Towards the architecture of the human inner kinetochore

Im Rahmen der Dissertation „Towards the architecture of the human inner kinetochore“ wurden Proteininteraktionen im humanen inneren Kinetochor untersucht. Dazu wurden die fluoreszenzmarkierten Kinetochorproteine CENP-A, CENP-B, CENP-C, CENP-I und Histone H1, H2A, H3 und H4 mittels AB-FRET (acceptor bleaching-fluorescence resonance energy tranfer) und FLIM (fluorescence lifetime imaging) in lebenden Hep2-Zellen analysiert. Weiterhin wurde mittels RNAi knock down die Expression des humanen Kinetochor-proteins CENP-H herunterreguliert und seine Funktion im Kinetochor untersucht

Automatic Segmentation of Fluorescence Lifetime Microscopy Images of Cells Using Multi-Resolution Community Detection

We have developed an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells inspired by a multi-resolution community detection (MCD) based network segmentation method. The image processing problem is framed as identifying segments with respective average FLTs against a background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network composed using image pixels as the nodes and similarity between the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments and high network resolution leads to smaller segments. Further, the mean-square error (MSE) in estimating the FLT segments in a FLIM image using the proposed method was found to be consistently decreasing with increasing resolution of the corresponding network. The proposed MCD method outperformed a popular spectral clustering based method in performing FLIM image segmentation. The spectral segmentation method introduced noisy segments in its output at high resolution. It was unable to offer a consistent decrease in MSE with increasing resolution.Comment: 21 pages, 6 figure

The CENP-T C-terminus is exclusively proximal to H3.1 and not to H3.2 or H3.3.

The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere-kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1(C96A) and H3.1(C110A) nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2

Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins

The vertebrate kinetochore complex assembles at the centromere on α-satellite DNA. In humans, α-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to α-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1° and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins

Prognostic relevance of Centromere protein H expression in esophageal carcinoma

<p>Abstract</p> <p>Background</p> <p>Many kinetochore proteins have been shown to be associated with human cancers. The aim of the present study was to clarify the expression of Centromere protein H (CENP-H), one of the fundamental components of the human active kinetochore, in esophageal carcinoma and its correlation with clinicopathological features.</p> <p>Methods</p> <p>We examined the expression of CENP-H in immortalized esophageal epithelial cells as well as in esophageal carcinoma cells, and in 12 cases of esophageal carcinoma tissues and the paired normal esophageal tissues by RT-PCR and Western blot analysis. In addition, we analyzed CENP-H protein expression in 177 clinicopathologically characterized esophageal carcinoma cases by immunohistochemistry. Statistical analyses were applied to test for prognostic and diagnostic associations.</p> <p>Results</p> <p>The level of CENP-H mRNA and protein were higher in the immortalized cells, cancer cell lines and most cancer tissues than in normal control tissues. Immunohistochemistry showed that CENP-H was expressed in 127 of 171 ESCC cases (74.3%) and in 3 of 6 esophageal adenocarcinoma cases (50%). Statistical analysis of ESCC cases showed that there was a significant difference of CENP-H expression in patients categorized according to gender (<it>P </it>= 0.013), stage (<it>P </it>= 0.023) and T classification (<it>P </it>= 0.019). Patients with lower CENP-H expression had longer overall survival time than those with higher CENP-H expression. Multivariate analysis suggested that CENP-H expression was an independent prognostic marker for esophageal carcinoma patients. A prognostic value of CENP-H was also found in the subgroup of T3~T4 and N0 tumor classification.</p> <p>Conclusion</p> <p>Our results suggest that CENP-H protein is a valuable marker of esophageal carcinoma progression. CENP-H might be used as a valuable prognostic marker for esophageal carcinoma patients.</p

Erstellung von Einstrahlungskarten durch eine Webkamera von einem Bergrücken aus

Die Solarthermie beschäftigt sich damit wie die Bestrahlung durch die Sonne zu bündeln ist um Wärme zu produzieren. Für jedes produzierende Gewerbe ist es wichtig, dass die Produktionsrate berechenbar ist. Diese Planbarkeit ist im Bereich der Solarthermie schwierig. Dies liegt daran, dass äußere Faktoren eine große Rolle spielen. Zum Beispiel wird die Produktionsrate gehemmt durch Staub in der Atmosphäre. Besonders groß ist der Einfluss von Wolkenschatten die zu Verschattungen von Teilen der Anlage führen wodurch nicht produziert werden kann. Dies kann bis zu einem gesamten erliegen der Produktion führen. Durch ihre hohe Dynamik und Vielseitigkeit sind die Verschattungen durch Wolken schwer einzuschätzen. Es gibt Wolkenschatten die kaum eine Verschattung am Boden hervorrufen z.B. Zirrus Wolken und somit die Produktion gering bis gar nicht einschränken. Andererseits gibt es Verschattungen bei denen keine Produktion möglich ist z.B. wenn eine geschlossene Wolkendecke sich ausgebildet hat. Um die Planbarkeit der bereitgestellten Wärme zu verbessern wird daran gearbeitet die Bewegung von Wolken und ihre Verschattung vorauszusagen. Sowie aus den Aufnahmen abzuleiten wie hoch die Bestrahlung ist. Ein System dafür ist das Schattenkamerasystem. Bei diesem System werden Aufnahmen vom Boden erstellt. Daraus wird in Echtzeit ermittelt wie stark die Bestrahlung in einem vorher bestimmten Gebiet ist. Das Schattenkamerasystem wurde auf der Plataforma Solar de Almería (PSA) bereits erfolgreich auf einer Fläche von 4 km² erprobt. In dieser Arbeit wird das Verfahren auf eine auszuwertende Fläche von ca. 288 km² angewendet. Hierdurch ergeben sich neue Schwierigkeiten welche in dieser Arbeit untersucht werden. Hierzu zählt die Streuung des Lichtes auf dem Weg zwischen Sonne und Bodenelement. Ebenso zählt dazu die Abschwächung der Bestrahlung zwischen Bodenelement und Kamera

Revisiting Constituents' Reflections on the Incorporation of Day-one Losses into IFRS 9

IFRS 9 requires the recognition of expected credit losses from the inception of a financial instrument, resulting in so-called day-one losses. The incorporation of day-one losses caused considerable controversy among the IASB members and its constituents. With a focus on the constituents' positions and reasoning, this study portrays the discussions held in the comment letters received by the IASB during the drafting process. We find that most constituents initially rejected day-one losses as conceptually unsound and/or as inappropriately affecting investors' and preparers' decision-making. Despite these continuing concerns, the majority of constituents eventually accepted day-one losses as a pragmatic approximation of expected credit losses in the absence of superior alternatives. Considering the technical and political nature of standard setting, our analysis provides insights into the constituents' assessment of departures from the Conceptual Framework and the constituents' views on the standard setters' responsibilities regarding financial stability after the financial crisis

Linker histone H1 is present in centromeric

chromatin of living human cells next to inner kinetochore protein

Functional Complementation of Human Centromere Protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae

We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans