Construction and characterization of an artificial kinetochore in budding yeast

Eukaryotische Zellen teilen ihre duplizierten Chromosomen waehrend der Mitose praezise zwischen den beiden Spindelpolen auf. Dieser Prozess gewaehrleistet die Integritaet des genetischen Materials ueber viele Generation. Sollte es dennoch vorkommen, dass Chromosomen falsch segregieren, kann dies zu Aneuploiditaet fuehren – ein charakteristisches Merkmal vieler humaner Tumore. Im Laufe der Zellteilung muessen die Chromosomen mit dem Spindelapparat verknuepft werden. Dies geschieht mit Hilfe von Kinetochoren. Kinetochore sind makromolekulare Maschinen, die an einer definierten Chromosomenregion, dem so genannten Centromer assemblieren und so als eine Art Plattform fuer die Plus-Enden der Spindelmikrotubuli dienen. Kinetochore bestehen aus einer Vielzahl an Proteinen, welche weiters in verschiedene Subkomplexe untergliedert werden. Diese Komplexe bauen sich hierarchisch auf dem Centromer auf und bilden so eine definierte Topologie die gewoehnlich als trilaminare Kinetochorstruktur bezeichnet wird. Im Gegensatz zu hoeheren Eukaryoten erstreckt sich das Centromer in S. Cerevisiae ueber einen Bereich von 125 Basenpaaren und ist mit nur einem einzelnen Mikrotubulus verknuepft. Diese vereinfachenden Umstaende machen die Hefe zu einem idealen Modelorganismus um die Organisation und Funktion von komplexen Kinetochoren zu untersuchen. Dennoch bestehen selbst in S. Cerevisiae die einzelnen Kinetochore aus mehr als 80 verschiedenen Proteinen was die genaue Charakterisierung von individuellen Subkomplexen erschwert. Waehrend meiner Doktorarbeit entwickelte ich ein System, mit Hilfe dessen individuelle Kinetochorproteine artifiziell durch das TetR-tetO Transkriptionssystems zu einem Hefen Mini-Chromosom rekrutiert werden koennen. Dies erlaubt die Beantwortung der Frage, ob die artifiziell rekrutierten Proteine in der Lage sind, die Funktion eines nativen Kinetochores zu uebernehmen. Im Zuge dessen konnte ich zeigen, dass die artifizielle Rekrutierung des Dam1 Komplexes ausreicht, um ein azentrisches Mini-Chromosom fehlerfrei zu segregieren – unabhaengig von inneren und zentralen Kinetochorproteinen. Dieses artifizielle Segregationssystem benoetigt die konservierte Kinase Ipl1, aber unterliegt nicht mehr der Kontrolle des allgemeinen Spindel Checkpoints. Des weiteren konnte das System effizient auf native Hefechromosomen uebertragen werden. Meine Daten zeigen, dass ein einfaches eukaryotisches Chromosomensegregationssystem kuenstlich nachgebaut werden kann, indem man Microtubuli-bindende Proteine rekrutiert, die die mechanische Energie, die waehrend der Depolymerisierung der MT entsteht, in Bewegung von Chromosomen umwandeln koennen. Diese Strategie wird auch von primitiven Bakterien genutzt, um ihre Erbinformation auf zwei Zellen aufzuteilen

Characterization of a naturally-occurring p27 mutation predisposing to multiple endocrine tumors

<p>Abstract</p> <p>Background</p> <p>p27Kip1 (p27) is an important negative regulator of the cell cycle and a putative tumor suppressor. The finding that a spontaneous germline frameshift mutation in <it>Cdkn1b </it>(encoding p27) causes the MENX multiple endocrine neoplasia syndrome in the rat provided the first evidence that <it>Cdkn1b </it>is a tumor susceptibility gene for endocrine tumors. Noteworthy, germline p27 mutations were also identified in human patients presenting with endocrine tumors. At present, it is not clear which features of p27 are crucial for this tissue-specific tumor predisposition in both rats and humans. It was shown that the MENX-associated <it>Cdkn1b </it>mutation causes reduced expression of the encoded protein, but the molecular mechanisms are unknown. To better understand the role of p27 in tumor predisposition and to characterize the MENX animal model at the molecular level, a prerequisite for future preclinical studies, we set out to assess the functional properties of the MENX-associated p27 mutant protein (named p27fs177) <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p><it>In vitro</it>, p27fs177 retains some properties of the wild-type p27 (p27wt) protein: it localizes to the nucleus; it interacts with cyclin-dependent kinases and, to lower extent, with cyclins. In contrast to p27wt, p27fs177 is highly unstable and rapidly degraded in every phase of the cell-cycle, including quiescence. It is in part degraded by Skp2-dependent proteasomal proteolysis, similarly to p27wt. Photobleaching studies showed reduced motility of p27fs177 in the nucleus compared to p27wt, suggesting that in this compartment p27fs177 is part of a multi-protein complex, likely together with the degradation machinery. Studies of primary rat newborn fibroblasts (RNF) established from normal and MENX-affected littermates confirmed the rapid degradation of p27fs177 <it>in vivo </it>which can be rescued by Bortezomib (proteasome inhibitor drug). Overexpression of the negative regulators microRNA-221/222 plays no role in regulating the amount of p27fs177 in RNFs and rat tissues.</p> <p>Conclusion</p> <p>Our findings show that reduced p27 levels, not newly acquired properties, trigger tumor formation in rats, similarly to what has been observed in mice. The molecular characteristics of p27fs177 establish MENX as a useful preclinical model to evaluate compounds that inhibit p27 degradation for their efficacy against endocrine tumors.</p

Microtubules control cellular shape and coherence in amoeboid migrating cells

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence

The central role of the centrosome

The centrosome decides which branch extending from the body of microglia will successfully engulf and clear away dead neurons

N-linked glycosylation of folded proteins by the bacterial oligosaccharyltransferase

N-linked protein glycosylation is found in all domains of life. In eukaryotes, it is the most abundant protein modification of secretory and membrane proteins, and the process is coupled to protein translocation and folding. We found that in bacteria, N-glycosylation can occur independently of the protein translocation machinery. In an in vitro assay, bacterial oligosaccharyltransferase glycosylated a folded endogenous substrate protein with high efficiency and folded bovine ribonuclease A with low efficiency. Unfolding the eukaryotic substrate greatly increased glycosylation. We propose that in the bacterial system, glycosylation sites are located in flexible parts of folded proteins, whereas the eukaryotic cotranslational glycosylation evolved to a mechanism presenting the substrate in a flexible form before folding

Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells

Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis

Polysialylation controls dendritic cell trafficking by regulating chemokine recognition

The addition of polysialic acid to N- and/or O-linked glycans, referred to as polysialylation, is a rare posttranslational modification that is mainly known to control the developmental plasticity of the nervous system. Here we show that CCR7, the central chemokine receptor controlling immune cell trafficking to secondary lymphatic organs, carries polysialic acid. This modification is essential for the recognition of the CCR7 ligand CCL21. As a consequence, dendritic cell trafficking is abrogated in polysialyltransferase-deficient mice, manifesting as disturbed lymph node homeostasis and unresponsiveness to inflammatory stimuli. Structure-function analysis of chemokine-receptor interactions reveals that CCL21 adopts an autoinhibited conformation, which is released upon interaction with polysialic acid. Thus, we describe a glycosylation-mediated immune cell trafficking disorder and its mechanistic basis

Solution structure of CCL19 and identification of overlapping CCR7 and PSGL-1 binding sites

CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1's enhancement of resting T-cell recruitment are discussed