A bacterial expression cloning screen reveals single-stranded DNA-binding proteins as potent desicco-protectants.

Desiccation kills most cells. Some proteins have been identified to help certain cells survive desiccation, but many protein protectants are likely to be unknown. Moreover, the mechanisms ensuring protection of key cellular components are incompletely understood.

Transient crosslinking controls the condensate formation pathway within chromatin networks.

The network structure of densely packed chromatin within the nucleus of eukaryotic cells acts in concert with nonequilibrium processes. Using statistical physics simulations, we explore the control provided by transient crosslinking of the chromatin network by structural-maintenance-of-chromosome (SMC) proteins over (i) the physical properties of the chromatin network and (ii) condensate formation of embedded molecular species. We find that the density and lifetime of transient SMC crosslinks regulate structural relaxation modes and tune the sol-vs-gel state of the chromatin network, which imparts control over the kinetic pathway to condensate formation.

Misregulation of cell cycle-dependent methylation of budding yeast CENP-A contributes to chromosomal instability.

Centromere () identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved -specific histone H3 variant CENP-A (Cse4 in , CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation.

R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast.

R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric ( chromatin in wild-type budding yeast.

Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset.

Anaphase onset is an irreversible cell cycle transition that is triggered by the activation of the protease Separase. Separase cleaves the Mcd1 (also known as Scc1) subunit of Cohesin, a complex of proteins that physically links sister chromatids, triggering sister chromatid separation. Separase is regulated by the degradation of the anaphase inhibitor Securin which liberates Separase from inhibitory Securin/Separase complexes.

Centromeric heterochromatin: the primordial segregation machine.

Centromeres are specialized domains of heterochromatin that provide the foundation for the kinetochore. Centromeric heterochromatin is characterized by specific histone modifications, a centromere-specific histone H3 variant (CENP-A), and the enrichment of cohesin, condensin, and topoisomerase II. Centromere DNA varies orders of magnitude in size from 125 bp (budding yeast) to several megabases (human).

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis.

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles.

Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome.

Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome.

Mechanisms of force generation by end-on kinetochore-microtubule attachments.

Generation of motile force is one of the main functions of the eukaryotic kinetochore during cell division. In recent years, the KMN network of proteins (Ndc80 complex, Mis12 complex, and KNL-1 complex) has emerged as a highly conserved core microtubule-binding complex at the kinetochore. It plays a major role in coupling force generation to microtubule plus-end polymerization and depolymerization.

Design features of a mitotic spindle: balancing tension and compression at a single microtubule kinetochore interface in budding yeast.

Accurate segregation of duplicated chromosomes ensures that daughter cells get one and only one copy of each chromosome. Errors in chromosome segregation result in aneuploidy and have severe consequences on human health. Incorrect chromosome number and chromosomal instability are hallmarks of tumor cells.

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.

Pericentric chromatin is organized into an intramolecular loop in mitosis.

Background: Cohesin proteins link sister chromatids and provide the basis for tension between bioriented sister chomatids in mitosis. Cohesin is concentrated at the centromere region of the chromosome despite the fact that sister centromeres can be separated by 800 nm in vivo. The function of cohesin at sites of separated DNA is unknown.

FluoroSim: A Visual Problem-Solving Environment for Fluorescence Microscopy.

Fluorescence microscopy provides a powerful method for localization of structures in biological specimens. However, aspects of the image formation process such as noise and blur from the microscope's point-spread function combine to produce an unintuitive image transformation on the true structure of the fluorescing molecules in the specimen, hindering qualitative and quantitative analysis of even simple structures in unprocessed images. We introduce FluoroSim, an interactive fluorescence microscope simulator that can be used to train scientists who use fluorescence microscopy to understand the artifacts that arise from the image formation process, to determine the appropriateness of fluorescence microscopy as an imaging modality in an experiment, and to test and refine hypotheses of model specimens by comparing the output of the simulator to experimental data.

Counting kinetochore protein numbers in budding yeast using genetically encoded fluorescent proteins.

Genetically encoded fluorescent proteins are an essential tool in cell biology, widely used for investigating cellular processes with molecular specificity. Direct uses of fluorescent proteins include studies of the in vivo cellular localization and dynamics of a protein, as well as measurement of its in vivo concentration. In this chapter, we focus on the use of genetically encoded fluorescent protein as an accurate reporter of in vivo protein numbers.

Molecular architecture of a kinetochore-microtubule attachment site.

Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes. Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function.

The kinetochore protein Ndc10p is required for spindle stability and cytokinesis in yeast.

The budding yeast kinetochore is comprised of >60 proteins and associates with 120 bp of centromeric (CEN) DNA. Kinetochore proteins are highly dynamic and exhibit programmed cell cycle changes in localization. The CEN-specific histone, Cse4p, is one of a few stable kinetochore components and remains associated with CEN DNA throughout mitosis.

Mechanisms of microtubule-based kinetochore positioning in the yeast metaphase spindle.

It has been hypothesized that spatial gradients in kMT dynamic instability facilitate mitotic spindle formation and chromosome movement. To test this hypothesis requires the analysis of kMT dynamics, which have not been resolved at the single kMT level in living cells. The budding yeast spindle offers an attractive system in which to study kMT dynamics because, in contrast to animal cells, there is only one kMT per kinetochore.

Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae.

Dicentric chromosomes undergo a breakage-fusion-bridge cycle as a consequence of having two centromeres on the same chromatid attach to opposite spindle poles in mitosis. Suppression of dicentric chromosome breakage reflects loss of kinetochore function at the kinetochore-microtubule or the kinetochore-DNA interface. Using a conditionally functional dicentric chromosome in vivo, we demonstrate that kinetochore mutants exhibit quantitative differences in their degree of chromosome breakage.

beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast.

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules.