Measuring Tubulin Content in Toxoplasma Gondii: a Comparison of Laser-scanning Confocal and Wide-field Fluorescence Microscopy

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2002  |  Pubmed ID: 11830634

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-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-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 approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 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.

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

Traffic (Copenhagen, Denmark). Jan, 2002  |  Pubmed ID: 11872140

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.

Cajal Body Dynamics and Association with Chromatin Are ATP-dependent

Nature Cell Biology. Jul, 2002  |  Pubmed ID: 12068306

Cajal bodies (CBs) are nuclear organelles that contain factors required for splicing, ribosome biogenesis and transcription. Our previous analysis in living cells showed that CBs are dynamic structures. Here, we show that CB mobility is described by anomalous diffusion and that bodies alternate between association with chromatin and diffusion within the interchromatin space. CB mobility increases after ATP depletion and inhibition of transcription, suggesting that the association of CB and chromatin requires ATP and active transcription. This behaviour is fundamentally different from the ATP-dependent mobility observed for chromatin and suggests that a novel mechanism governs CB, and possibly other, nuclear body dynamics.

Macromolecular Mobility Inside the Cell Nucleus

Trends in Cell Biology. Nov, 2002  |  Pubmed ID: 12446102

Live Cell Imaging Using Wide-field Microscopy and Deconvolution

Cell Structure and Function. Oct, 2002  |  Pubmed ID: 12502887

The use of fluorescence imaging methods, most recently based on fluorescent protein technology, and the availability of high quality fluorescence imaging systems have driven a revolution in cell and molecular biology. Live cell imaging, especially using fluorescence, is now used in a wide variety of assays in academic and commercial laboratories. The use of this technology requires particular attention to be paid to cell engineering, the design of the image acquisition system, the imaging protocol, and subsequent processing and analytic methods. In this review, we discuss each of these steps, highlighting practical techniques developed by us and others.

Time-lapse Imaging Reveals Dynamic Relocalization of PP1gamma Throughout the Mammalian Cell Cycle

Molecular Biology of the Cell. Jan, 2003  |  Pubmed ID: 12529430

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, alpha, beta/delta, and gamma1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1gamma or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1gamma to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1gamma shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1gamma also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1gamma revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.

The Making of the Mitotic Chromosome: Modern Insights into Classical Questions

Molecular Cell. Mar, 2003  |  Pubmed ID: 12667441

The condensation of mitotic chromosomes is essential for the faithful segregation of sister chromatids in anaphase. An emerging view is that chromosome assembly is an active and dynamic process of chromatin reorganization in which two ATP hydrolyzing enzymes, topoisomerase II and the condensin complex, play central roles. In this review, we discuss recent work that sheds new light on the molecular and structural dynamics of mitotic chromosomes.

Informatics and Quantitative Analysis in Biological Imaging

Science (New York, N.Y.). Apr, 2003  |  Pubmed ID: 12677061

Biological imaging is now a quantitative technique for probing cellular structure and dynamics and is increasingly used for cell-based screens. However, the bioinformatics tools required for hypothesis-driven analysis of digital images are still immature. We are developing the Open Microscopy Environment (OME) as an informatics solution for the storage and analysis of optical microscope image data. OME aims to automate image analysis, modeling, and mining of large sets of images and specifies a flexible data model, a relational database, and an XML-encoded file standard that is usable by potentially any software tool. With this design, OME provides a first step toward biological image informatics.

Human CLASP1 is an Outer Kinetochore Component That Regulates Spindle Microtubule Dynamics

Cell. Jun, 2003  |  Pubmed ID: 12837247

One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. Here, we show that CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that we term the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. Our data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior.

Mitotic Mechanics: the Auroras Come into View

Current Opinion in Cell Biology. Dec, 2003  |  Pubmed ID: 14644191

Aurora kinases have recently taken centre stage in the regulation of key cell cycle processes. Aurora A is emerging as a critical regulator of centrosome and spindle function. Aurora B mediates chromosome segregation by ensuring proper biorientation of sister chromatids, possibly through the regulation of microtubule dynamics. This enzyme also functions in cytokinesis apparently by interacting with a critical GTPase and a kinesin-like protein. Recent work on both kinases has revealed functional links between Aurora kinase activity and the mechanics of cell division.

