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.

Sister cohesion and structural axis components mediate homolog bias of meiotic recombination.

Meiotic double-strand break (DSB)-initiated recombination must occur between homologous maternal and paternal chromosomes ("homolog bias"), even though sister chromatids are present. Through physical recombination analyses, we show that sister cohesion, normally mediated by meiotic cohesin Rec8, promotes "sister bias"; that meiosis-specific axis components Red1/Mek1kinase counteract this effect, thereby satisfying an essential precondition for homolog bias; and that other components, probably recombinosome-related, directly ensure homolog partner selection. Later, Rec8 acts positively to ensure maintenance of bias. These complexities mirror opposing dictates for global sister cohesion versus local separation and differentiation of sisters at recombination sites. Our findings support DSB formation within axis-tethered recombinosomes containing both sisters and ensuing programmed sequential release of "first" and "second" DSB ends. First-end release would create a homology-searching "tentacle." Rec8 and Red1/Mek1 also independently license recombinational progression and abundantly localize to different domains. These domains could comprise complementary environments that integrate inputs from DSB repair and mitotic chromosome morphogenesis into the complete meiotic program.

Rapid Homolog Juxtaposition During Meiotic Chromosome Pairing.

A central basic feature of meiosis is pairing of homologous maternal and paternal chromosomes ("homologs") intimately along their lengths. Recognition between homologs and their juxtaposition in space are mediated by axis-associated DNA recombination complexes. Additional effects ensure that pairing occurs without ultimately giving entanglements among unrelated chromosomes. Here we examine the process of homolog juxtaposition in real time by 4D fluorescence imaging of tagged chromosomal loci at high spatio-temporal resolution in budding yeast. We discover that corresponding loci start coming together from a quite large distance (∼1.8 µm) and progress to completion of pairing in a very short time, usually less than six minutes (thus, "rapid homolog juxtaposition" or "RHJ"). Juxtaposition initiates by motion-mediated extension of a nascent interhomolog DNA linkage, raising the possibility of a tension-mediated trigger. In a first transition, homolog loci move rapidly together (in ∼30 sec, at speeds of up to ∼60 nm/sec) into a discrete intermediate state corresponding to canonical ∼400 nm axis distance coalignment. Then, after a short pause, crossover/noncrossover differentiation (crossover interference) mediates a second short, rapid transition that brings homologs even closer together. If synaptonemal complex (SC) component Zip1 is present, this transition concomitantly gives final close pairing by axis juxtaposition at ∼100 nm, the "SC distance". We also find that: (i) RHJ occurs after chromosomes acquire their prophase chromosome organization; (ii) is nearly synchronously over thirds (or more) of chromosome lengths; but (iii) is asynchronous throughout the genome. Furthermore, cytoskeleton-mediated movement is important for the timing and distance of RHJ onset and also for ensuring normal progression. Potential implications for local and global aspects of pairing are discussed.

Crossover Interference Mediates Multiscale Patterning Along Meiotic Chromosomes.

The classical phenomenon of crossover interference is a one-dimensional spatial patterning process that produces evenly spaced crossovers during meiosis. Quantitative analysis of diagnostic molecules along budding yeast chromosomes reveals that this process also sets up a second, interdigitated pattern of related but longer periodicity, in a "two-tiered" patterning process. The second tier corresponds to a previously mysterious minority set of crossovers. Thus, in toto, the two tiers account for all detected crossover events. Both tiers of patterning set up spatially clustered assemblies of three types of molecules ("triads") representing the three major components of meiotic chromosomes (crossover recombination complexes and chromosome axis and synaptonemal complex components), and give focal and domainal signals, respectively. Roles are suggested. All observed effects are economically and synthetically explained if crossover patterning is mediated by mechanical forces along prophase chromosomes. Intensity levels of domainal triad components are further modulated, dynamically, by the conserved protein remodeler Pch2/TRIP13.

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

PR65 is the two-layered (alpha-alpha 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.

High Temporal Resolution 3D Live-Cell Imaging of Budding Yeast Meiosis Defines Discontinuous Actin/Telomere-Mediated Chromosome Motion, Correlated Nuclear Envelope Deformation and Actin Filament Dynamics.

Chromosome movement is prominent at mid-meiotic prophase and is proposed to enhance the efficiency and/or stringency of homolog pairing and/or to help prevent or resolve topological entanglements. Here, we combine fluorescent repressor operator system (FROS) labeling with three-dimensional (3D) live-cell imaging at high spatio-temporal resolution to define the detailed kinetics of mid-meiotic prophase motion for a single telomere-proximal locus in budding yeast. Telomere motions can be grouped into three general categories: (i) pauses, in which the telomere "jiggles in place"; (ii) rapid, straight/curvilinear motion which reflects Myo2/actin-mediated transport of the monitored telomere; and (iii) slower directional motions, most of which likely reflect indirectly promoted motion of the monitored telomere in coordination with actin-mediated motion of an unmarked telomere. These and other findings highlight the importance of dynamic assembly/disassembly of telomere/LINC/actin ensembles and also suggest important roles for nuclear envelope deformations promoted by actin-mediated telomere/LINC movement. The presented low-SNR (signal-to-noise ratio) imaging methodology provides opportunities for future exploration of homolog pairing and related phenomena.

Tsr-GFP accumulates linearly with time at cell poles, and can be used to differentiate 'old' versus 'new' poles, in Escherichia coli.

SUMMARY: 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.

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.

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.