Xiangwei He

Huntingtin toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntingtons disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1–151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1–85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.

Transient Sister Chromatid Separation and Elastic Deformation of Chromosomes during Mitosis in Budding Yeast

The accurate segregation of chromosomes at mitosis requires that all pairs of chromatids bind correctly to microtubules prior to the dissolution of sister cohesion and the initiation of anaphase. By analyzing the motion of GFP-tagged S. cerevisiae chromosomes, we show that kinetochore-microtubule attachments impose sufficient tension on sisters during prometaphase to transiently separate centromeric chromatin toward opposite sides of the spindle. Transient separations of 2-10 min duration occur in the absence of cohesin proteolysis, are characterized by independent motion of the sisters along the spindle, and are followed by the apparent reestablishment of sister linkages. The existence of transient sister separation in yeast explains the unusual bilobed localization of kinetochore proteins and supports an alternative model for spindle structure. By analogy with animal cells, we propose that yeast centromeric chromatin acts as a tensiometer.

Molecular Analysis of Kinetochore-Microtubule Attachment in Budding Yeast

The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5′-to-3′ degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

DANPOS: Dynamic analysis of nucleosome position and occupancy by sequencing

Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome.

Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres

Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site.

A high-resolution map of nucleosome positioning on a fission yeast centromere

A key element for defining the centromere identity is the incorporation of a specific histone H3, CENPA, known as Cnp1p in Schizosaccharomyces pombe. Previous studies have suggested that functional S. pombe centromeres lack regularly positioned nucleosomes and may involve chromatin remodeling as a key step of kinetochore assembly. We used tiling microarrays to show that nucleosomes are, in fact, positioned in regular intervals in the core of centromere 2, providing the first high-resolution map of regional centromere chromatin. Nucleosome locations are not disrupted by mutations in kinetochore protein genes cnp1, mis18, mis12, nuf2, mal2; overexpression of cnp1; or the deletion of ams2, which encodes a GATA-like factor participating in CENPA incorporation. Bioinformatics analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence bias in nucleosome positioning. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites of Ams2p. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence.

Detecting Apoptotic Cells and Monitoring Their Clearance in the Nematode Caenorhabditis elegans

Apoptosis is a genetically controlled process of cell suicide that plays an important role in animal development and in maintaining homeostasis. The nematode Caenorhabditis elegans has proven to be an excellent model organism for studying the mechanisms controlling apoptosis and the subsequent clearance of apoptotic cells, aided with cell-biological and genetic tools. In particular, the transparent nature of worm bodies and eggshells makes C. elegans particularly amiable for live cell microscopy. Here we describe a few methods for identifying apoptotic cells in living C. elegans embryos and adults and for monitoring their clearance during embryonic development. These methods are based on Differential Interference Contrast microscopy and on fluorescence microscopy using GFP-based reporters.

Quantitative microscopy of green fluorescent protein-labeled yeast.

Publisher Summary This chapter discusses the quantitative microscopy of green fluorescent protein-labeled yeast. With the development of methods to tag proteins using green fluorescent protein (GFP), fluorescence microscopy has become increasingly important for characterizing protein function in yeast. The chapter describes the use of three-dimensional (3-D) deconvolution microscopy to perform fixed- and live-cell analysis of cells carrying GFP-tagged proteins. The chapter also discusses the process of deconvolution in fluorescence microscopy. Despite the focus on high-performance imaging, the methods described in the chapter are applicable to a wide range of experiments using conventional wide-field microscopes. The chapter describes the various steps for preparing biological samples, such as strain construction, mounting cells for microscopy, preparing slides with agar pads for live-cell microscopy, mounting cells on agar pads, mounting live cells without agar pads, fixing cells with paraformaldehyde and mounting, optimizing microscope optics, selecting filters, selecting cover glass, temperature control, minimizing spherical aberration through oil matching, acquiring images, and viewing and printing the image.

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

The Dam1 complex is a kinetochore component that couples chromosomes to the dynamic ends of kinetochore microtubules (kMTs). Work in the budding yeast Saccharomyces cerevisiae has shown that the Dam1 complex forms a 16-unit ring encircling and tracking the tip of a MT in vitro, consistent with its cellular function as a coupler. Dam1 also forms smaller, nonring patches in vitro that track the dynamic ends of MTs. However, the identity of Dam1’s functional form in vivo remains unknown. Here we report a comprehensive in vivo characterization of Dam1 in the fission yeast Schizosaccharomyces pombe. In addition to their dense localizations on kinetochores and spindle MTs during mitosis, we identify that Dam1 is also localized onto cytoplasmic MTs as discrete spots in interphase, providing the unique opportunity to analyze Dam1 oligomers at the single-particle resolution in live cells. Such analysis shows that each oligomer contains one to five copies of Dam1, and is able to “switch-rail” while moving along MTs, precluding the possibility of a 16-unit encircling structure. Dam1 patches track the plus ends of the shortening, but not the elongating, MTs and retard MT depolymerization. Together with Mal3, the EB1-like MT-interacting protein, cytoplasmic Dam1 plays an important role in maintaining proper cell shape. In mitosis, kinetochore-associated Dam1 appears to facilitate kMT depolymerization. Together, our findings suggest that patches, instead of rings, are the physiologically functional forms of Dam1 in pombe. Our findings help establish the benchmark parameters of the Dam1 coupler and elucidate the mechanism of its functions.