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Rebecca Heald

Publications and source records attributed to Rebecca Heald.

At least 19 recordsLinked to original sources
PubMed2006

Essential roles for cohesin in kinetochore and spindle function in Xenopus egg extracts.

To facilitate their accurate distribution by the mitotic spindle, sister chromatids are tethered during DNA replication, attached by their kinetochores and bi-oriented on the spindle, and then simultaneously released at the metaphase to anaphase transition, allowing for their segregation to opposite spindle poles. The highly conserved cohesin complex is fundamental to this process, yet its role in mitosis is not fully understood. We show that depletion of cohesin from Xenopus egg extracts impairs sister chromatid cohesion and kinetochore-microtubule interactions, causing defective spindle attachments and chromosome alignment during metaphase and mis-segregation during anaphase. In the absence of cohesin, sister kinetochore pairing and centromeric localization of chromosomal passenger proteins INCENP and aurora B were lost upon bipolar spindle attachment. However, kinetochores remained paired with normal passenger localization if bipolar spindle formation was prevented by inhibiting the kinesin-5 motor (Eg5). These observations indicate that cohesin is not required to establish sister association, but is necessary to maintain cohesion in the presence of bipolar spindle forces. Co-depletion of cohesin together with another major SMC complex, condensin, revealed cumulative effects on spindle assembly and chromosome architecture. These data underscore the essential requirement for cohesin in sister chromatid cohesion, kinetochore and spindle function.

Adenosine Triphosphatases↗
PubMed2006

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics.

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.

Adenosine Triphosphate↗
PubMed2006

Analysis of a RanGTP-regulated gradient in mitotic somatic cells.

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran-importin-beta-cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

Animals↗
PubMed2006

The long and the short of it: linker histone H1 is required for metaphase chromosome compaction.

Faithful distribution of the genome to daughter cells requires that replicated chromatids are compacted and resolved from one another, and that functional kinetochores assemble at their centromeres to mediate spindle interactions. A molecular and mechanistic understanding of these critical processes is far from complete. We discovered that linker histone H1, a major chromatin component, is required for longitudinal compaction of replicated sperm chromosome arms in Xenopus laevis egg extracts that recapitulate the cell cycle in vitro. Due to their elongated structure, H1-depleted chromosomes could not be aligned or segregated by the spindle despite apparently normal kinetochore assembly. Interestingly, enrichment of H1 onto chromatin required passage through interphase, when DNA replication takes place. Here we show that interphase-dependent loading is also a feature of kinetochore components including Ndc80. Thus, linker histone H1 contributes to chromosome condensation in vertebrates, and important molecular determinants of mitotic chromosome and kinetochore structure and function are established during interphase.

Animals↗
PubMed2006

Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle.

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.

Anaphase↗
PubMed2006

Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.

Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and "cycling" of the reaction through interphase and back into metaphase promotes formation of bipolar spindles capable of segregating their duplicated chromosomes. Reactions can be "spun down" onto cover slips for immunofluorescence analysis. High-speed extracts support mitotic chromosome condensation, which can be observed live by fluorescence time-lapse video microscopy. Because of the biochemically accessible nature of the egg extract system, a wide array of biochemical techniques can be combined with spindle and chromosome assembly reactions to evaluate the roles of specific proteins in these processes.

Animals↗
PubMed2006

Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts.

Microtubules are dynamic polymers that participate in multiple cellular processes such as vesicular transport and cell division. Microtubule dynamics alter dramatically during the cell cycle. An excellent system to study microtubule dynamics is Xenopus egg extracts since it is a system that is open to manipulation. The extracts can be cycled between mitosis and interphase allowing the study of microtubules in these phases as well as during cell cycle transitions. Here, we provide simple assays to study microtubules in extracts and in vitro using purified components. Protocols are provided for the purification of frog tubulin, microtubule pelleting from extracts and in vitro, assembly of microtubule structures in extracts, and isolation of microtubule-associated proteins from extract. These methods can be used to analyze the effect of a protein of interest on the microtubule cytoskeleton.

Animals↗
PubMed2006

Challenges facing the biologist doing chemical genetics.

The goal of high-throughput screening (HTS) from the perspective of the biologist is to identify a highly specific small molecule that can be used to inhibit a protein in its normal biological context. Although several useful small molecules have been identified with HTS, there are many challenges to be considered when contemplating a screen, especially by those unfamiliar with chemical biology.

Biological Assay↗
PubMed2005

The perichromosomal layer.

In addition to genetic information, mitotic chromosomes transmit essential components for nuclear assembly and function in a new cell cycle. A specialized chromosome domain, called the perichromosomal layer, perichromosomal sheath, chromosomal coat, or chromosome surface domain, contains proteins required for a variety of cellular processes, including the synthesis of messenger RNA, assembly of ribosomes, repair of DNA double-strand breaks, telomere maintenance, and apoptosis regulation. The layer also contains many proteins of unknown function and is a major target in autoimmune disease. Perichromosomal proteins are found along the entire length of chromosomes, excluding centromeres, where sister chromatids are paired and spindle microtubules attach. Targeting of proteins to the perichromosomal layer occurs primarily during prophase, and they generally remain associated until telophase. During interphase, perichromosomal proteins localize to nucleoli, the nuclear envelope, nucleoplasm, heterochromatin, centromeres, telomeres, and/or the cytoplasm. It has been suggested that the perichromosomal layer may contribute to chromosome structure, as several of the associated proteins have functions in chromatin remodeling during interphase. We review the identified proteins associated with this chromosome domain and briefly discuss their known functions during interphase and mitosis.

