localized to the base of prototypic primary cilia in cultured cells and the modified primary cilia of retinal photoreceptors, where it interacts with TRiC/CCT chaperonins and the Bardet Biedl syndrome (BBS) chaperonin-like proteins. Knockdown of ADGRV1, CCT2 and 3, and BBS6 result in common ciliogenesis phenotypes, namely reduced ciliated cells combined with shorter primary cilia. In addition, the localization of ADGRV1 to primary cilia depends on the activity of a co-complex of TRiC/CCT chaperonins and the BBS chaperonin-like proteins. In the absence of components of the TRiC/CCT-BBS chaperonin co-complex, ADGRV1 is depleted from the base of the primary cilium and degraded via the proteasome. Defects in the TRiC/CCT-BBS chaperonin may lead to an overload of proteasomal degradation processes and imbalanced proteostasis. Dysfunction or absence of ADGRV1 from primary cilia may underly the pathophysiology of human Usher syndrome type 2 and epilepsy caused by mutations in ADGRV1.

cell biology↗

bioRxiv2024

The adhesion GPCR ADGRV1 controls glutamate homeostasis in hippocampal astrocytes supporting neuron development: First insights into to pathophysiology of ADGRV1-associated epilepsy

ADGRV1 is the largest member of adhesion G protein-coupled receptor (aGPCR) family. In the cell, aGPCRs have dual roles in cell adhesion and signal transduction. Mutations in ADGRV1 have been linked not only to Usher syndrome (USH), which causes deaf-blindness, but recently also to various forms of epilepsy. While the USH defects are attributed to the loss of fiber links between membranes formed by the extracellular domain of ADGRV1, the pathomechanisms leading to epilepsy remain elusive to date. Here, we study the specific functions of ADGRV1 in astrocytes where it is highest expressed in the nervous system. Affinity proteomics showed the interaction of ADRGV1 with proteins enriched in astrocytes. Dysregulations of cellular processes important in astrocyte function were indicated by the different transcriptomes of patient-derived cells and Adgrv1-deficent mouse hippocampi compared to appropriate controls. Alteration in morphology and reduced numbers of astrocytes in the hippocampus of Adgrv1-deficent mice. Monitoring the glutamate uptake in colorimetric assay and by live cell imaging of a genetic glutamate reporter consistently showed that glutamate uptake from the extracellular environment is significantly reduced in Adgrv1-deficent astrocytes. Expression analyses of key enzymes of the glutamate-glutamine cycle in astrocytes and the glutamate metabolism indicated imbalanced glutamate homeostasis in Adgrv1-deficient astrocytes. Finally, we provide evidence that the supportive function of astrocytes in neuronal development also relies on ADGRV1 expression in astrocytes. Our data collectively provides first insights into the molecular pathophysiology underlying the development of epilepsy associated with mutations in ADGRV1. HighlightsO_LIADGRV1 deficiency reduces the number of astrocytes in CA1 and changes the morphology of astrocytes in the hippocampus. C_LIO_LIADGRV1 interacts with numerous proteins enriched in astrocytes. C_LIO_LIDifferential transcriptomes revealed differential expression of genes related to glutamate homeostasis and epilepsy in ADGRV1 deficient models. C_LIO_LIADGRV1 controls glutamate uptake and regulates homeostasis in astrocytes. C_LIO_LIADGRV1 in astrocytes is vital for neuron morphogenesis. C_LIO_LIFirst insights into the molecular pathophysiology underlying the development of epilepsy associated with mutations in ADGRV1. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/591120v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1f1f749org.highwire.dtl.DTLVardef@a1ea01org.highwire.dtl.DTLVardef@a0be1org.highwire.dtl.DTLVardef@191f96a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E

The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogs in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in MEFs results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 KO in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other IFT proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi-cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi. SummaryPhylogenetic analyses of ARF family GTPases predict that ARL16 is linked to cilia. This was confirmed using MEFs deleted for ARL16, resulting in defects in Golgi to cilium traffic, with accumulation of IFT140 and INPP5E at Golgi.

cell biology↗

bioRxiv2021

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and Cilia to Regulate Ciliogenesis and Ciliary Protein Traffic

ELMODs are a family of three mammalian paralogs that display GTPase activating protein (GAP) activity towards a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogs ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon expression of activating mutants of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions. Thus, we believe that ELMOD1 and ELMOD3 perform multiple functions in cells, most prominently linked to ciliary biology and Golgi-ciliary traffic, and likely acting from more than one cellular location.

cell biology↗

bioRxiv2021

Early Disruption of Photoreceptor Cell Architecture and Loss of Vision in a Humanized Pig Model of Usher Syndrome

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and, so far, there are no suitable animal models for testing therapeutic strategies. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Primary cells from those animals and USH1C patients showed significantly elongated primary cilia, compared to wild-type, confirming the nature of USH as a true and general ciliopathy and proving the therapeutic capacity of gene supplementation and gene repair approaches.

genetics↗

bioRxiv2021

Roles for ELMOD2 and Rootletin in Ciliogenesis

ELMOD2 is a GTPase activating protein (GAP) with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway support a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.

cell biology↗