Patrick Heun

Drosophila CENH3 Is Sufficient for Centromere Formation

A specific histone is sufficient for the formation of a functional and heritable centromere in the fruit fly. CENH3 is a centromere-specific histone H3 variant essential for kinetochore assembly. Despite its central role in centromere function, there has been no conclusive evidence supporting CENH3 as sufficient to determine centromere identity. To address this question, we artificially targeted Drosophila CENH3 (CENP-A/CID) as a CID-GFP-LacI fusion protein to stably integrated lac operator (lacO) arrays. This ectopic CID focus assembles a functional kinetochore and directs incorporation of CID molecules without the LacI-anchor, providing evidence for the self-propagation of the epigenetic mark. CID-GFP-LacI–bound extrachromosomal lacO plasmids can assemble kinetochore proteins and bind microtubules, resulting in their stable transmission for several cell generations even after eliminating CID-GFP-LacI. We conclude that CID is both necessary and sufficient to serve as an epigenetic centromere mark and nucleate heritable centromere function.

Heterochromatin boundaries are hotspots for de novo kinetochore formation

The centromere-specific histone H3 variant CENH3 (also known as CENP-A) is considered to be an epigenetic mark for establishment and propagation of centromere identity. Pulse induction of CENH3 (Drosophila CID) in Schneider S2 cells leads to its incorporation into non-centromeric regions and generates CID islands that resist clearing from chromosome arms for multiple cell generations. We demonstrate that CID islands represent functional ectopic kinetochores, which are non-randomly distributed on the chromosome and show a preferential localization near telomeres and pericentric heterochromatin in transcriptionally silent, intergenic chromatin domains. Although overexpression of heterochromatin protein 1 (HP1) or increasing histone acetylation interferes with CID island formation on a global scale, induction of a locally defined region of synthetic heterochromatin by targeting HP1–LacI fusions to stably integrated Lac operator arrays produces a proximal hotspot for CID deposition. These data indicate that the characteristics of regions bordering heterochromatin promote de novo kinetochore assembly and thereby contribute to centromere identity.

CAL1 is the Drosophila CENP-A assembly factor

Representing a unique family of histone assembly factors, CAL1 assembles the histone H3 variant CENP-A on centromeric DNA in Drosophila.

The Nucleoplasmin Homolog NLP Mediates Centromere Clustering and Anchoring to the Nucleolus

Centromere clustering during interphase is a phenomenon known to occur in many different organisms and cell types, yet neither the factors involved nor their physiological relevance is well understood. Using Drosophila tissue culture cells and flies, we identified a network of proteins, including the nucleoplasmin-like protein (NLP), the insulator protein CTCF, and the nucleolus protein Modulo, to be essential for the positioning of centromeres. Artificial targeting further demonstrated that NLP and CTCF are sufficient for clustering, while Modulo serves as the anchor to the nucleolus. Centromere clustering was found to depend on centric chromatin rather than specific DNA sequences. Moreover, unclustering of centromeres results in the spatial destabilization of pericentric heterochromatin organization, leading to partial defects in the silencing of repetitive elements, defects during chromosome segregation, and genome instability.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

A Pair of Centromeric Proteins Mediates Reproductive Isolation in Drosophila Species

Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. However, the molecular basis of hybrid lethality and the evolutionary driving forces that provoke it remain largely elusive. The hybrid male rescue (Hmr) and the lethal hybrid rescue (Lhr) genes serve as a model to study speciation in Drosophilids because their interaction causes lethality in male hybrid offspring. Here, we show that HMR and LHR form a centromeric complex necessary for proper chromosome segregation. We find that the Hmr expression level is substantially higher in Drosophila melanogaster, whereas Lhr expression levels are increased in Drosophila simulans. The resulting elevated amount of HMR/LHR complex in hybrids results in an extensive mislocalization of the complex, an interference with the regulation of transposable elements, and an impairment of cell proliferation. Our findings provide evidence for a major role of centromere divergence in the generation of biodiversity.

Identification of novel Drosophila centromere-associated proteins

Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere‐specific histone H3‐variant centromere protein A (CENP‐A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere‐associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila melanogaster (D.mel), the conserved CCAN member CENP‐C. To gain further insights into D.mel centromere composition and biology, we analyzed affinity‐purified chromatin prepared from D.mel cell lines expressing green fluorescent protein tagged histone three variants by MS. In addition to already‐known centromeric proteins, we identified novel factors that were repeatedly enriched in affinity purification‐MS experiments. We analyzed the cellular localization of selected candidates by immunocytochemistry and confirmed localization to the centromere and other genomic regions for ten factors. Furthermore, RNA interference mediated depletion of CG2051, CG14480, and hyperplastic discs, three of our strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP‐ACID loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, we provide a comprehensive analysis of the proteomic composition of Drosophila centromeres. All MS data have been deposited in the ProteomeXchange with identifier PXD000758 (http://proteomecentral.proteomexchange.org/dataset/PXD000758).

Blind Deconvolution of Widefield Fluorescence Microscopic Data by Regularization of the Optical Transfer Function (OTF)

With volumetric data from wide field fluorescence microscopy, many emerging questions in biological and biomedical research are being investigated. Data can be recorded with high temporal resolution while the specimen is only exposed to a low amount of photo toxicity. These advantages come at the cost of strong recording blur caused by the infinitely extended point spread function (PSF). For wide field microscopy, its magnitude only decays with the square of the distance to the focal point and consists of an airy bessel pattern which is intricate to describe in the spatial domain. However, the Fourier transform of the incoherent PSF (denoted as Optical Transfer Function (OTF)) is well localized and smooth. In this paper, we present a blind deconvolution method that improves results of state-of-the-art deconvolution methods on wide field data by exploiting the properties of the wide field OTF.

3D Deformable Surfaces with Locally Self-Adjusting Parameters - A Robust Method to Determine Cell Nucleus Shapes

When using deformable models for the segmentation of biological data, the choice of the best weighting parameters for the internal and external forces is crucial. Especially when dealing with 3D fluorescence microscopic data and cells within dense tissue, object boundaries are sometimes not visible. In these cases, one weighting parameter set for the whole contour is not desirable. We are presenting a method for the dynamic adjustment of the weighting parameters, that is only depending on the underlying data and does not need any prior information. The method is especially apt to handle blurred, noisy, and deficient data, as it is often the case in biological microscopy.

A 3D Active Surface Model for the Accurate Segmentation of Drosophila Schneider Cell Nuclei and Nucleoli

We present an active surface model designed for the segmentation of Drosophila Schneider cell nuclei and nucleoli from wide-field microscopic data. The imaging technique as well as the biological application impose some major challenges to the segmentation. On the one hand, we have to deal with strong blurring of the 3D data, especially in z-direction. On the other hand, concerning the biological application, we have to deal with non-closed object boundaries and touching objects. To cope with these problems, we have designed a fully 3D active surface model. Our model prefers roundish object shapes and especially imposes roughly spherical surfaces where there is little gradient information. We have adapted an external force field for this model, which is based on gradient vector flow (