Michael D. Blower

Conserved organization of centromeric chromatin in flies and humans

Recent studies have highlighted the importance of centromere-specific histone H3-like (CENP-A) proteins in centromere function. We show that Drosophila CID and human CENP-A appear at metaphase as a three-dimensional structure that lacks histone H3. However, blocks of CID/CENP-A and H3 nucleosomes are linearly interspersed on extended chromatin fibers, and CID is close to H3 nucleosomes in polynucleosomal preparations. When CID is depleted by RNAi, it is replaced by H3, demonstrating flexibility of centromeric chromatin organization. Finally, contrary to models proposing that H3 and CID/CENP-A nucleosomes are replicated at different times in S phase, we show that interspersed H3 and CID/CENP-A chromatin are replicated concurrently during S phase in humans and flies. We propose that the unique structural arrangement of CID/CENP-A and H3 nucleosomes presents centromeric chromatin to the poleward face of the condensing mitotic chromosome.

The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions.

Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Here we show that CID, the Drosophila homologue of the CENP-A centromere-specific H3-like proteins, colocalizes with molecular-genetically defined functional centromeres in minichromosomes. Injection of CID antibodies into early embryos, as well as RNA interference in tissue-culture cells, showed that CID is required for several mitotic processes. Deconvolution fluorescence microscopy showed that CID chromatin is physically separate from proteins involved in sister cohesion (MEI-S332), centric condensation (PROD), kinetochore function (ROD, ZW10 and BUB1) and heterochromatin structure (HP1). CID localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of POLO, CENP-meta, ROD, BUB1 and MEI-S332, but not PROD or HP1, depends on the presence of functional CID. We conclude that the centromere and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that CID is required for normal kinetochore formation and function, as well as cell-cycle progression.

Determining centromere identity: cyclical stories and forking paths

The centromere is the genetic locus required for chromosome segregation. It is the site of spindle attachment to the chromosomes and is crucial for the transfer of genetic information between cell and organismal generations. Although the centromere was first recognized more than 120 years ago, little is known about what determines its site(s) of activity, and how it contributes to kinetochore formation and spindle attachment. Recent work in this field has supported the hypothesis that most eukaryotic centromeres are determined epigenetically rather than by primary DNA sequence. Here, we review recent studies that have elucidated the organization and functions of centromeric chromatin, and evaluate present-day models for how centromere identity and propagation are determined.

A Rae1-Containing Ribonucleoprotein Complex Is Required for Mitotic Spindle Assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.

Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules.

RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we comprehensively surveyed all messenger RNAs (mRNAs) that bound to microtubules during metaphase in both Xenopus laevis egg extracts and mitotic human cell extracts. We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis. Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes. Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules. Our results represent the first genome-wide survey of mRNAs localized to a specific cytoskeletal component and suggest that microtubule localization of specific mRNAs is likely to function in mitotic regulation and mRNA segregation during cell division.

Systematic and single-cell analysis of Xenopus Piwi-interacting RNAs and Xiwi.

Piwi proteins and Piwi‐interacting RNAs (piRNAs) are essential for germ cell development, but analysis of the molecular mechanisms of these ribonucleoproteins remains challenging in most animal germ cells. To address this challenge, we systematically characterized Xiwi, a Xenopus Piwi homologue, and piRNAs from Xenopus eggs and oocytes. We used the large size of Xenopus eggs to analyze small RNAs at the single cell level, and find abundant piRNAs and large piRNA clusters in the Xenopus tropicalis genome, some of which resemble the Drosophila piRNA‐generating flamenco locus. Although most piRNA clusters are expressed simultaneously in an egg, individual frogs show distinct profiles of cluster expression. Xiwi is associated with microtubules and the meiotic spindle, and is localized to the germ plasm—a cytoplasmic determinant of germ cell formation. Xiwi associates with translational regulators in an RNA‐dependent manner, but Xenopus tudor interacts with Xiwi independently of RNA. Our study adds insight to piRNA transcription regulation by showing that individual animals can have differential piRNA expression profiles. We suggest that in addition to regulating transposable elements, Xiwi may function in specifying RNA localization in vertebrate oocytes.

Reconstitution of the mitochondrial calcium uniporter in yeast

Significance The mitochondrial uniporter is a highly selective calcium channel found in many diverse eukaryotes, but absent in the yeast Saccharomyces cerevisiae. Although the uniporter’s existence was recognized more than 50 y ago, its molecular components have been identified only recently. Here we use yeast as a facile reconstitution system to identify the minimal components sufficient for in vivo uniporter activity. We describe the simplified calcium uniporter of slime mold, consisting of one transmembrane component, DdMCU, which alone is sufficient for robust calcium uptake in yeast mitochondria. Intriguingly, the human uniporter requires two proteins, MCU and the animal-specific protein EMRE, that together are sufficient for uniporter activity. Our work provides a powerful reconstitution system for studying the evolution and function of this channel. The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits –mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)– and two soluble regulatory components–MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.

Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle

The African clawed frog Xenopus laevis has been instrumental to investigations of both development and cell biology, but the utility of this model organism for genetic and proteomic studies is limited by its long generation time and unsequenced pseudotetraploid genome. Xenopus tropicalis, which is a small, faster-breeding relative of X. laevis, has recently been adopted for research in developmental genetics and functional genomics, and has been chosen for genome sequencing. We show that X. tropicalis egg extracts reconstitute the fundamental cell cycle events of nuclear formation and bipolar spindle assembly around exogenously added sperm nuclei. Interestingly, X. tropicalis spindles were ∼30% shorter than X. laevis spindles, and mixing experiments revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors. Measurements of microtubule dynamics revealed that microtubules polymerized slower in X. tropicalis extracts compared to X. laevis, but that this difference is unlikely to account for differences in spindle size. Thus, in addition to expanding the range of developmental and cell biological experiments, the use of X. tropicalis provides novel insight into the complex mechanisms that govern spindle morphogenesis.

The endoplasmic reticulum: structure, function and response to cellular signaling

The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, sheets and the nuclear envelope. Several proteins that contribute to the overall architecture and dynamics of the ER have been identified, but many questions remain as to how the ER changes shape in response to cellular cues, cell type, cell cycle state and during development of the organism. Here we discuss what is known about the dynamics of the ER, what questions remain, and how coordinated responses add to the layers of regulation in this dynamic organelle.

Functional analysis of the microtubule-interacting transcriptome

A combination of bioinformatic and RNA interference analysis of Xenopus tropicalis RNA-seq data shows that the identification of microtubule-associated (MT) mRNAs can be used for discovering novel factors in the processes of spindle pole organization and centrosome structure. MT-RNAs are likely to contribute to spindle-localized mitotic translation.