Quantitative Fluorescence Microscopy and Image Deconvolution

Methods in Cell Biology. 2003  |  Pubmed ID: 14719340

Aurora B Regulates MCAK at the Mitotic Centromere

Developmental Cell. Feb, 2004  |  Pubmed ID: 14960279

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 MCAK's 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 MCAK's activity and its localization at the centromere and kinetochore.

Phosphorylation Regulates the Dynamic Interaction of RCC1 with Chromosomes During Mitosis

Current Biology : CB. Jun, 2004  |  Pubmed ID: 15203004

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.

Chromosome Condensation: DNA Compaction in Real Time

Current Biology : CB. Jul, 2004  |  Pubmed ID: 15268872

Mitotic chromosomes must be organised into a highly ordered and compacted form to allow proper segregation of DNA during each round of cell division. Two new studies report observations of DNA compaction by eukaryotic and bacterial condensin molecules in real time using magnetic and optical trapping micromanipulation techniques.

Optimisation of the Two-dimensional Gel Electrophoresis Protocol Using the Taguchi Approach

Proteome Science. Sep, 2004  |  Pubmed ID: 15357868

BACKGROUND: Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE) for separation and characterisation of complex protein mixtures. Among the difficulties associated with this approach is the solubilisation of protein mixtures for isoelectric focusing (IEF). To find the optimal formulation of the multi-component IEF rehydration buffer (RB) we applied the Taguchi method, a widely used approach for the robust optimisation of complex industrial processes, to determine optimal concentrations for the detergents, carrier ampholytes and reducing agents in RB for 2DE using commercially supplied immobilised pH gradient (IPG) gel strips. RESULTS: Our optimisation resulted in increased protein solubility, improved resolution and reproducibility of 2D gels, using a wide variety of samples. With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved. Moreover, with improved sample loading and resolution, analysis of individual spots by immunoblotting and mass spectrometry revealed previously uncharacterised posttranscriptional modifications in a variety of chromatin proteins. CONCLUSIONS: While the optimised RB (oRB) is specific to the gels and analysis approach we use, our use of the Taguchi method should be generally applicable to a broad range of electrophoresis and analysis systems.

The Open Microscopy Environment (OME) Data Model and XML File: Open Tools for Informatics and Quantitative Analysis in Biological Imaging

Genome Biology. 2005  |  Pubmed ID: 15892875

The Open Microscopy Environment (OME) defines a data model and a software implementation to serve as an informatics framework for imaging in biological microscopy experiments, including representation of acquisition parameters, annotations and image analysis results. OME is designed to support high-content cell-based screening as well as traditional image analysis applications. The OME Data Model, expressed in Extensible Markup Language (XML) and realized in a traditional database, is both extensible and self-describing, allowing it to meet emerging imaging and analysis needs.

Modelling Data Across Labs, Genomes, Space and Time

Nature Cell Biology. Nov, 2006  |  Pubmed ID: 17060903

Logical models and physical specifications provide the foundation for storage, management and analysis of complex sets of data, and describe the relationships between measured data elements and metadata - the contextual descriptors that define the primary data. Here, we use imaging applications to illustrate the purpose of the various implementations of data specifications and the requirement for open, standardized, data formats to facilitate the sharing of critical digital data and metadata.

Phosphorylation by Aurora-B Negatively Regulates Survivin Function During Mitosis

Cell Cycle (Georgetown, Tex.). May, 2007  |  Pubmed ID: 17457057

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, survivin(T117A), can substitute for the wild type protein, a phosphomimic, survivin(T117E) 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.

Mitotic Spindle Orientation Distinguishes Stem Cell and Terminal Modes of Neuron Production in the Early Spinal Cord

Development (Cambridge, England). May, 2007  |  Pubmed ID: 17470968

Despite great insight into the molecular mechanisms that specify neuronal cell type in the spinal cord, cell behaviour underlying neuron production in this tissue is largely unknown. In other neuroepithelia, divisions with a perpendicular cleavage plane at the apical surface generate symmetrical cell fates, whereas a parallel cleavage plane generates asymmetric daughters, a neuron and a progenitor in a stem cell mode, and has been linked to the acquisition of neuron-generating ability. Using a novel long-term imaging assay, we have monitored single cells in chick spinal cord as they transit mitosis and daughter cells become neurons or divide again. We reveal new morphologies accompanying neuron birth and show that neurons are generated concurrently by asymmetric and terminal symmetric divisions. Strikingly, divisions that generate two progenitors or a progenitor and a neuron both exhibit a wide range of cleavage plane orientations and only divisions that produce two neurons have an exclusively perpendicular orientation. Neuron-generating progenitors are also distinguished by lengthening cell cycle times, a finding supported by cell cycle acceleration on exposure to fibroblast growth factor (FGF), an inhibitor of neuronal differentiation. This study provides a novel, dynamic view of spinal cord neurogenesis and supports a model in which cleavage plane orientation/mitotic spindle position does not assign neuron-generating ability, but functions subsequent to this step to distinguish stem cell and terminal modes of neuron production.

