File:Meiotic-Chromosome-Pairing-Is-Promoted-by-Telomere-Led-Chromosome-Movements-Independent-of-Bouquet-pgen.1002730.s005.ogv

English: Time-lapse images of wild-type, mps3-dCC , mps3-dAR and ndj1 Δ at 4 hours. Shown are fields containing 20–25 cells/nuclei with each of the 4 genotypes, where the real time between movie frames is 1 second. Each nucleus contains a larger, fuzzier spot that marks the spindle pole-associated microtubules and 1 or 2 smaller, discrete spots that mark a pair of homologous telomeres (1 spot following pairing/synapsis that causes fusion of the 2 spots). Telomere movements tend to be more vigorous following pairing/synapsis, i. e. , in the 1-telomere-spot versus the 2-telomere-spots nuclei, and in wild-type cells as compared with mps3-dCC , mps3-dAR and, most noticeably, ndj1 Δ. Quantification generally is required to make clear distinctions between different movement types, mutants and time-points. Strains used: wild-type (MDY1560XMDY1567), mps3-dCC (MDY2580XMDY2759), mps3-dAR (MDY2523XMDY2756), ndj1 Δ (MDY2294XMDY1560). Method: Concatemers containing 256 copies of the lacI binding site from LacO were inserted adjacent to the telomeres of both chromosome 4 right arms and were decorated with lacI-GFP to produce discrete fluorescent spots marking the positions of the telomeres in living cells. Microtubules were labeled with Tub1-GFP which marks the position of the spindle pole as a fuzzy spot that generally is larger than the telomere-adjacent spots. Images were acquired every second for 1 minute using a Zeiss Axioplan 2ie fitted with a 100× 1.4NA objective, Roper CoolSnap camera and custom-written software to acquire “thru-focus” images which were deconvolved using a constrained iterative algorithm [46] . Movies were analyzed using custom-written software [27] . (MOV)