Paul D. Andrews

Aurora B Regulates MCAK at the Mitotic Centromere

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAKs microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAKs activity and its localization at the centromere and kinetochore.

Phosphorylation of threonine 156 of the μ2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo

The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells.

Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-α-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-α-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for ≈10 microtubule segments of 0.5-μm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains ≈1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

Mechanical stress induces profound remodelling of keratin filaments and cell junctions in epidermolysis bullosa simplex keratinocytes.

The outer epidermal layer of the skin is an epithelium with remarkable protective barrier functions, which is subject to pronounced physical stress in its day-to-day function. A major candidate component for absorbing this stress is the K5/K14 keratin intermediate filament network. To investigate the part played by keratins in stress resilience, keratinocyte cell lines were subjected to mechanical stress. Repeated stretch and relaxation cycles over increasing time produced reproducible changes in the configuration of the keratin network. When wild-type cells were compared with cells carrying a keratin mutation associated with severe epidermolysis bullosa simplex-type skin fragility, the mutant keratin filaments were unable to withstand the mechanical stress and progressively fragmented yielding aggregates and novel ring structures. The cell junctions into which the keratin filaments are normally anchored also progressively disassembled, with all components tested of the cytoplasmic plaques becoming relocated away from the membrane and onto the keratin rings, while integral membrane receptors integrins and cadherins remained at the plasma membrane. The results suggest that maintenance of desmosomes and hemidesmosomes may require some tension, normally mediated by keratin attachments.

Phosphorylation Regulates the Dynamic Interaction of RCC1 with Chromosomes during Mitosis

The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.

To 5D and Beyond: Quantitative Fluorescence Microscopy in the Postgenomic Era

Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide‐field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large‐scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.

Phosphorylation by Aurora-B Negatively Regulates Survivin Function During Mitosis

Survivin operates in a complex with aurora B kinase and is phosphorylated by it on threonine 117 in vitro. Here we ask whether phosphorylation of survivin by aurora B kinase regulates its function during mitosis in vivo. Using a phospho-specific antibody we first establish that survivin is phosphorylated at T117 during mitosis and is present at the midbody during cytokinesis. Next we use two independent RNAi complementation approaches to investigate threonine 117 mutants in survivin depleted cells. Our data suggest that while non-phosphorylatable survivin, survivinT117A, can substitute for the wild type protein, a phosphomimic, survivinT117E cannot restore viability, nor can it complement chromosome congression and spindle checkpoint defects that arise due to depletion of endogenous survivin. Fluorescence imaging and fluorescence recovery after photobleaching analysis suggest that the phosphomimic has reduced affinity for centromeres compared with the non-phosphorylatable form. We conclude that survivin is phosphorylated at T117 during mitosis, and once phosphorylated, dephosphorylation is crucial for chromosome congression and progression into anaphase.

17-P003 Discovery of small molecules to control human embryonic stem cell fate

A common feature of early vertebrate embryos is that they are surrounded by a simple protective epithelial cell layer. However, it is currently not well understood how the differentiation of this epithelium is controlled. We present data showing that BMP4 is sufficient to promote differentiation of this epithelium in diverse vertebrates. In mouse embryos the outer epithelium forms the trophectoderm which overlies the pluripotent inner cell mass. Xenopus embryos do not have a trophectoderm or inner cell mass but they do have outer epithelial cells and inner deep cells, which follow distinct fates. We have examined the role of BMP4 in the Xenopus epidermis and found that it is sufficient and required for differentiation of the outer epithelial cells. In contrast, BMP4 inhibits genes normally expressed in the deep cells, arguing that BMP4 specifically promotes differentiation of the outer epithelial cells. We then examined the role of BMP4 signalling in mouse ES cells. We found that in the absence of additional factors BMP4 is able to induce ES cells to form a trophectoderm like cell type. Thus, it appears that BMP4 acts to promote differentiation of the first epithelium in both Xenopus and mouse. Our data also suggest that, despite differences between vertebrates, there are conserved pathways which control differentiation of the first epithelial cells.

In Vivo Imaging of Mammalian Cells: Image Acquisition and Analysis

Live cell imaging provides a powerful technique for the analysis of molecular dynamics within cells. Advances in imaging technology and probe design have established this approach as an important tool in modern biology. It is now possible to obtain commercial turnkey systems for digital imaging using a number of different imaging modalities. Nevertheless, it still requires considerable technical care and expertise to conduct a successful experiment. To perform a successful imaging experiment, it is important to maximize the signal-to-noise ratio (S:N) while minimizing damage to the cells. In this article, we focus on the use of fluorescence microscopy in live cell imaging, although most of the points discussed are relevant to any type of imaging. We describe many of the methods and considerations that are required for performing a successful imaging experiment in living cells. However, we do not provide a single recipe for success: The approach is much too empirical and depends on careful observation of the particular cells under investigation.

In Vivo Imaging of Mammalian Cells: Cell Engineering and Viability

Cold Spring Harb Protoc Jason R. Swedlow, Paul D. Andrews and Melpomeni Platani In Vivo Imaging of Mammalian Cells: Cell Engineering and Viability Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (338 articles) Visualization, general (458 articles) Visualization (150 articles) Video Imaging / Time Lapse Imaging (226 articles) Live Cell Imaging (272 articles) Labeling for Imaging (235 articles) In Vivo Imaging (520 articles) Imaging/Microscopy, general (218 articles) Imaging for Neuroscience (192 articles) Imaging Development (200 articles) Fluorescent Proteins (347 articles) Fluorescence (565 articles) Developmental Biology (435 articles) Cell Imaging (241 articles) Cell Culture (989 articles) Cell Biology, general