Quantitative Analysis of Digital Microscope Images

Methods in Cell Biology. 2007  |  Pubmed ID: 17519175

Quantitative Fluorescence Microscopy and Image Deconvolution

Methods in Cell Biology. 2007  |  Pubmed ID: 17519179

RNA Polymerase II Transcription in Living Color

Nature Structural & Molecular Biology. Sep, 2007  |  Pubmed ID: 17823607

ELYS/MEL-28 Chromatin Association Coordinates Nuclear Pore Complex Assembly and Replication Licensing

Current Biology : CB. Oct, 2007  |  Pubmed ID: 17825564

Xenopus egg extract supports all the major cell-cycle transitions in vitro. We have used a proteomics approach to identify proteins whose abundance on chromatin changes during the course of an in vitro cell cycle. One of the proteins we identified was ELYS/MEL-28, which has recently been described as the earliest-acting factor known to be required for nuclear pore complex (NPC) assembly [1-4]. ELYS interacts with the Nup107-160 complex and is required for its association with chromatin. ELYS contains an AT-hook domain, which we show binds to chromatin with a high affinity. This domain can compete with full-length ELYS for chromatin association, thereby blocking NPC assembly. This provides evidence that ELYS interacts directly with chromatin and that this interaction is essential for NPC assembly and compartmentalization of chromosomal DNA within the cell. Furthermore, we detected a physical association on chromatin between ELYS and the Mcm2-7 replication-licensing proteins. ELYS chromatin loading, NPC assembly, and nuclear growth were delayed when Mcm2-7 was prevented from loading onto chromatin. Because nuclear assembly is required to shut down licensing prior to entry into S phase, our results suggest a mechanism by which these two early cell-cycle events are coordinated with one another.

Bod1, a Novel Kinetochore Protein Required for Chromosome Biorientation

The Journal of Cell Biology. Oct, 2007  |  Pubmed ID: 17938248

We have combined the proteomic analysis of Xenopus laevis in vitro-assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule-kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.

Evaluating Performance in Three-dimensional Fluorescence Microscopy

Journal of Microscopy. Dec, 2007  |  Pubmed ID: 18045334

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is 'better', in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging.

Open Tools for Storage and Management of Quantitative Image Data

Methods in Cell Biology. 2008  |  Pubmed ID: 18155479

The explosion in quantitative imaging has driven the need to develop tools for storing, managing, analyzing, and viewing large sets of data. In this chapter, we discuss tools we have built for storing large data sets for the lifetime of a typical research project. As part of the Open Microscopy Environment (OME) Consortium, we have built a series of open-source tools that support the manipulation and visualization of large sets of complex image data. Images from a number of proprietary file formats can be imported and then accessed from a single server running in a laboratory or imaging facility. We discuss the capabilities of the OME Server, a Perl-based data management system that is designed for large-scale analysis of image data using a web browser-based user interface. In addition, we have recently released a lighter weight Java-based OME Remote Objects Server that supports remote applications for managing and viewing image data. Together these systems provide a suite of tools for large-scale quantitative imaging that is now commonly used throughout cell and developmental biology.

Temporal Profiling of the Chromatin Proteome Reveals System-wide Responses to Replication Inhibition

Current Biology : CB. Jun, 2008  |  Pubmed ID: 18514518

Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic. Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either replication licensing or S phase CDK activity. This revealed an unexpectedly broad system-wide effect on the chromatin proteome, indicating that the response to replication inhibition extends to many other functional modules in addition to the replication machinery. Several proteins that respond to replication inhibition (including nuclear pore proteins) coprecipitated with the Mcm2-7 licensing complex on chromatin, suggesting that Mcm2-7 play a central role in coordinating nuclear structure with DNA replication.

