Biology Reference
In-Depth Information
Tips
- Cell density should be kept below 50% of confluence to prevent massive
cell detachment during the incubations, which is greatly enhanced by the
incubation with tubulin.
- Gelatin coating not only helps keeping cell attachment to coverslips but
also prevents excessive tubulin adsorption to the substratum and keeps
tubulin staining background to reasonable levels.
- Microtubule number and length are limited by the concentration of
tubulin added during the elongation phase. Tubulin concentration should
be kept lower than 0.5 g/L to prevent excessive protein adsorption on
cellular ghosts. To visualize internal GTP islands, growing microtubules
to steady state is sufficient. To visualize GTP caps, labeling with MB11
should be performed while microtubules are still in a growth phase. This
is usually obtained within the first 10-15 min of incubation, yielding
an important amount of short microtubules. Note that some microtubules
may also detach (spontaneously or mechanically during incubations and
washes) and be observed in various cellular or even extracellular
locations (
Fig. 10.2
).
FIGURE 10.2
GTP-tubulin labeling in microtubules nucleated from cellular centrosomes or elongated
from cellular microtubules. (A) After complete microtubule disassembly with nocodazole and
cold, RPE-1 cells were permeabilized and microtubules were grown from cellular
centrosomes with GTP. Note the quite high tubulin staining in cell ghosts and the presence of
detached microtubules (inset). Images were acquired on a Leica DMLB microscope. (B)
HeLa cells were permeabilized and treated with ATP prior to extracellular microtubule
elongation using purified tubulin and GTP. Images are confocal planes acquired using a Zeiss
LSM510 microscope. Note that the cell from the right panel detached after fixation, but
extracellularly elongated microtubules remained visible. Microtubules were stained for
GTP-tubulin using MB11 (red) and total tubulin (green) using anti alpha-tubulin
antibody (clone DM1A). GTP caps and internal GTP islands are indicated with arrows and
arrowheads, respectively. Scale bar
10 mm. Signals have been artificially increased for
better visualization in the black and white version of the figure.
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