CAPÍTULO 5: Modelización del motor Stirling
5.3. Desarrollo de un prototipo
5.3.1. Máquina de dos bloques
Immunoblotting was performed as described 37. Immunofluorescence was essentially performed as described 11. Images were acquired using a Zeiss Meta confocal microscope with a 63X 1.3 N.A. objective. For Aurora-B/CREST ratio calculations, images with multiple Z-planes were taken. For each plane in a Z-stack, CREST staining was used to define kinetochore/centro- mere regions and CREST intensity was used as constant reference for the kinetochore/centro- mere signal. The average CREST staining was similar on aligned and unaligned chromosomes. Background fluorescence from regions within the cell devoid of kinetochores was subtracted. For inter-kinetochore distance measurements, cells were stained with CENP-A, and images with multiple z-planes were captured at intervals of 0.25 μm. The center-to-center distance between paired CENP-A signals that fell within the same focal planes were obtained by using Zeiss LSM Meta 510 software. On average 75 CENP-A pairs in a total of 10 cells were acquired per condition.
Acknowledgements
We thank C. Cruijsen, G. Kops, A. Lindqvist and B. van de Weerdt for helpful discus- sions and comments on the manuscript. We are grateful to S. Taylor, N. Galjart, S. Wheatley and H. Masumoto for sharing reagents. We acknowl- edge AstraZeneca for supplying ZM447439. This work is supported by Dutch Cancer Society (NKI 2002-2764) and the Netherlands Organization for Scientific Research (Vidi 917.66.332).
Chapter 5
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0 Chapter 5
Supplementary Figure 1: (a) U2OS
cells grown on coverslips were transfected with indicated plasmids in combination with H2B-GFP. Cells were synchronised using thymidine. Fourteen hours after thy- midine release cells were fixed and stained for Aurora-B and vsv. DNA was stained with DAPI. Arrowheads indicate residual localization of Aurora-B to the central spindle during anaphase. (b) U2OS cells grown on coverslips and were synchro- nised using thymidine. Cell were released from thymidine and treated with 25 μg/l Nocodazole to induce minor misalign- ments. Fourteen hours after thymidine release cells were fixed and stained for CENP-A. Non-aligned chromosomes (see arrowheads) were defined as chromosomes clearly outside a metaphase plate. Aligned chromosomes were defined as chromosomes within a metaphase plate. Inter-kinetochore distances (± st. dev) were determined. See Material and Methods for more detail on how inter-kinetochore distances of kinetochore pairs were determined. (c) U2OS cells were grown on coverslips and were synchronised using thymidine. Cells were released from thymidine and treated with 25 μg/l Nocodazole to induce minor misalignments. Fourteen hours after thymidine release cells were fixed and stained for Aurora-B (a-rabbit) and CENP-A (upper panel), Borealin and CENP-A (middle panel), and Survivin and CENP-A (lower panel). aligned non- aligned 0.4 0.8 1.2 n=15 n=19 0 inter kinet ochore dist ance ( mm)
b
c
Aurora-B(rabbit) CENP-A Mergew/DNA
Borealin CENP-A Mergew/DNA
Survivin CENP-A Merge w/DNA Aurora-B CENP-A Borealin CENP-A Survivin CENP-A prometaphase anaphase anaphase prometaphase merge w/DNA Aurora-B vsv vsv- CENP B vsv-CENPB INCENP
a
1
Aurora-B restriction upon bi-orientation
20 10 0 0.2 0.5 2.0 µg a c empty vector vsv-CENP-B vsv-CENP-B INCENP mitotic cells (%) 20 10 0 mitotic cells (%) mock siRN A INCENP siRN A mock siRN A INCENP siRN A mock siRN A INCENP siRN A vsv-CENP-B INCENP vsv-CENP-B empty vector b 40 10 0 30 20 -Taxol +Taxol mock siRNA empty vector empty vector INCENP siRNA vsv-CENP-B INCENP mitotic cells (%) wt-INCENP a 30 20 10 0 ZM447439 DMSO vsv-CENP-B INCENP vsv-CENP-B mitotic cells (%)
Supplementary Figure 3: (a) U2OS cells were transfected with the indicated plasmid
and GFP-Spectrin. Cells were synchronised using thymidine. DMSO or ZM447439 (2 μM) was added upon release from the thymidine block and eighteen hours after thymidine re- lease cells were harvested and mitotic percentages were determined by MPM-2 staining and FACS analysis.
Supplementary Figure 2:(a)-(c) U2OS cells were
transfected with the indicated plasmids and GFP-Spec- trin. Cells were synchronised using thymidine. Cells were released from the thymidine block and eighteen hours after thymidine release cells were harvested and mitotic percentages were determined by MPM-2 stain- ing and FACS analysis. (a) Taxol was added to half of the samples upon release from the thymidine block.
Chapter 6
4 Chapter 6
After the initial description of the strik- ing and dynamic localization pattern of the chro- mosomal passenger proteins 1, the function of this protein complex during mitosis has been intimately linked with its localization at specific structures throughout the mitotic cell. Early in mitosis, the Chromosomal Passenger Complex (CPC) local- ises to the chromosome arms, where it controls mitotic chromosome structure and organization. Concentration at the centromeres during promet- aphase reflects its essential function in between the paired kinetochores to control and regulate proper microtubule-kinetochore interactions. Relocalisa- tion of the CPC to the central spindle and the equatorial cell cortex during anaphase is essential for the proper positioning and function of the con- tractile ring that ensures cytoplasmic division. Evi- dently, proper localisation of the CPC at the right time is essential for a faithful execution of mitosis. However, at the onset of the studies described in this thesis, the underlying mechanisms that control CPC localisation were poorly understood.