Cdk1 phosphorylation sites on Cdc27 are required for correct chromosomal localisation and APC/C function in syncytial Drosophila embryos

J.-Y. Huang; G. Morley; D. Li; M. Whitaker

doi:10.1242/jcs.006833

Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140191 ◽

1995 ◽

Vol 53 ◽

pp. 764-765

Author(s):

W.F. Marshall ◽

A.F. Dernburg ◽

B. Harmon ◽

J.W. Sedat

Keyword(s):

Nuclear Envelope ◽

Chromatin Condensation ◽

Three Dimensional ◽

Physiological Role ◽

Nuclear Surface ◽

Intact Cells ◽

Molecular Determinants ◽

Surface Harmonic ◽

Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

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Laser scanning confocal microscopic analysis of cytoskeletal and nuclear reorganization in live Drosophila embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146710 ◽

1993 ◽

Vol 51 ◽

pp. 174-175

Author(s):

William Theurkauf

Keyword(s):

Laser Scanning ◽

Microscopic Analysis ◽

Large Cell ◽

Early Embryo ◽

Drosophila Embryo ◽

Cortical Actin ◽

Nuclear Reorganization ◽

Cytoskeletal Elements ◽

Microtubule Structure ◽

Drosophila Embryos

Cell division in eucaryotes depends on coordinated changes in nuclear and cytoskeletal components. In Drosophila melanogaster embryos, the first 13 nuclear divisions occur without cytokinesis. During the final four divisions, nuclei divide in a uniform monolayer at the surface of the embryo. These surface divisions are accompanied by dramatic changes in cortical actin and microtubule structure (Karr and Alberts, 1986), and inhibitor studies indicate that these changes are essential to orderly mitosis (Zalokar and Erk, 1976). Because the early embryo is syncytial, fluorescent probes introduced by microinjection are incorporated in structures associated with all of the nuclei in the blastoderm. In addition, the nuclei divide synchronously every 10 to 20 min. These characteristics make the syncytial blastoderm embryo an excellent system for the analysis of mitotic reorganization of both nuclear and cytoskeletal elements. However, the Drosophila embryo is a large cell, and resolution of cytoskeletal filaments and nuclear structure is hampered by out-of focus signal.

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Specificity determinants of phosphoprotein phosphatases controlling kinetochore functions

Essays in Biochemistry ◽

10.1042/ebc20190065 ◽

2020 ◽

Vol 64 (2) ◽

pp. 325-336 ◽

Cited By ~ 1

Author(s):

Dimitriya H. Garvanska ◽

Jakob Nilsson

Keyword(s):

Spindle Assembly Checkpoint ◽

Activity Level ◽

Phosphorylation Sites ◽

Binding Affinities ◽

Mitotic Kinases ◽

Anaphase Onset ◽

Phosphoprotein Phosphatases ◽

Linear Motifs ◽

Temporal And Spatial ◽

Kinetochore Function

Abstract Kinetochores are instrumental for accurate chromosome segregation by binding to microtubules in order to move chromosomes and by delaying anaphase onset through the spindle assembly checkpoint (SAC). Dynamic phosphorylation of kinetochore components is key to control these activities and is tightly regulated by temporal and spatial recruitment of kinases and phosphoprotein phosphatases (PPPs). Here we focus on PP1, PP2A-B56 and PP2A-B55, three PPPs that are important regulators of mitosis. Despite the fact that these PPPs share a very similar active site, they target unique ser/thr phosphorylation sites to control kinetochore function. Specificity is in part achieved by PPPs binding to short linear motifs (SLiMs) that guide their substrate specificity. SLiMs bind to conserved pockets on PPPs and are degenerate in nature, giving rise to a range of binding affinities. These SLiMs control the assembly of numerous substrate specifying complexes and their position and binding strength allow PPPs to target specific phosphorylation sites. In addition, the activity of PPPs is regulated by mitotic kinases and inhibitors, either directly at the activity level or through affecting PPP–SLiM interactions. Here, we discuss recent progress in understanding the regulation of PPP specificity and activity and how this controls kinetochore biology.

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THE DYNACTOME: INVESTIGATING DYNAMIC KINASE NETWORKS IN CELLULAR DECISION-MAKING

Clinical & Investigative Medicine ◽

10.25011/cim.v31i4.4827 ◽

2008 ◽

Vol 31 (4) ◽

pp. 22

Author(s):

Jonathan So ◽

Kelly Elder ◽

Anna Dai ◽

Claus Jorgensen ◽

Rune Linding ◽

...

Keyword(s):

Spectrometric Analysis ◽

Phosphorylation Sites ◽

Complex Samples ◽

Downstream Signaling ◽

Through Flow ◽

Colorectal Cancer Cells ◽

Induced Apoptosis ◽

Cell Phenotypes ◽

Kinase Phosphorylation ◽

Kinase Expression

Networks of kinases play a role in the transmission and integration of signals from the membrane to the nucleus. We aim to elucidate kinase phosphorylation and interaction partners in these networks through the immuno-precipitation and mass spectrometric analysis of a representative set of 100 Flag-tagged kinases stably expressed in human colorectal cancer cells. The goal is to generate a comprehensive set of interactions and dynamic phosphorylation sites which correlate with cell phenotypes such as apoptosis and proliferation. The techniques of mass-spectrometry have allowed for the identification of proteins and their phosphorylation sites in complex samples. Various labeling methods such as iTRAQ has enabled the relative quantification of these sites as afunction of time (White et al. PNAS, 2007). However, kinases usually work in the context of particular signaling stimuli. We aim to characterize the role of these over-expressed kinases in the context of Trail-induced apoptosis. This isparticularly relevant to tumorigenesis in that many cancers are resistant to apoptosis and recombinant Trail therapies are currently undergoing clinical trials. We present assays to correlate the proliferative ability and sensitivity to apoptosis of various stable cell lines with kinase expression levels through flow cytometry. We also present efforts to trace downstream signaling through the monitoring of MAP kinase phosphorylation using a high-throughput bead array.

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