Bioimage Informatics for Experimental Biology

Annual Review of Biophysics. 2009  |  Pubmed ID: 19416072

Over the past twenty years there have been great advances in light microscopy with the result that multidimensional imaging has driven a revolution in modern biology. The development of new approaches of data acquisition is reported frequently, and yet the significant data management and analysis challenges presented by these new complex datasets remain largely unsolved. As in the well-developed field of genome bioinformatics, central repositories are and will be key resources, but there is a critical need for informatics tools in individual laboratories to help manage, share, visualize, and analyze image data. In this article we present the recent efforts by the bioimage informatics community to tackle these challenges, and discuss our own vision for future development of bioimage informatics solutions.

Building a Scientific Data Grid with DiGS

Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences. Jun, 2009  |  Pubmed ID: 19451103

We provide an insight into the challenge of building and supporting a scientific data infrastructure with reference to our experience working with scientists from computational particle physics and molecular biology. We illustrate how, with modern high-performance computing resources, even small scientific groups can generate huge volumes (petabytes) of valuable scientific data and explain how grid technology can be used to manage, publish, share and curate these data. We describe the DiGS software application, which we have developed to meet the needs of smaller communities and we have highlighted the key elements of its functionality.

Open Source Bioimage Informatics for Cell Biology

Trends in Cell Biology. Nov, 2009  |  Pubmed ID: 19833518

Significant technical advances in imaging, molecular biology and genomics have fueled a revolution in cell biology, in that the molecular and structural processes of the cell are now visualized and measured routinely. Driving much of this recent development has been the advent of computational tools for the acquisition, visualization, analysis and dissemination of these datasets. These tools collectively make up a new subfield of computational biology called bioimage informatics, which is facilitated by open source approaches. We discuss why open source tools for image informatics in cell biology are needed, some of the key general attributes of what make an open source imaging application successful, and point to opportunities for further operability that should greatly accelerate future cell biology discovery.

The Nup107-160 Nucleoporin Complex Promotes Mitotic Events Via Control of the Localization State of the Chromosome Passenger Complex

Molecular Biology of the Cell. Dec, 2009  |  Pubmed ID: 19864462

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.

In Vivo Imaging of Mammalian Cells: Cell Engineering and Viability

Cold Spring Harbor Protocols. Sep, 2009  |  Pubmed ID: 20147260

In Vivo Imaging of Mammalian Cells: Image Acquisition and Analysis

Cold Spring Harbor Protocols. Sep, 2009  |  Pubmed ID: 20147261

Kinetochore Alignment Within the Metaphase Plate is Regulated by Centromere Stiffness and Microtubule Depolymerases

The Journal of Cell Biology. Mar, 2010  |  Pubmed ID: 20212316

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion.

Cell Biology Forum: Genome-wide View of Mitosis

Nature. Apr, 2010  |  Pubmed ID: 20360724

Metadata Matters: Access to Image Data in the Real World

The Journal of Cell Biology. May, 2010  |  Pubmed ID: 20513764

Data sharing is important in the biological sciences to prevent duplication of effort, to promote scientific integrity, and to facilitate and disseminate scientific discovery. Sharing requires centralized repositories, and submission to and utility of these resources require common data formats. This is particularly challenging for multidimensional microscopy image data, which are acquired from a variety of platforms with a myriad of proprietary file formats (PFFs). In this paper, we describe an open standard format that we have developed for microscopy image data. We call on the community to use open image data standards and to insist that all imaging platforms support these file formats. This will build the foundation for an open image data repository.

Advanced Hardware and Software Tools for Fast Multidimensional Imaging of Living Cells

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2010  |  Pubmed ID: 20807743

Sds22 Regulates Aurora B Activity and Microtubule-kinetochore Interactions at Mitosis

The Journal of Cell Biology. Oct, 2010  |  Pubmed ID: 20921135

We have studied Sds22, a conserved regulator of protein phosphatase 1 (PP1) activity, and determined its role in modulating the activity of aurora B kinase and kinetochore-microtubule interactions. Sds22 is required for proper progression through mitosis and localization of PP1 to mitotic kinetochores. Depletion of Sds22 increases aurora B T-loop phosphorylation and the rate of recovery from monastrol arrest. Phospho-aurora B accumulates at kinetochores in Sds22-depleted cells juxtaposed to critical kinetochore substrates. Sds22 modulates sister kinetochore distance and the interaction between Hec1 and the microtubule lattice and, thus, the activation of the spindle assembly checkpoint. These results demonstrate that Sds22 specifically defines PP1 function and localization in mitosis. Sds22 regulates PP1 targeting to the kinetochore, accumulation of phospho-aurora B, and force generation at the kinetochore-microtubule interface.

Finding an Image in a Haystack: the Case for Public Image Repositories

Nature Cell Biology. Mar, 2011  |  Pubmed ID: 21364564

Image repositories are growing. Their potential impact is huge; they require support and proper funding.

How to Be a Mitotic Chromosome

Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology. Apr, 2011  |  Pubmed ID: 21461697

Proper mitotic chromosome structure is essential for faithful chromosome segregation. Mounting evidence suggests that mitotic chromosome assembly is a progressive, dynamic process that requires topoisomerase II, condensins and cohesin and the activity of several signalling molecules. Current results suggest how these different activities might interact to achieve the familiar form of the mitotic chromosome.

Channeling the Data Deluge

Nature Methods. Jun, 2011  |  Pubmed ID: 21623351

OMX: a New Platform for Multimodal, Multichannel Wide-field Imaging

Cold Spring Harbor Protocols. Aug, 2011  |  Pubmed ID: 21807861

A Novel Reporter of Notch Signalling Indicates Regulated and Random Notch Activation During Vertebrate Neurogenesis

BMC Biology. 2011  |  Pubmed ID: 21880129

Building the complex vertebrate nervous system involves the regulated production of neurons and glia while maintaining a progenitor cell population. Neurogenesis starts asynchronously in different regions of the embryo and occurs over a long period of time, allowing progenitor cells to be exposed to multiple extrinsic signals that regulate the production of different cell types. Notch-mediated cell-cell signalling is one of the mechanisms that maintain the progenitor pool, however, little is known about how the timing of Notch activation is related to the cell cycle and the distinct modes of cell division that generate neurons. An essential tool with which to investigate the role of Notch signalling on cell by cell basis is the development a faithful reporter of Notch activity.

Software Tools, Data Structures, and Interfaces for Microscope Imaging

Cold Spring Harbor Protocols. Jan, 2012  |  Pubmed ID: 22194261

The arrival of electronic photodetectors in biological microscopy has led to a revolution in the application of imaging in cell and developmental biology. The extreme photosensitivity of electronic photodetectors has enabled the routine use of multidimensional data acquisition spanning space and time and spectral range in live cell and tissue imaging. These techniques have provided key insights into the molecular and structural dynamics of living biology. However, digital photodetectors offer another advantage-they provide a linear mapping between the photon flux coming from the sample and the electronic sample they produce. Thus, an image presented as a visual representation of the sample is also a quantitative measurement of photon flux. These quantitative measurements are the basis of subsequent processing and analysis to improve signal contrast, to compare changes in the concentration of signal, and to reveal changes in cell structure and dynamics. For this reason, many laboratories and companies have committed their resources to software development, resulting in the availability of a large number of image-processing and analysis packages. In this article, we review the software tools for image data analysis that are now available and give some examples of their use in imaging experiments to reveal new insights into biological mechanisms. In our final section, we highlight some of the new directions for image analysis that are significant unmet challenges and present our own ideas for future directions.

OMERO: Flexible, Model-driven Data Management for Experimental Biology

Nature Methods. Mar, 2012  |  Pubmed ID: 22373911

Data-intensive research depends on tools that manage multidimensional, heterogeneous datasets. We built OME Remote Objects (OMERO), a software platform that enables access to and use of a wide range of biological data. OMERO uses a server-based middleware application to provide a unified interface for images, matrices and tables. OMERO's design and flexibility have enabled its use for light-microscopy, high-content-screening, electron-microscopy and even non-image-genotype data. OMERO is open-source software, available at http://openmicroscopy.org/.

Innovation in Biological Microscopy: Current Status and Future Directions

BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Mar, 2012  |  Pubmed ID: 22408015

The current revolution in biological microscopy stems from the realisation that advances in optics and computational tools and automation make the modern microscope an instrument that can access all scales relevant to modern biology - from individual molecules all the way to whole tissues and organisms and from single snapshots to time-lapse recordings sampling from milliseconds to days. As these and more new technologies appear, the challenges of delivering them to the community grows as well. I discuss some of these challenges, and the examples where openly shared technology have made an impact on the field.