Laser ablation experiments challenge century-old belief
Comparative fluorescence and electron microscopy suggest new role for cellular material.
David L. Shenkenberg
The centriole is an unusual organelle that does not have an outer membrane and that exists freely in the cytoplasm. Its outer wall is a trademark cylindrical bundle of nine microtubules that are easily noticeable with an electron microscope. Its symmetrical arrangement has only contributed to the structure’s mystique.
“The centriole has been called ‘a central enigma of cell biology,’ ” said cell biologist Alexey Khodjakov, referring to the title of a book by D.N. Wheatley. “The centriole is involved in cell division and has other functions. We don’t really know what those functions are.”
Researchers investigated the mechanism of daughter centriole formation using laser ablation and comparative light and electron microscopy. This image shows a cell beginning mitosis. The tiny magenta spots on either side of the blue chromosomes are the centrosomes, each containing one mother and one daughter centriole.
At the Wadsworth Center in Albany, N.Y., a state Department of Health laboratory, Khodjakov and colleagues ablated centrioles and observed the results with correlative fluorescence and electron microscopy. Their findings conflict with the prevailing belief that the existing “mother” centriole governs the formation of two new “daughter” centrioles that eventually end up in separate cells as a result of cell division. The team’s work suggests a previously unrecognized role for the pericentriolar material, a poorly defined mass of numerous proteins surrounding the centriole.
Although the prevailing belief is based on a century of research, this past research involved mostly passive observations using light and electron microscopy, whereas the scientists in Albany modify the cell and observe what happens.
“Unless you modify it, how else are you going to see how the system reacts?” Khodjakov said. The procedure cleanly removes the centriole but leaves the cells otherwise intact, alive and healthy.
The researchers performed laser ablation with 8-ns pulses at a 20-Hz repetition rate at 532 nm using an Nd:YAG laser from Thales Laser of Orsay, France, that was Q-switched, a common procedure that enables the production of light pulses with the peak power necessary for ablation — 2.5 μJ at the laser and roughly a few hundred nanojoules at the specimen.
The researchers delivered the laser beam through the 100×, 1.4-NA lens of a Nikon confocal microscope. “You need a high-NA lens because you want to focus the beam on a tiny little spot,” Khodjakov said. The researchers aligned each centriole with the laser beam by moving the microscope stage, while the beam remained fixed in position. About three to five laser pulses destroyed a centriole. The researchers marked the cells with the ablated centrioles by scratching the glass slides with diamond.
This is a model of the centrosome in a Chinese hamster ovary cell arrested in the phase of the cell cycle in which daughter centrioles normally form. Centrin-GFP (green) is on the distal end of each daughter centriole, SAS-6 (red) is on the proximal end of each daughter centriole, and gamma-tubulin (blue) is in the pericentriolar material.
An hour later, they imaged the cells using the same Nikon microscope with a Photometrics back-illuminated electron-multiplying CCD camera. An integrated Yokogawa spinning disk, fast mechanical shutters from Vincent Associates and acousto-optical tunable filters from Solamere Technology Group helped keep the cells alive by reducing unnecessary exposure to light. “Cells without [centrioles] are more sensitive to light,” Khodjakov said.
Fluorescence microscopy enabled the researchers to detect GFP-labeled centrioles in living cells. On the other hand, electron microscopy typically is incompatible with living cells, but its much higher resolution enabled them to inspect the morphology of the centriole and pericentriolar material. They performed serial-section transmission electron microscopy at 100 kV with a Zeiss microscope.
As reported in the March 2008 issue of Nature Cell Biology, the investigators ablated centrioles in HeLa cervical cancer cells and Chinese hamster ovary cells, both overexpressing centrin-GFP and both arrested in the phase of the cell cycle in which daughter centrioles normally form. Centrin is present in the centriole and in the pericentriolar material.
When the scientists ablated a daughter centriole in HeLa cells, the mother developed a new daughter that appeared normal under the light and electron microscopes. When they destroyed the mother centriole, the daughter centriole did not form a new mother centriole. Several of the orphaned daughter centrioles deteriorated and were not surrounded by pericentriolar material at the one-hour observation period, suggesting that these daughter centrioles disintegrated because of lost association with the pericentriolar material.
The investigators found that Chinese hamster ovary cells could form a new daughter centriole to replace a destroyed one, just as HeLa cells did. However, in contrast to those in HeLa cells, the two daughter centrioles in Chinese hamster ovary cells did not deteriorate but moved in a coordinated fashion and even created a new mother centriole.
The researchers also overexpressed pericentrin in Chinese hamster ovary cells overexpressing centrin-GFP. The overexpression of pericentrin resulted in large clouds of pericentriolar material and in an abnormally high number of centrioles in the pericentriolar material, which suggested a link between the amount of pericentriolar material and centriole production.
The researchers concluded that the pericentriolar material actively participates in the assembly of the daughter centriole; on the other hand, the mother centriole serves primarily to shepherd the pericentriolar material.
As an extension of this work, they plan to perform laser ablations in cells that lack certain proteins involved in the process of creating daughter centrioles.
MORE FROM PHOTONICS MEDIA