SUMMARY Mammalian mitotic chromosome morphogenesis was analyzed by 4D live cell and snapshot deconvolution fluorescence imaging. Prophase chromosomes, whose organization was previously unknown, are revealed to comprise co-oriented sister linear loop arrays displayed along a single, peripheral, regularly-kinked Topoisomerase II/cohesin/condensin II axis. Thereafter, rather than smooth, progressive compaction as generally envisioned, progression to metaphase is a discontinuous process involving chromosome expansion as well as compaction. At Late Prophase, dependent on Topoisomerase II and with concomitant cohesin release, chromosomes expand, axes split and straighten, and chromatin loops transit to a radial disposition around now-central axes. Finally, chromosomes globally compact, giving the metaphase state. These patterns are consistent with the hypothesis that the molecular events of chromosome morphogenesis are governed by accumulation and release of chromosome stress, created by chromatin compaction and expansion. Chromosome state could evolve analogously throughout the cell cycle.

SUMMARYVipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.

Vipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.

Three-dimensional organization of the genome is important for regulation of gene expression and maintenance of genomic stability. It also defines, and is defined by, contacts between different chromosomal loci. Interactions between loci positioned on different chromosomes, i.e. “trans” interactions are one type of such contacts. Here, we describe a case of inducible trans interaction in chromosomes of the budding yeast S. cerevisiae. Special DNA sequences, inserted in two ectopic chromosomal loci positioned in trans, pair with one another in an inducible manner. The spatial proximity diagnostic of pairing is observable by both chromosome capture analysis (3C) and epifluorescence microscopy in whole cells. Protein synthesis de novo appears to be required for this process. The three-dimensional organization of the yeast nucleus imposes a constraint on such pairing, presumably by dictating the probability with which the two sequences collide with one another.

Chromosomally-integrated arrays of lacO and tetO operator sites visualized by LacI and TetR repressor proteins fused with GFP (or other fluorescent proteins) are widely used to monitor the behavior of chromosomal loci in various systems. Yet, insertion of such arrays and expression of the corresponding proteins is known to perturb genomic architecture. In several cases, juxtaposition of such arrays located on different chromosomes has been inferred to reflect pairing of the corresponding loci. Here, we report that a version of TetR-GFP mutated to disrupt GFP dimerization (TetR-A206KGFP or “TetR-kGFP”), abolishes pairing of tetO-arrays in vivo and brings spatial proximity of chromosomal loci marked with those arrays back to the wild type level. These data argue that pairing of arrays is caused by GFP dimerization and thus presents an example of protein-assisted interaction in chromosomes. Arrays marked with another protein, TetR-tdTomato, which has a propensity to form intra-molecular dimers instead of inter-molecular dimers, also display reduced level of pairing, supporting this idea. TetR-kGFP provides an improved system for studying chromosomal loci with a low pairing background.