38 research outputs found
The Centrosomal Protein Pericentrin Identified at the Basal Body Complex of the Connecting Cilium in Mouse Photoreceptors
BACKGROUND: Pericentrin (Pcnt), a conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Recent studies indicate that Pcnt mutations are associated with a range of diseases including primordial dwarfism and ciliopathies. To date, three Pcnt splice variants from orthologous genes in mice and humans are known. PRINCIPAL FINDINGS: We generated a specific Pcnt antiserum detecting all known Pcnt splice variants and examined the cellular and subcellular distribution of Pcnt in ciliated tissues of the mouse, the olfactory epithelium and the retina. For the first time, we identified Pcnt and its centrosomal interaction partners at the basal body complex of mouse retinal photoreceptors. Photoreceptors are morphologically and functionally subdivided into the light sensitive outer segment and the inner segment comprising the metabolic function of the cell. The two compartments are linked via a modified, specialized, non-motile cilium, the connecting cilium. Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments. Surprisingly, photoreceptors expressed a small Pcnt splice transcript - most likely a modified variant of Pcnt S - which was not present in receptor neurons of the olfactory epithelium. CONCLUSIONS: Our findings suggest distinct functional roles of several Pcnt variants in different ciliated tissues and sensory neurons, like the olfactory epithelium and the retina of the mouse. The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene
Functional analyses of Pericentrin and Syne-2/Nesprin-2 interaction in ciliogenesis
Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt–Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration
Molekulare Charakterisierung der Centrin-Isoformen in der Retina von Säugetieren
Centrine sind Mitglieder einer hoch konservierten Ăśberfamilie von Ca2+-bindenden
Proteinen mit EF-Hand Motiven. Bislang sind vier Centrin-Isoformen bei Säugern
beschrieben worden, die in diversen Zellen in der
Regel mit Centriolen von Centrosomen
oder Centrosomen-verwandten Strukturen assoziiert sind. Im Rahmen der vorliegenden
Dissertation wurden die vier Centrin-Isoformen bezĂĽglich der Expression in verschiedenen
Geweben untersucht. Dabei lag der Hauptfokus auf Untersuchungen der Centrine in den
Photorezeptorzellen der Retina. Analysen auf subzellulärer Ebene brachten Klarheit über die
differenzielle Lokalisation der verschiedenen Isoformen in der Retina. Mit Hilfe von
verschiedenen Methoden konnten Wechselwirkungspartner in der Retina identifiziert
werden, die eine Rolle in der visuellen Signaltransduktionskaskade spielen. Dabei könnten
Centrine einem Regelmechanismus angehören, der wichtige Translokationsprozesse dieser
Proteine regelt.
In den Photorezeptorzellen der Säugetierretina werden die vier Isoformen exprimiert,
die in den Strukturen des Cilienapparates differenziell lokalisiert sind. Dabei beschränkt
sich ihre Lokalisation entweder auf den Basalkörper (Centrin 4),
auf das Verbindungscilium
(Centrin 1) oder sie sind in beiden Strukturen zu finden (Centrin 2 und 3). In den nicht-
Photorezeptorzellen der Retina sind die Isoformen Centrin 2 und 3 zudem an den Centriolen
der Centrosomen lokalisiert. In der vorliegenden Arbeit wurde zum ersten Mal gezeigt, dass
alle Centrin-Isoformen in ein und derselben Zelle, der Photorezeptorzelle, koexprimiert
werden und dabei subzellulär kolokalisiert sind. Im Weiteren konnte die ubiquitäre
Expression von Centrin 2 und 3 in allen untersuchten Geweben an Centrosomen bestätigt
werden. Centrin 1 und 4 hingegen werden nur in Geweben mit Cilien-tragenden Zellen
exprimiert.
Die Funktion der Centrine wird nicht nur durch Bindung von Ca2+, sondern auch durch
Phosphorylierungen reguliert. Alle Sequenzen der Centrine weisen diverse mögliche
Phosphorylierungsstellen für unterschiedliche Proteinkinasen auf. Die Ergebnisse aller durchgeführten in vitro und ex vivo Phosphorylierungs „Assays“ zeigen eine licht-
abhängige
Phosphorylierung der Centrin-Isoformen in der Retina. Dabei war in der dunkel-adaptierten
Retina die Phosphorylierung vor allem von Centrin 1 und 2 erhöht. Weiterführende
Experimente mit Kinase-Inhibitoren wiesen darauf hin, dass vor allem die Proteinkinase
CKII eine bedeutende Rolle bei der Centrin-Phosphorylierung in der Retina einnimmt.
Centrine sind die ersten Cytoskelettkomponenten, deren Phosphorylierungsgrad lichtabhängig
moduliert wird. Diese Ergebnisse weisen auf einen Signalweg, der zwischen der
visuellen Signaltransduktionskaskade und der Regulation der Centrin-Aktivität vermittelt,
hin.
Bei der Suche nach Centrin-Bindungspartnern gelang mit Hilfe von Centrin 1 Blot
„Overlay Assays“ der Durchbruch. Der neuartige Ansatz zeigte, dass ausschließlich
Ca2+-aktiviertes Centrin 1 mit Proteinen aus der Retina interagierte. Nach der Identifikation
eines 37 kDa-Proteins als die β-Untereinheit des visuellen G-Proteins Transducin wurden
die Untersuchungen auf
diesen Interaktionspartner fokussiert. Die Ergebnisse der hier
durchgefĂĽhrten biochemischen und biophysikalischen Protein-Protein
Interaktionsexperimente zeigen insgesamt folgendes:
⇒ Alle vier Centrine interagieren mit Transducin, wobei Centrin 3 die geringste
Affinität zu Transducin hat.
⇒ Die Assemblierung der Centrin•G-Protein-Komplexe ist strikt Ca2+-abhängig.
⇒ Die Centrine binden sowohl an das isolierte Gtβγ-Heterodimer als auch an den
heterotrimeren Gt-holo-Proteinkomplex, nicht aber an Gtα.
Die quantitativen immunoelektronenmikroskopischen Analysen zeigen im Weiteren, dass
sich die Komplexe aus Transducin und Centrin 1 bis 3 wahrscheinlich in einer Subdomäne
des Verbindungsciliums der Photorezeptorzellen ausbilden. Dabei dĂĽrfte die Ausbildung der
Komplexe an der Regulation der lichtinduzierten Translokation von Transducin zwischen
Innen- und AuĂźensegment der Photorezeptorzellen beteiligt sein. Dieser
Translokationsmechanismus wird als ein wichtiger Bestandteil
der Langzeitadaption der
Signaltransduktionskaskade der Säugerretina diskutiert. Der neuartige Regelmechanismus
der molekularen Translokationen, in dem Centrine involviert sind, ist außergewöhnlich und
dĂĽrfte ĂĽber die speziellen Photorezeptorzellen hinaus von weit reichender Bedeutung sein.Centrins are members of the superfamily of Ca2+-binding EF-hand proteins. In eukaryotic cells, up to 4 centrin isoforms are commonly associated with centrioles of centrosomes and spindle poles. Nevertheless, in ciliated cells, centrins are not only present in the centrioles of basal bodies but are also localized in the transition zone of cilia.
We have previously shown that centrins are prominent cytoskeletal components of the connecting cilium linking inner and outer segments of photoreceptor cells in the vertebrate retina. Here, we demonstrate that all 4 known centrin isoforms
are expressed in the mammalian retina. Immunocytochemical analysis using isoform specific antibodies against the centrins reveals differential subcellular localizations of the centrin isoforms in the photoreceptors: Centrin 1, 2 and 3 colocalize in the connecting cilium. In contrast, centrin 4 is exclusively found in the basal body, where it colocalizes with centrin 2 and 3.
In the search for centrin interacting proteins, we identified the β-subunit of visual G-protein transducin as a binding partner to centrin 1. Recent analyses reveal that all 4 centrins interact with transducin in a Ca2+-dependent way. Immunoelectron microscopy demonstrated that transducin colocalizes with centrin 1, 2 and 3 in the photoreceptor connecting cilium. Due to the fact that centrin 3 has much lower affinity to transducin than the other three centrin isoforms, centrin 1 and 2 remain as predominant candidates for the Ca2+-dependent interaction with transducin. The binding of both centrin isoforms to transducin may regulate the
previously described light-dependent movements through the photoreceptor cilium.
In general, the assembly of centrin-G-protein complexes are a novel aspect of the supply of signaling proteins in sensory cells, and potential links between molecular translocations and signal transduction.
Supports: DFG; FAUN-Stiftun
Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome
A strict regulation of protein expression during developmental stages and in response to environmental signals is essential to every cell and organism. Recent research has shown that the mammalian brain is particularly sensitive to alterations in expression patterns of specific proteins and cognitive deficits as well as autistic behaviours have been linked to dysregulated protein expression. An intellectual disability characterised by changes in the expression of a variety of proteins is the fragile X syndrome. Due to the loss of a single mRNA binding protein, the Fragile X Mental Retardation Protein FMRP, vast misregulation of the mRNA metabolism is taking place in the disease. Here, we present the identification and characterisation of a novel protein named Simiate, whose mRNA contains several FMRP recognition motifs and associates with FMRP upon co-precipitation. Sequence analysis revealed that the protein evolved app. 1.7 billion years ago when eukaryotes developed. Applying antibodies generated against Simiate, the protein is detected in a variety of tissues, including the mammalian brain. On the subcellular level, Simiate localises to somata and nuclear speckles. We show that Simiate and nuclear speckles experience specific alterations in FMR1-/- mice. An antibody-based block of endogenous Simiate revealed that the protein is essential for cell survival. These findings suggest not only an important role for Simiate in gene transcription and/or RNA splicing, but also provide evidence for a function of nuclear speckles in the fragile X syndrome. Indeed, transcription and splicing are two fundamental mechanisms to control protein expression, that underlie not only synaptic plasticity and memory formation, but are also affected in several diseases associated with mental disabilities
Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma
Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications
Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma
Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications
Laminin-511-E8 promotes efficient in vitro expansion of human limbal melanocytes
Abstract Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Here, we established a protocol for the efficient isolation and cultivation of pure populations of human limbal melanocytes, which could be expanded at high yield by using recombinant laminin (LN)-511-E8 as culture substrate. Co-cultivation of limbal melanocytes with limbal epithelial stem/progenitor cells on fibrin hydrogels pre-incubated with LN-511-E8 resulted in multilayered stratified epithelial constructs within ten days. By reproducing physiological cell–cell and cell–matrix interactions of the native niche environment, these biomimetic co-culture systems provide a promising experimental model for investigating the functional roles of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction