Beth M. Weiner

Chromosome pairing via multiple interstitial interactions before and during meiosis in yeast

Fluorescence in situ hybridization analysis reveals that homologous chromosomes are paired in yeast cells about to enter meiosis. Pairing involves multiple interstitial interactions, one per approximately 65 kb. These observations exclude several classes of models for somatic/premeiotic pairing. The number of t = 0 pairing interactions is about the same as the number of subsequent meiotic recombination events. As cells enter meiosis, pairing disappears concomitant with DNA replication and then reappears, independent of synaptonemal complex. Mutant phenotypes suggest that formation of an individual meiotic pairing connection does not require a meiosis-specific double-stranded break (DSB). Mutants defective in recombination before or after DSBs exhibit pairing defects. These and other observations can be united by a model in which premeiotic pairing and early meiotic pairing occur by closely related paranemic DNA-DNA interactions between intact duplexes, with early meiotic interactions subsequently converted directly to plectonemic recombination intermediates via DSBs.

Four-Dimensional Imaging of E. coli Nucleoid Organization and Dynamics in Living Cells

Visualization of living E. coli nucleoids, defined by HupA-mCherry, reveals a discrete, dynamic helical ellipsoid. Three basic features emerge. (1) Nucleoid density coalesces into longitudinal bundles, giving a stiff, low-DNA-density ellipsoid. (2) This ellipsoid is radially confined within the cell cylinder. Radial confinement gives helical shape and directs global nucleoid dynamics, including sister segregation. (3) Longitudinal density waves flux back and forth along the nucleoid, with 5%-10% of density shifting within 5 s, enhancing internal nucleoid mobility. Furthermore, sisters separate end-to-end in sequential discontinuous pulses, each elongating the nucleoid by 5%-15%. Pulses occur at 20 min intervals, at defined cell-cycle times. This progression includes sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intranucleoid mechanical stress. These effects could comprise a chromosome-based cell-cycle engine. Overall, the presented results suggest a general conceptual framework for bacterial nucleoid morphogenesis and dynamics.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis

PR65 is the two-layered (α-α solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix–helix separation along one layer (“fracturing”) leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme’s substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A’s response to spindle tension) or arise spontaneously (e.g., in PP2A’s interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer’s-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance.

Csm4, in Collaboration with Ndj1, Mediates Telomere-Led Chromosome Dynamics and Recombination during Yeast Meiosis

Chromosome movements are a general feature of mid-prophase of meiosis. In budding yeast, meiotic chromosomes exhibit dynamic movements, led by nuclear envelope (NE)-associated telomeres, throughout the zygotene and pachytene stages. Zygotene motion underlies the global tendency for colocalization of NE-associated chromosome ends in a “bouquet.” In this study, we identify Csm4 as a new molecular participant in these processes and show that, unlike the two previously identified components, Ndj1 and Mps3, Csm4 is not required for meiosis-specific telomere/NE association. Instead, it acts to couple telomere/NE ensembles to a force generation mechanism. Mutants lacking Csm4 and/or Ndj1 display the following closely related phenotypes: (i) elevated crossover (CO) frequencies and decreased CO interference without abrogation of normal pathways; (ii) delayed progression of recombination, and recombination-coupled chromosome morphogenesis, with resulting delays in the MI division; and (iii) nondisjunction of homologs at the MI division for some reason other than absence of (the obligatory) CO(s). The recombination effects are discussed in the context of a model where the underlying defect is chromosome movement, the absence of which results in persistence of inappropriate chromosome relationships that, in turn, results in the observed mutant phenotypes.

Tsr–GFP accumulates linearly with time at cell poles, and can be used to differentiate ‘old’ versus ‘new’ poles, in Escherichia coli

In Escherichia coli, the chemotaxis receptor protein Tsr localizes abundantly to cell poles. The current study, utilizing a Tsr–GFP fusion protein and time‐lapse fluorescence microscopy of individual cell lineages, demonstrates that Tsr accumulates approximately linearly with time at the cell poles and that, in consequence, more Tsr is present at the old pole of each cell than at its newborn pole. The rate of pole‐localized Tsr accumulation is large enough that old and new poles can always be reliably distinguished, even for cells whose old poles have had only one generation to accumulate signal. Correspondingly, Tsr–GFP can be reliably used to assign new and old poles to any cell without use of information regarding pole heritage, thus providing a useful tool to analyse cells whose prior history is not available. The absolute level of Tsr–GFP at the old pole of a cell also provides a rough estimate of pole (and thus cell) age.

Real-Time Imaging of Meiotic Chromosomes in Saccharomyces cerevisiae

Important information on cellular physiology can be obtained by directly observing living cells. The nucleus, and the chromatin within, is of particular interest to many researchers. Monitoring the behavior of specific DNA loci in the living cell is now commonly achieved through the insertion of binding sites for fluorescently tagged proteins at the sequence of interest (e.g. Ref 1). However, visualizing the behavior of full length chromosomes can only be achieved when they constitute discrete, relatively well individualized units. During meiotic mid-prophase, chromosomes of budding yeast are well-organized structures that present such characteristics, making them remarkably suited for visualization. Here we describe the optimized protocols and techniques that allow monitoring of chromosome behavior during meiotic prophase in budding yeast.

Assaying Chromosome Pairing by FISH Analysis of Spread Saccharomyces cerevisiae Nuclei

Fluorescent in situ hybridization (FISH) provides a powerful tool to study the localization of DNA sequences in relationship to one another. FISH has the advantage over other methods, notably use of GFP-tagged repressor/operator arrays, that an almost unlimited number of probes can be utilized without having to make new strains for each new locus one wants to study. Also, the number of sites that can be visualized at the same time is limited only by the number of fluorophores that are available and can be distinguished by the available microscope. Described here is a method for FISH analysis and its application to analysis of chromosome pairing during meiosis in S. cerevisiae.