Anthony A. Hyman

Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III.

Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of ∼96% of the ∼2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (∼6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that ∼47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the ∼21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.

Preparation of modified tubulins

Publisher Summary This chapter presents a collection of the various different ways by which tubulins are modified to generate probes for investigating microtubule (MT) dynamics in vitro and in vivo . Labeling with biotin and various fluorochromes is described, as well as the preparation of N-ethylmaleimide tubulin, which has been used to block minus-end growth in vitro . The use of GTP analogs to prepare stable labeled microtubules has proved very useful in a number of different experiments. The tubulin used in the presented methods was prepared from bovine brain by two cycles of temperature-dependent polymerization, followed by phosphocellulose chromatography. The cycling procedure described in the chapter selects active subunits and removes free nucleotide. This produces a tubulin preparation suitable for use in in vitro assays. The standard biotin-labeled tubulin preparation has been used to determine sites of microtubule elongation in vivo and in vitro . It is difficult to quantitate the stoichiometry of biotin labeling on a routine basis, but early work using radioactive N-hydroxysuccinimide (NHS)-biotin gave a labeling stochiometry of one to three biotins/tubulin dimer. The final yield of twice cycled biotin-tubulin is about 10% of the starting protein. Tetramethylrhodamine-labeled tubulin has been used to follow microtubules in living cells and it is also used for marking microtubules in real-time in vitro assays.

Dynamics and mechanics of the microtubule plus end

An important function of microtubules is to move cellular structures such as chromosomes, mitotic spindles and other organelles around inside cells. This is achieved by attaching the ends of microtubules to cellular structures; as the microtubules grow and shrink, the structures are pushed or pulled around the cell. How do the ends of microtubules couple to cellular structures, and how does this coupling regulate the stability and distribution of the microtubules? It is now clear that there are at least three properties of a microtubule end: it has alternate structures; it has a biochemical transition defined by GTP hydrolysis; and it forms a distinct target for the binding of specific proteins. These different properties can be unified by thinking of the microtubule as a molecular machine, which switches between growing and shrinking modes. Each mode is associated with a specific end structure on which end-binding proteins can assemble to modulate dynamics and couple the dynamic properties of microtubules to the movement of cellular structures.

Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation

P Granule Conundrum In many organisms, the presumptive germ cells can be distinguished from somatic cells by the presence of distinctive cytoplasmic granules. In Caenorhabditis elegans, these P granules are more or less uniformly distributed in the oocyte and one-cell stage of the fertilized egg. By the end of the first cleavage, however, the anterior cell is essentially free of P granules, whereas the posterior cell still displays a prominent population of granules. Exactly how this process occurs and whether it involves directed migration of the granules is unclear. Now Brangwynne et al. (p. 1729, published online 21 May; see the Perspective by Le Goff and Lecuit) provide evidence that localization occurs by a quite different mechanism, controlled dissolution and condensation of granule components. This type of cytoplasmic remodeling by physicochemical mechanisms can now be looked for in other cellular and developmental systems. Localization of RNA and protein-rich germ-cell granules occurs by controlled dissolution and condensation. In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs. In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension. As with other liquids, P granules rapidly dissolved and condensed. Localization occurred by a biased increase in P granule condensation at the posterior. This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell. Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm.

BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.NOTE: In the version of this article initially published online, the name of one individual was misspelled in the Acknowledgments. The second sentence of the Acknowledgments paragraph should read, “We thank I. Cheesman for helpful discussions.” The error has been corrected for all versions of the article.

Rab5 regulates motility of early endosomes on microtubules

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.

The spindle: a dynamic assembly of microtubules and motors

In all eukaryotes, a microtubule-based structure known as the spindle is responsible for accurate chromosome segregation during cell division. Spindle assembly and function require localized regulation of microtubule dynamics and the activity of a variety of microtubule-based motor proteins. Recent work has begun to uncover the molecular mechanisms that underpin this process. Here we describe the structural and dynamic properties of the spindle, and introduce the current concepts regarding how a bipolar spindle is assembled and how it functions to segregate chromosomes.

Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3β phosphorylation

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.