Christian Hoischen

Dynamics of inner kinetochore assembly and maintenance in living cells

To investigate the dynamics of centromere organization, we have assessed the exchange rates of inner centromere proteins (CENPs) by quantitative microscopy throughout the cell cycle in human cells. CENP-A and CENP-I are stable centromere components that are incorporated into centromeres via a “loading-only” mechanism in G1 and S phase, respectively. A subfraction of CENP-H also stays stably bound to centromeres. In contrast, CENP-B, CENP-C, and some CENP-H and hMis12 exhibit distinct and cell cycle–specific centromere binding stabilities, with residence times ranging from seconds to hours. CENP-C and CENP-H are immobilized at centromeres specifically during replication. In mitosis, all inner CENPs become completely immobilized. CENPs are highly mobile throughout bulk chromatin, which is consistent with a binding-diffusion behavior as the mechanism to scan for vacant high-affinity binding sites at centromeres. Our data reveal a wide range of cell cycle–specific assembly plasticity of the centromere that provides both stability through sustained binding of some components and flexibility through dynamic exchange of other components.

Dynamics of component exchange at PML nuclear bodies.

PML nuclear bodies (NBs) are involved in the regulation of key nuclear pathways but their biochemical function in nuclear metabolism is unknown. In this study PML NB assembly dynamics were assessed by live cell imaging and mathematic modeling of its major component parts. We show that all six nuclear PML isoforms exhibit individual exchange rates at NBs and identify PML V as a scaffold subunit. SP100 exchanges at least five times faster at NBs than PML proteins. Turnover dynamics of PML and SP100 at NBs is modulated by SUMOylation. Exchange is not temperature-dependent but depletion of cellular ATP levels induces protein immobilization at NBs. The PML-RARα oncogene exhibits a strong NB retention effect on wild-type PML proteins. HIPK2 requires an active kinase for PML NB targeting and elevated levels of PML IV increase its residence time. DAXX and BLM turn over rapidly and completely at PML NBs within seconds. These findings provide a kinetics model for factor exchange at PML NBs and highlight potential mechanisms to regulate intranuclear trafficking of specific factors at these domains.

CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin

Centromeres are important structural constituents of chromosomes that ensure proper chromosome segregation during mitosis by providing defined sites for kinetochore attachment. In higher eukaryotes, centromeres have no specific DNA sequence and thus, they are rather determined through epigenetic mechanisms. A fundamental process in centromere establishment is the incorporation of the histone variant CENP-A into centromeric chromatin, which provides a binding platform for the other centromeric proteins. The Mis18 complex, and, in particular, its member M18BP1 was shown to be essential for both incorporation and maintenance of CENP-A. Here we show that M18BP1 displays a cell cycle-regulated association with centromeric chromatin in mouse embryonic stem cells. M18BP1 is highly enriched at centromeric regions from late anaphase through to G1 phase. An interaction screen against 16 core centromeric proteins revealed a novel interaction of M18BP1 with CENP-C. We mapped the interaction domain in M18BP1 to a central region containing a conserved SANT domain and in CENP-C to the C-terminus. Knock-down of CENP-C leads to reduced M18BP1 association and lower CENP-A levels at centromeres, suggesting that CENP-C works as an important factor for centromeric M18BP1 recruitment and thus for maintaining centromeric CENP-A.

Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State

Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.

Dynamics of CENP-N kinetochore binding during the cell cycle

Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

Use of cell wall-less bacteria (L-forms) for efficient expression and secretion of heterologous gene products

In spite of many efforts and achievements to optimize the prokaryotic expression systems, there are still general and specific problems in obtaining sufficient yields of the functionally active gene products. The main problems concern the formation of inclusion bodies, incorrect folding, toxicity for the producer cells and degradation by proteases. One way to overcome these problems is with expression systems alternative to those of Escherichia coli. For the first time, cell wall-less L-form bacteria were used to establish such an alternative expression system and test its practicability. The results showed that various recombinant proteins can be synthesized in considerable amounts as soluble, functionally active products with these cell wall-less strains.

Assembly of the Inner Kinetochore Proteins CENP-A and CENP-B in Living Human Cells

DNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. A subkinetochore complex, the “inner kinetochore”, is present at the centromere during the entire cell cycle. Its location seems to be defined by the settlement of CENP‐A (CENH3), which replaces histone H3 in centromeric nucleosomes. This suggests that CENP‐A can recruit further inner kinetochore proteins by direct binding. Surprisingly, intense in vitro studies could not identify an interaction of CENP‐A with any other inner kinetochore protein. Instead, centromere identity seems to be maintained by a unique nucleosome, which might have a modified structure or epigenetic state that serves to distinguish the centromere from the rest of the chromosome. We investigated the association of CENP‐A and CENP‐B by fluorescence intensity and lifetime‐based FRET measurements in living human HEp‐2 cells. We observed Förster resonance energy transfer (FRET) between CENP‐A and CENP‐B at centromere locations; this indicates that these proteins are in the molecular vicinity (<10 nm) of each other. In addition, we analysed protein–protein interactions within the centromeric nucleosome. We could detect energy transfer between CENP‐A and histone H4 as well as between CENP‐A molecules themselves. On the other hand, no FRET was detected between CENP‐A and H2A.1 or H3.1. Our data support the view that two CENP‐A molecules are packed with H4, but not with H3, in a single centromeric nucleosome.

Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of Proteus mirabilis and Escherichia coli

ABSTRACT We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 μg ml−1, with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.

Live-cell imaging reveals sustained centromere binding of CENP-T via CENP-A and CENP-B

At the centromere, a network of proteins, the kinetochore, assembles in order to grant correct chromatin segregation. In this study the dynamics and molecular interactions of the inner kinetochore protein CENP-T were analyzed employing a variety of fluorescence microscopy techniques in living human cells. Acceptor-bleaching FRET indicates that CENP-T directly associates with CENP-A and CENP-B. CENP-T exchange into centromeres is restricted to the S-phase of the cell cycle as revealed by FRAP, suggesting a coreplicational loading mechanism, as we have recently also demonstrated for CENP-I. These properties make CENP-T one of the basic inner kinetochore proteins with most further proteins binding downstream, suggesting a fundamental role of CENP-T in kinetochore function.

Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex

Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.