Analysen zur Assemblierung von Centromeren und PML-Kernkörperchen in lebenden Zellen

Essenzielle Funktionen im eukaryotischen Zellkern wie RNA-Transkription, DNA-Replikation und DNA-Reparatur werden von makromolekularen Proteinkomplexen bewerkstelligt. Ziel der vorliegenden Arbeit war es, neue Einblicke in die Formation und Funktion von zwei sehr unterschiedlichen Mutiproteinkomplexen zu gewinnen: des Centromer-/Kinetochorkomplexes und der so genannten PML-Kernkörperchen (PML: Promyelozytische Leukämie). Dazu wurde die Mobilität von sechs Kinetochorproteinen und sechs PML-Proteinisoformen mit Hilfe von Fluoreszenz-Bleichtechniken (FRAP: „fluorescence recovery after photobleaching“) und Fluoreszenz-Korrelations-Spektroskopie (FCS) in lebenden Zellen gemessen. Außerdem wurde die funktionelle Rolle von PML-Kernkörperchen bei der DNA-Doppelstrangbruch-Reparatur untersucht

Moderation of Arabidopsis Root Stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 Receptor Kinase Complexes

SummaryBackgroundThe root system of higher plants originates from the activity of a root meristem, which comprises a group of highly specialized and long-lasting stem cells. Their maintenance and number is controlled by the quiescent center (QC) cells and by feedback signaling from differentiated cells. Root meristems may have evolved from structurally distinct shoot meristems; however, no common player acting in stemness control has been found so far.ResultsWe show that CLAVATA1 (CLV1), a key receptor kinase in shoot stemness maintenance, performs a similar but distinct role in root meristems. We report that CLV1 is signaling, activated by the peptide ligand CLAVATA3/EMBRYO SURROUNDING REGION40 (CLE40), together with the receptor kinase ARABIDOPSIS CRINKLY4 (ACR4) to restrict root stemness. Both CLV1 and ACR4 overlap in their expression domains in the distal root meristem and localize to the plasma membrane (PM) and plasmodesmata (PDs), where ACR4 preferentially accumulates. Using multiparameter fluorescence image spectroscopy (MFIS), we show that CLV1 and ACR4 can form homo- and heteromeric complexes that differ in their composition depending on their subcellular localization.ConclusionsWe hypothesize that these homo- and heteromeric complexes may differentially regulate distal root meristem maintenance. We conclude that essential components of the ancestral shoot stemness regulatory system also act in the root and that the specific interaction of CLV1 with ACR4 serves to moderate and control stemness homeostasis in the root meristem. The structural differences between these two meristem types may have necessitated this recruitment of ACR4 for signaling by CLV1

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics

A Potential Lock-Type Mechanism for Unconventional Secretion in Fungi

Protein export in eukaryotes can either occur via the classical pathway traversing the endomembrane system or exploit alternative routes summarized as unconventional secretion. Besides multiple examples in higher eukaryotes, unconventional secretion has also been described for fungal proteins with diverse functions in important processes such as development or virulence. Accumulating molecular insights into the different export pathways suggest that unconventional secretion in fungal microorganisms does not follow a common scheme but has evolved multiple times independently. In this study, we review the most prominent examples with a focus on the chitinase Cts1 from the corn smut Ustilago maydis. Cts1 participates in cell separation during budding growth. Recent evidence indicates that the enzyme might be actively translocated into the fragmentation zone connecting dividing mother and daughter cells, where it supports cell division by the degradation of remnant chitin. Importantly, a functional fragmentation zone is prerequisite for Cts1 release. We summarize in detail what is currently known about this potential lock-type mechanism of Cts1 secretion and its connection to the complex regulation of fragmentation zone assembly and cell separation

Replication of centromeric heterochromatin in mouse fibroblasts takes place in early, middle, and late S phase.

The replication of eukaryotic chromosomes takes place throughout S phase, but little is known how this process is organized in space and time. Early and late replicating chromosomal domains appear to localize to distinct spatial compartments of the nucleus where DNA synthesis can take place at defined times during S phase. In general, transcriptionally active chromatin replicates early in S phase whereas transcriptionally inactive chromatin replicates later. Here we provide evidence for significant deviation from this dogma in mouse NIH3T3 cells. While the bulk pericentromeric heterochromatin replicates exclusively during mid to late S phase, centromeric DNA domains associated with constitutive kinetochore proteins are replicated throughout all stages of S phase. On an average, 12+/-4% of centromeres replicate in early S phase. Early replication of a subset of centromeres was also detected in living C2C12 murine cells. Thus, in contrast to expectation, late replication is not an obligatory feature of centromeric heterochromatin in murine cells and it does not determine their 'heterochromatic state'

Stem Cell Signaling in Arabidopsis Requires CRN to Localize CLV2 to the Plasma Membrane1[W][OA]

Stem cell number in shoot and floral meristems of Arabidopsis (Arabidopsis thaliana) is regulated by the CLAVATA3 (CLV3) signaling pathway. Perception of the CLV3 peptide requires the receptor kinase CLV1, the receptor-like protein CLV2, and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags and their capacities for interaction via efficiency of fluorescence resonance energy transfer. We found that CLV2 and CRN require each other for export from the endoplasmic reticulum and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN

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