Andrei Pozniakovsky

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.

Systematic Analysis of Human Protein Complexes Identifies Chromosome Segregation Proteins

Division Machinery Tagged An international consortium of labs has been testing the feasibility of large-scale screening for insights into the function of mammalian proteins by expressing a tagged version of proteins from bacterial artificial chromosomes harbored in mammalian cells. Depending on the tag used, Hutchins et al. (p. 593, published online 1 April) were able to monitor localization of tagged proteins by microscopy or to isolate interacting proteins and subsequently identify the binding partners by mass spectrometry. Applying the technology to proteins implicated in control of cell division revealed about 100 protein machines required for mitosis. A strategy designed to decipher the function of proteins identified in RNA interference screens reveals new insights into mitosis. Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification–mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the γ-tubulin ring complex—large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

Systematic Localization and Purification of Human Protein Complexes Identifies Chromosome Segregation Proteins

Division Machinery Tagged An international consortium of labs has been testing the feasibility of large-scale screening for insights into the function of mammalian proteins by expressing a tagged version of proteins from bacterial artificial chromosomes harbored in mammalian cells. Depending on the tag used, Hutchins et al. (p. 593, published online 1 April) were able to monitor localization of tagged proteins by microscopy or to isolate interacting proteins and subsequently identify the binding partners by mass spectrometry. Applying the technology to proteins implicated in control of cell division revealed about 100 protein machines required for mitosis. A strategy designed to decipher the function of proteins identified in RNA interference screens reveals new insights into mitosis. Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification–mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the γ-tubulin ring complex—large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast

Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s). In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD. Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar. We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD. For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic [Ca2+], a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria. A novel mitochondrial protein required for thread-grain transition is identified.

XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region

XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215’s overall affinity for free tubulin. The mutants’ overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a “bonsai” protein, with two TOG domains and a basic region, that has almost full polymerase activity.

XMAP215 regulates microtubule dynamics through two distinct domains.

XMAP215 belongs to a family of proteins involved in the regulation of microtubule dynamics. In this study we analyze the function of different parts of XMAP215 in vivo and in Xenopus egg extracts. XMAP215 has been divided into three fragments, FrN, FrM and FrC (for N‐terminal, middle and C‐terminal, respectively). FrN co‐localizes with microtubules in egg extracts but not in cells, FrC co‐ localizes with microtubules and centrosomes both in egg extracts and in cells, while FrM does not co‐ localize with either centrosomes or microtubules. In Xenopus egg extracts, FrN stimulates microtubule growth at plus‐ends by inhibiting catastrophes, while FrM has no effect, and FrC suppresses microtubule growth by promoting catastrophes. Our results suggest that XMAP215 is targeted to centrosomes and microtubules mainly through its C‐terminal domain, while the evolutionarily conserved N‐terminal domain contains its microtubule‐stabilizing activity.

Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon

We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (∼60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

Identification of substrates for cyclin dependent kinases

Protein phosphorylation is a key regulatory mechanism for cell cycle control in eukaryotes. From yeast to humans, cell cycle progression and cell division require the activation of a group of serine– threonine protein kinases called cyclin dependent kinases (CDKs) (Morgan, 1997), which initiate and coordinate these processes by orderly phosphorylation of their targets. CDKs require association with a cyclin subunit to activate them,whichprovides substrate specificity (Russo,1997; Solomonet al.,1992), and phosphorylation by an activating protein kinase (CAK) at a conserved threonine residue (Krek and Nigg,1992; Lorca et al., 1992; Solomon et al., 1992). The concentration of cyclins oscillate during the cell cycle and their abundance is regulated at several levels: transcriptional, translational and at the level of protein stability. In particular, at the end of mitosis, ubiquitin-mediated proteolysis results in the disappearance of mitotic cyclins, which results in inactivation of CDKs (Zachariae and Nasmyth, 1999). The key cell cycle transitions involving the activity of CDKs occur at the initiation of S-phase and entry into mitosis. Loss of CDK activity at the end of mitosis permits the return to interphase, and is presumably accompanied by dephosphorylation of the majority of target proteins that were modified when the cells entered mitosis. There is rather specific control of the different phases of the cell cycle, so that cells do not enter mitosis with unreplicated or damaged DNA, and do not exit mitosis until all the chromosomes are correctly aligned on the metaphase plate. There is a theoretical conundrum, however, in explaining how it is that activation of S-phase CDKs, such as cyclin E/CDK2 and cyclin A/ CDK2 in vertebrate cells promotes entry into S-phase and not into mitosis, or why activation of the Mphase CDKs does not promote a further round of DNA replication. One hypothesis to explain why cells enter S-phase, rather than mitosis, when the S-phase CDK (CDK2-cyclin E in higher eukaryotes) is activated would be a restricted substrate specificity for this particular CDK, yet there is very little information on this point. Another possibility is that CDK2-cyclin E resides in the nucleus, and entry

Regulated assembly of a supramolecular centrosome scaffold in vitro

A little bit of this and a little bit of that Centrosomes are the major microtubule-organizing centers in animal cells. Key to this function is the somewhat mysterious pericentriolar material (PCM). Woodruff et al. describe the in vitro reconstitution of PCM assembly. In cells, PCM is recruited by centrioles to form centrosomes that nucleate and anchor microtubules. SPD-5, the main component of the PCM matrix in Caenorhabiditis elegans, polymerized in vitro to form micrometer-sized porous networks. SPD-5 polymerization was directly controlled by the polo family kinase Plk1 and Cep192/SPD-2, two conserved regulators that control PCM assembly across metazoans. Science, this issue p. 808 Centrosome assembly in Caenorhabditis elegans involves self-assembly of an interconnected, micrometer-scale network of proteins. The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules

Metaphase spindles are microtubule-based structures that use a multitude of proteins to modulate their morphology and function. Today, we understand many details of microtubule assembly, the role of microtubule-associated proteins, and the action of molecular motors. Ultimately, the challenge remains to understand how the collective behaviour of these nanometre-scale processes gives rise to a properly sized spindle on the micrometre scale. By systematically engineering the enzymatic activity of XMAP215, a processive microtubule polymerase, we show that Xenopus laevis spindle length increases linearly with microtubule growth velocity, whereas other parameters of spindle organization, such as microtubule density, lifetime and spindle shape, remain constant. We further show that mass balance can be used to link the global property of spindle size to individual microtubule dynamic parameters. We propose that spindle length is set by a balance of non-uniform nucleation and global microtubule disassembly in a liquid-crystal-like arrangement of microtubules.