Zulin Yu

Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication

Duplication of the yeast centrosome (called the spindle pole body, SPB) is thought to occur through a series of discrete steps that culminate in insertion of the new SPB into the nuclear envelope (NE). To better understand this process, we developed a novel two-color structured illumination microscopy with single-particle averaging (SPA-SIM) approach to study the localization of all 18 SPB components during duplication using endogenously expressed fluorescent protein derivatives. The increased resolution and quantitative intensity information obtained using this method allowed us to demonstrate that SPB duplication begins by formation of an asymmetric Sfi1 filament at mitotic exit followed by Mps1-dependent assembly of a Spc29- and Spc42-dependent complex at its tip. Our observation that proteins involved in membrane insertion, such as Mps2, Bbp1, and Ndc1, also accumulate at the new SPB early in duplication suggests that SPB assembly and NE insertion are coupled events during SPB formation in wild-type cells. DOI: http://dx.doi.org/10.7554/eLife.08586.001

Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1

Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1.

Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex

Significance Because inaccurate chromosome segregation during meiosis is a leading cause of miscarriage in humans, we seek to understand how homologous chromosomes segregate properly. Meiotic chromosome segregation occurs with fidelity only in the presence of the synaptonemal complex (SC), a protein structure that assembles between homologs and facilitates the occurrence of crossing over. Although some functions of the SC are evolutionarily conserved, the mechanisms underlying its multiple roles during meiosis, as well as organizational variances among different organisms, remain under investigation. Here we combine superresolution and expansion microscopy and find strong evidence that the Drosophila SC comprises two visually distinct layers, perhaps suggesting that each layer connects one sister chromatid from each homologous chromosome. The synaptonemal complex (SC), a structure highly conserved from yeast to mammals, assembles between homologous chromosomes and is essential for accurate chromosome segregation at the first meiotic division. In Drosophila melanogaster, many SC components and their general positions within the complex have been dissected through a combination of genetic analyses, superresolution microscopy, and electron microscopy. Although these studies provide a 2D understanding of SC structure in Drosophila, the inability to optically resolve the minute distances between proteins in the complex has precluded its 3D characterization. A recently described technology termed expansion microscopy (ExM) uniformly increases the size of a biological sample, thereby circumventing the limits of optical resolution. By adapting the ExM protocol to render it compatible with structured illumination microscopy, we can examine the 3D organization of several known Drosophila SC components. These data provide evidence that two layers of SC are assembled. We further speculate that each SC layer may connect two nonsister chromatids, and present a 3D model of the Drosophila SC based on these findings.

Ribosomal DNA copy number loss and sequence variation in cancer

Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

Molecular model of fission yeast centrosome assembly determined by superresolution imaging

Microtubule-organizing centers (MTOCs), known as centrosomes in animals and spindle pole bodies (SPBs) in fungi, are important for the faithful distribution of chromosomes between daughter cells during mitosis as well as for other cellular functions. The cytoplasmic duplication cycle and regulation of the Schizosaccharomyces pombe SPB is analogous to centrosomes, making it an ideal model to study MTOC assembly. Here, we use superresolution structured illumination microscopy with single-particle averaging to localize 14 S. pombe SPB components and regulators, determining both the relationship of proteins to each other within the SPB and how each protein is assembled into a new structure during SPB duplication. These data enabled us to build the first comprehensive molecular model of the S. pombe SPB, resulting in structural and functional insights not ascertained through investigations of individual subunits, including functional similarities between Ppc89 and the budding yeast SPB scaffold Spc42, distribution of Sad1 to a ring-like structure and multiple modes of Mto1 recruitment.

Immediate perception of a reward is distinct from the reward's long-term salience

Reward perception guides all aspects of animal behavior. However, the relationship between the perceived value of a reward, the latent value of a reward, and the behavioral response remains unclear. Here we report that, given a choice between two sweet and chemically similar sugars—L- and D-arabinose—Drosophila melanogaster prefers D- over L- arabinose, but forms long-term memories of L-arabinose more reliably. Behavioral assays indicate that L-arabinose-generated memories require sugar receptor Gr43a, and calcium imaging and electrophysiological recordings indicate that L- and D-arabinose differentially activate Gr43a-expressing neurons. We posit that the immediate valence of a reward is not always predictive of the long-term reinforcement value of that reward, and that a subset of sugar-sensing neurons may generate distinct representations of similar sugars, allowing for rapid assessment of the salient features of various sugar rewards and generation of reward-specific behaviors. However, how sensory neurons communicate information about L-arabinose quality and concentration—features relevant for long-term memory—remains unknown. DOI: http://dx.doi.org/10.7554/eLife.22283.001

Synaptonemal complex architecture facilitates the chromosome-specific regulation of recombination in Drosophila

