motorisation of centrosome separation
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Motorisation of Centrosome Separation. Steve Norton. The Centrosome. Microtubule organising centre during mitosis One positioned either side of the mitotic spindle Microtubules used to pull kinetochores apart – divide DNA between daughter cells. The Centrosome Cycle. The Question…. - PowerPoint PPT PresentationTRANSCRIPT
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Motorisation of Centrosome Separation
Steve Norton
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The Centrosome
• Microtubule organising centre during mitosis
• One positioned either side of the mitotic spindle
• Microtubules used to pull kinetochores apart – divide DNA between daughter cells
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The Centrosome Cycle
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The Question…
• How do centrosomes get from their position together, next to the nucleus, to being spread 10 μm apart around the spindle?
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Pathways to Separation
• Centrosomes can separate at different times - before, or after nuclear envelope breakdown - the prophase and prometaphase pathways
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Aims of the Project
• To observe centrosome movements in live cells during prophase
• To observe patterns in centrosome separation behaviour
• To make preliminary deductions about motors involved in the process
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Methods
• HeLa cells cultured
• Expressing Centrin fused to GFP and α-tubulin fused to mCherry
• Live-cell imaging: capture stacks through single cells at approx. 15 second intervals
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Choosing the Cells
• Centrosomes and early asters should be visible…
• But the nucleus should still be intact
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Movies Generated
• Able to observe whole separation process in some cells.
• Saw cells rounding up, centrosomes moving around nucleus, NEBD, centrosomes positioning themselves at spindle poles.
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At surface of LabTek
Away from surface
Time
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Separation over Time• Over the course of the movie, each cell’s
centrosome separation was measured
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Separation is Discontinuous• Separation happens in phases. • Not the same in every cell, but all cells show some phases of
growth in separation, reduction of separation and phases of constant distance.
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• Cells 3 and 9 were prometaphase pathway (centrosomes keep growing apart long after NEBD).
• All other cells prophase pathway.
• Prophase pathway cells converged on separation distance of 7.41 μm (s.d. = 0.18 μm) 15 time steps after NEBD initiation. This isn’t the spindle length!
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• After converging to the 7.41 μm average separation, the centrosomes continue to move together and apart until they end of imaging. Average spindle length was 9.106 μm
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Automated Tracking• Applied Ed’s work on kinetochore tracking to centrosomes,
with some success:
• However, tracks were incomplete and not as good in all dimensions
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Validation of Computer Measurements
• Compared Cell 3 and Cell 6 centrosome distance measurements done manually, to those calculated by MATLAB:
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Conclusions
• Centrosomes separate discontinuously, not smoothly.
• After NEBD they tend to a separation distance of 7.41 μm, though why is unclear.
• After this convergence the centrosomes continue to move together and apart.
• Computational tracking of centrosomes should be the best way to further these experiments in the future, perhaps needing better quality movies to work well.