Chromosome Spindle Attachments in Dividing Cells
Using spinning disk confocal microscopy
Mitotic cell division relies upon the ability of cells to properly distribute sister chromatids
into forming cells. To reliably perform the function cells use internal editing mechanisms to
correct inaccurate chromosome / spindle fiber attachments. Paired kinetochores are usually
aligned to properly attach spindle fibers and segregate chromatin, however, erroneous and
misaligned kinetochores do result. Cells contain specialized editing mechanisms to prevent
and correct these misaligned kinetochore pairs, although the exact mechanism is not well
understood 1,2 . It is postulated by Tatiana Moutinho-Santos at the Universidade do Porto,
Portugal's Instituto de Biologia Molecular e Celular (IBMC) that the presence of POLO
kinase is necessary to promote chromosome bi-orientation (termed amphitelic arrangement)
and thereby preserve the proper kinetochore alignment.
To better understand its role in regulating kinetochore development,
Dr. Moutinho dos Santos studied depleted Drosophila POLO kinase
within live cells to substantiate POLO's involvement in the editing
functions required to maintain amphitelic kinetochore arrangement.
In this case, syntelic arrangement (sister kinetochores attached to
microtubules emanating from the same spindle) was studied. Timelapse
analysis of mitosis was performed on Drosophila S2 cells stably
expressing CID-mCherry for visualisation of the centromeres, and
GFP-a-tubulin for the microtubules. Visualization of kinetochores
is demanding due to their small size, low fluorescent signal and
brief appearance during cell division. Confocal fluorescence
microscopy is often used to image kinetochores, however, even in
this environment, visualization remains difficult. The kinetochores
are very small (300 nm)3, close to the lateral resolving capabilities
of the light microscope, and exceed the microscope's axial resolving
ability. Moreover, imaging fluorescent markers within living cells
poses potential phototoxic and photobleaching effects. Intense laser
illumination can create phototoxicity issues, especially damaging for
living, dividing cells.
Andor's Revolution XD spinning disk confocal microscopy is
employed to overcome Dr. Moutinho dos Santo's unique challenge
associated with the observation of kinetochores. In this case, four
separate factors must be simultaneously addressed to adequately
visualize the dynamic cellular processes:
- Speed of acquisition
- Spatial and temporal resolving capability
- The ability to detect very low fluorescence intensity levels
- Management of available fluorescence signal
These themes of spatial, temporal and intensity resolution recur
frequently with fluorescence microscopy and are often at odds
with experiments involving the observation of cell viability. For
example, long camera exposures and extended periods of high
intensity illumination generate the phototoxic effects that damage or
destroy living cells. Simultaneously, lightly labeled microstructures
demand longer exposure to visualize but can be adversely affected
by photobleaching. Compounding the issue is the need to combine
traditional three-dimensional data with that of time. Finally, it
remains necessary to discern signal from background noise and outof-
Speed of acquisition is of particular importance to Dr. Mountinho dos
Santos. Control S2 cells exhibit a division cycle of approximately
30 minutes. However, the POLO depleted S2 cells utilized in her
experiments showed an arrested period in excess of eight hours.
Recording the POLO depleted cell division requires collection of
between 7,200 and 12,000 image sets (two fluorochromes imaged
every thirty seconds for one to five hours, acquired at 0.5 micron
axial steps at up to 20 steps for axial kinetochore resolution).
Conventional fluorescence microscopy and laser illumination will
permit neither the gentle illumination requirements necessary nor the
speed of acquisition to complete this time-sensitive task. For example
laser rastering in traditional confocal microscopy requires longer
time periods time to collect signal from the sample. Spinning disk
confocal systems are used to overcome these traditional barriers and
to reveal new insights into molecular kinetochore editing techniques.
Compared to conventional widefield fluorescence systems, the spatial
resolution of a spinning disk confocal is superior in both lateral (x
and y) and axial (z) dimensions. Through the constant scanning of the
pinhole array, samples can be viewed in real-time at high contrast,
providing clear images at the diffraction limits of the microscope's
optics. This enables the 3D visualization and understanding of the
dynamic kinetochore behavior in relation to the microtubules of the
spindle. Kinetochore and centromere lateral resolution of 300nm and
spindle fiber resolution of 300-500 nm were reported and are detailed
in Figure A.
Visualizing the fluorescently labeled kinetochores associated with
each centromere places additional demands on the acquisition
system. The centromeres and kinetochores under study are primarily
visible only during cell division interphase. As the cells pass through
prophase, the centromeres have already resolved themselves into
Drosophila’s typical twelve chromosome pairs. Managing the
available light budget during lengthy acquisition necessitates gentle
illumination and high resolution capable by spinning disk techniques.
The apertures within the spinning disk unit provide these benefits.
Because light excites fluorophores only when an aperture is present,
phototoxic effects are minimized. While counterintuitive, a reduced
light budget does not indicate faint, hard-to-detect objects. Intensity
resolution is increased through the spinning disk's exclusion of out of focus signal and subsequent signal amplification by an EMCCD
camera architecture. This creates the new possibility to generate
higher contrast images revealing the development and alignment of
punctate objects such as kinetochores.
The use of spinning disk technology in Dr. Mountinho dos
Santos live cell application led to an important observation. In
the absence of the POLO kinase, Drosophila cells were shown to
lack the corrective mechanisms necessary to maintain amphitelic
chromosome arrangement. The presence of POLO was shown to
provide the right environment for correct centromere architecture,
while simultaneously ensuring proper chromosome bi-orientation.
Cultured Drosophila cells undergoing mitosis in the absence of
POLO kinase chromosomes attach to spindle fibers with syntelic
orientation, i.e., with sister kinetochores attached to microtubules that
come from a single spindle pole.
This conclusion was drawn by careful analysis of 4D fluorescence
microscopy in cells expressing fluorescently labeled centromere/
kinetochore marker and microtubules. (Figure 1).