In Drosophila, meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs), which occur within the context of the synaptonemal complex (SC). To better understand the role of the SC in mediating recombination we created an in-frame deletion mutant in c(3)G (deleting amino acids L340 to N550, denoted as c(3)GccΔ1), which encodes the major transverse filament protein of the SC. Although c(3)GccΔ1 oocytes assemble ribbon-like SC and exhibit normal DSB formation, the euchromatic SC precociously disassembles into fragments that persist until mid to late pachytene in both c(3)GccΔ1 heterozygotes and homozygotes. Centromeric SC, however, is unaffected in both genotypes. Thus, c(3)GccΔ1 is a separation-of-function mutant that establishes different functional and structural requirements between euchromatic and centromeric SC. Our data also demonstrate that the chromosome arms differ in their sensitivity to c(3)GccΔ1-induced perturbations in the SC. The X chromosome is distinctly sensitive to these perturbations, such that euchromatic pairing and crossing over are altered in c(3)GccΔ1 heterozygotes and severely reduced in c(3)GccΔ1 homozygotes. On the autosomes, crossovers are shifted to centromere-proximal regions and crossover interference is defective in both c(3)GccΔ1 homozygotes and heterozygotes. However, only c(3)GccΔ1 homozygotes display a progressive loss of euchromatic pairing in distal autosomal regions, suggesting that discontinuity in the euchromatic SC—rather than failed pairing—might cause the altered crossover distribution. These phenotypes reveal that different chromatin states or regions have differing requirements to maintain both the SC and homologous pairing. Furthermore, c(3)GccΔ1 is the first mutant in Drosophila to demonstrate that the SC appears to facilitate the regulation of recombination frequency and distribution differently on each chromosome. Author Summary Chromosome segregation errors during meiosis are the leading cause of miscarriage and birth defects in humans. To prevent these errors from occurring, meiotic cells have evolved multiple mechanisms to ensure that each gamete receives exactly half the number of chromosomes. During meiosis I, this is accomplished by forming a crossover between homologous chromosomes, which is facilitated by a large protein complex called the synaptonemal complex (SC). The SC is assembled between homologous chromosomes during early prophase I, and it is unclear how the SC regulates the position and number of crossovers each homolog receives. To better understand the role of the SC in mediating recombination, we created an in-frame deletion mutant in Drosophila melanogaster in the gene encoding the C(3)G protein, the major transverse filament protein of the SC. Although mutant oocytes assemble ribbon-like SC, the SC along the chromosome arms precociously disassembles in early meiosis. Surprisingly, the SC around the centromeres is unaffected in these mutants, suggesting that the requirements for SC formation may differ depending on where the SC is located along the chromosomes. Our data also demonstrate that the chromosome arms differ in their sensitivity to the mutant-induced perturbations of the SC in both crossing over and homolog pairing.Abstract The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double strand breaks (DSBs) into crossovers or gene conversions. The removal of any central region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct three in-frame deletions within the predicted coiled-coil region of the C(3)G protein. These three deletion mutants disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, allowing us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently than the autosomes.

Membrane insertion function for SUN-KASH complex revealed by high resolution analysis of yeast centrosomes

Bipolar spindle formation in yeast requires insertion of centrosomes (known as spindle pole bodies (SPBs)) into fenestrated regions of the nuclear envelope (NE). Using structured-illumination microscopy and bimolecular fluorescence complementation, we map protein distribution at SPB fenestra and interrogate protein-protein interactions with high spatial resolution. We find that the Sad1-UNC-84 (SUN) protein Mps3 forms a ring-like structure around the SPB, similar to toroids seen for components of the SPB insertion network (SPIN). Mps3 and the SPIN component Mps2 (a Klarsicht-ANC-1-Syne-1 domain (KASH)-like protein) form a novel non-canonical linker of nucleoskeleton and cytoskeleton (LINC) complex that is connected in both luminal and extraluminal domains. This hairpin-like LINC complex forms during SPB insertion, suggesting it functions in NE reorganization at the pore membrane. The LINC complex also controls the distribution of a soluble SPIN component Bbp1. Taken together our work shows that Mps3 is a fifth SPIN component and suggests both direct and indirect roles for the LINC complex in NE remodeling.

Combined expansion microscopy with structured illumination microscopy for analyzing protein complexes

Biologists have long been fascinated with the organization and function of intricate protein complexes. Therefore, techniques for precisely imaging protein complexes and the location of proteins within these complexes are critically important and often require multidisciplinary collaboration. A challenge in these explorations is the limited resolution of conventional light microscopy. However, a new microscopic technique has circumvented this resolution limit by making the biological sample larger, thus allowing for super-resolution of the enlarged structure. This ‘expansion’ is accomplished by embedding the sample in a hydrogel that, when exposed to water, uniformly expands. Here, we present a protocol that transforms thick expansion microscopy (ExM) hydrogels into sections that are physically expanded four times, creating samples that are compatible with the super-resolution technique structured illumination microscopy (SIM). This super-resolution ExM method (ExM–SIM) allows the analysis of the three-dimensional (3D) organization of multiprotein complexes at ~30-nm lateral (xy) resolution. This protocol details the steps necessary for analysis of protein localization using ExM–SIM, including antibody labeling, hydrogel preparation, protease digestion, post-digestion antibody labeling, hydrogel embedding with tissue-freezing medium (TFM), cryosectioning, expansion, image alignment, and particle averaging. We have used this approach for 3D mapping of in situ protein localization in the Drosophila synaptonemal complex (SC), but it can be readily adapted to study thick tissues such as brain and organs in various model systems. This procedure can be completed in 5 d.This protocol describes how to combine expansion microscopy (ExM) with structured illumination microscopy (SIM). ExM–SIM is exemplified by super-resolution analysis of the synaptonemal complex (SC) and single-particle averaging of SC proteins.

The budding yeast RSC complex maintains ploidy by promoting spindle pole body insertion

Ploidy is tightly regulated in eukaryotic cells and is critical for cell function and survival. Cells coordinate multiple pathways to ensure replicated DNA is segregated accurately to prevent abnormal changes in chromosome number. In this study, we characterize an unanticipated role for the Saccharomyces cerevisiae “remodels the structure of chromatin” (RSC) complex in ploidy maintenance. We show that deletion of any of six nonessential RSC genes causes a rapid transition from haploid to diploid DNA content because of nondisjunction events. Diploidization is accompanied by diagnostic changes in cell morphology and is stably maintained without further ploidy increases. We find that RSC promotes chromosome segregation by facilitating spindle pole body (SPB) duplication. More specifically, RSC plays a role in distributing two SPB insertion factors, Nbp1 and Ndc1, to the new SPB. Thus, we provide insight into a role for a SWI/SNF family complex in SPB duplication and ploidy maintenance.