Animals↗
PubMed2005

Xnf7 contributes to spindle integrity through its microtubule-bundling activity.

Regulation of microtubule dynamics and organization in mitosis by a number of microtubule-associated proteins (MAPs) is required for proper bipolar spindle assembly, yet the precise mechanisms by which many MAPs function are poorly understood. One interesting class of MAPs is known to localize to the nucleus during interphase yet fulfill important spindle functions during mitosis. We have identified Xenopus nuclear factor 7 (Xnf7), a developmental regulator of dorsal-ventral patterning, as a microtubule-binding protein that also associates with the nuclear import receptor importin alpha/beta. Xnf7 localized to interphase nuclei and metaphase spindles both in Xenopus egg extracts and cultured cells. Xnf7-depleted spindles were hypersensitive to microtubule-depolymerizing agents. Functional characterization of Xnf7 revealed that it binds directly to microtubules, exhibits RING-finger-dependent E3-ubiquitin-ligase activity, and has C-terminal-dependent microtubule-bundling activity. The minimal microtubule-bundling domain of Xnf7 was sufficient to rescue the spindle-hypersensitivity phenotype. Thus, we have identified Xnf7 as a nuclear MAP whose microtubule-bundling activity, but not E3-ligase activity, contributes to microtubule organization and spindle integrity. Characterization of the multiple activities of Xnf7 may have implications for understanding human diseases caused by mutations in related proteins.

Animals↗
PubMed2005

Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts.

During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis.

Adenosine Triphosphatases↗
PubMed2005

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.

Animals↗
PubMed2005

Scratch n' screen for inhibitors of cell migration.

Yarrow et al. have identified a small molecule inhibitor of cell migration, 3-(4-pyridyl)indole ("Rockout"), that targets Rho-kinase via a novel screening method using a scratch wound healing assay adapted to a high-throughput format and automated microscopy.

Animals↗
PubMed2005

Chromosome congression: another fine mesh we've gotten into.

Collecting chromosomes prior to their accurate distribution by the mitotic spindle is widely believed to be a microtubule-driven process. However, a recent study in Nature by Lénart et al. (2005) has revealed that a contractile actin network makes an essential contribution to chromosome capture in animal oocytes.

Animals↗
PubMed2004

Adenomatous polyposis coli associates with the microtubule-destabilizing protein XMCAK.

During cell division, the proper formation of a bipolar spindle and its function to segregate chromosomes requires precise coordination of microtubule-stabilizing and destabilizing activities. Globally destabilized, dynamic microtubules radiating from duplicated centrosomes are locally regulated by chromosomes. Proteins at the kinetochore of each sister chromatid mediate a dynamic attachment, allowing chromosome movement coupled to microtubule polymerization/depolymerization and error-correction mechanisms for improperly attached chromosomes. The tumor suppressor protein adenomatous polyposis coli (APC) stabilizes microtubules both in vitro and in vivo and is implicated in mitosis, although its mechanisms of action are not well characterized. Here, we show that in mitotic Xenopus egg extracts, the carboxyl-terminus of APC can associate with the amino terminus of the microtubule-destabilizing KinI, Xenopus mitotic centromere-associated kinesin (XMCAK), in a cytoplasmic complex. We find that like XMCAK, APC can localize to the centromere as well as the kinetochore region of mitotic chromosomes and does not require microtubules for chromosomal targeting in Xenopus egg extracts. We propose that the presence of these proteins in a complex brings together both positive and negative microtubule effectors, whose opposing activities may be regulated by additional factors, thereby providing precise control of both global and local microtubule dynamics.

Adenomatous Polyposis Coli Protein↗
PubMed2004

Mechanisms and molecules of the mitotic spindle.

In all eukaryotes, morphogenesis of the microtubule cytoskeleton into a bipolar spindle is required for the faithful transmission of the genome to the two daughter cells during division. This process is facilitated by the intrinsic polarity and dynamic properties of microtubules and involves many proteins that modulate microtubule organization and stability. Recent work has begun to uncover the molecular mechanisms behind these dynamic events. Here we describe current models and discuss some of the complex repertoire of factors required for spindle assembly and chromosome segregation.

Cell Cycle↗
PubMed2004

Centromere glue provides spindle cue.

During cell division, accurate distribution of the genome by the mitotic spindle requires that sister chromatids remain tethered until the right moment. Studies of an uncharacterized vertebrate protein, Sgo (Salic et al., 2004 [this issue of Cell]), reveal dual roles as a chromosome cohesion factor and a regulator of spindle microtubule dynamics.

Animals↗