One Of The Most Disregarded Fact About DEF6

Stretched bivalents were defined as the chromosomes that reach >70% of their maximal interkinetochore distance. If a stretched bivalent maintained the angle with the estimated spindle axis within Epigenetics inhibitor when the diameter of the disassembling nucleolus becomes less than 5?��m. The other is time after metaphase entry, which is defined as the time when half of chromosomes established orientation along the spindle axis. All calculations were automated by an in-house developed Java (Sun Microsystems) program and R (http://www.r-project.org/). The 2D and the 3D plots were generated by the plotting software Gnuplot (http://www.gnuplot.info/) or Prism (GraphPad) and the ray-tracing software Pov-Ray (http://www.povray.org/), respectively, Selleckchem MK-4827 controlled by scripts generated from the Java program. Extended Experimental Procedures Culture of Oocytes and Microinjection Oocytes were collected from ?8-week-old FVB mice according to the guidelines of EMBL Laboratory Animal Resources and cultured as described (Schuh and Ellenberg, 2007). Microinjection of in?vitro-transcribed RNAs to the oocytes was performed (Jaffe and Terasaki, 2004) with 1pl EGFP-CENP-C, 0.2pl mCherry-H2B, 1pl 3mCherry-CENP-C, 2pl EGFP-MAP4, 1pl Mad2-EGFP of 1?��g/��l mRNAs. Time-lapse image acquisitions were performed using a customized Zeiss LSM510 Meta confocal microscope equipped with the 40x C-Apochromat 1.2NA water immersion objective lens (Carl Zeiss) with an in-house-developed 3D multilocation tracking macro (Rabut and Ellenberg, 2004). We recorded the subvolume centered around chromosomes or kinetochores by 3D time-lapse (4D) imaging for 3�C5 cells in parallel. For kinetochore tracking with EGFP-CENP-C, we imaged 17 z-confocal sections (every 1.5 or 2.0?��m) of 256?�� 256 or 512?�� 512 pixel xy images, in total covering at least 32.0?��m �� 32.0?��m �� 25.5?��m volume, at time interval of 90?s for 9?hr after induction of maturation, covering NEBD to anaphase onset of meiosis I. For 3mCherry-CENP-C, we imaged 17 z-confocal sections (every 2.0?��m) of 256?�� 256 pixel xy images, in total covering a 37.5?��m �� 37.5?��m �� 34.0?��m volume, at time interval of 90?s from 1?hr to 6?hr after NEBD, covering DEF6 phase 2, phase 3, and the first part of phase 4. For peak enhancement and background subtraction for kinetochore signals, we subtracted the kinetochore images processed by a ten-pixel Gaussian blur from the same images processed by a two-pixel Gaussian blur. We reversed the time sequence of the movie because it was more error-free to perform kinetochore tracking reversely from late metaphase, when homologous kinetochore pairs are obvious because of chromosome biorientation. These image processing tasks were automated by an in-house developed macro in ImageJ.