Cellular mitosis depends in part on small organelles that extend spindles to pull apart chromosome pairs. Before they can perform this and other essential tasks, these tiny cylindrical structures — known as centrioles in animals and spindle pole bodies (SPBs) in yeast — must themselves duplicate.
However, much about this nanoscale process has remained veiled by the limits of current microscopy. Optical approaches cannot resolve objects below certain wavelength limits, while non-optical approaches like electron microscopy (EM) can only study nonliving cells.
Now, a team of researchers from the Stowers Institute for Medical Research and the University of Colorado Boulder has devised a novel optical technique — a combination of structured illumination microscopy (SIM) and single-particle averaging (SPA) — to resolve individual components of SPB duplication in living yeast cells. In the process, they have uncovered surprising facts in what many once considered well-trodden ground. More than that, they have opened up new possibilities in the field of cellular imaging.
“The use of SIM to study SPB structure completely changes the types of questions we can ask and answer, since sample size is no longer limiting and it is likely that SIM will work in living cells,” says Sue Jaspersen, Ph.D., an associate investigator at the Institute who led the investigation.
The study will be published in the online journal eLife on September 15, 2015.
For years, EM, which uses a beam of electrons to achieve molecular and even atomic resolutions, has been the go-to technique for studying SPBs, which at less than 200 nanometers (nm) in size fall below the wavelength limit of what is observable using visible light. However, EM carries with it significant limits, including the fact that it does not work on living cells.
The research team turned to SIM as an optical alternative. SIM uses a laser-generated field of horizontal lines to project an interference pattern onto a sample. According to Jay Unruh, Ph.D., a Stowers research advisor and co-author, analyzing these patterns enables researchers to effectively double their resolution.
“Basically, finely structured objects interfere with the line pattern in a way that makes a new pattern, which is larger in size.”
For all of its advantages, SIM still involves sifting through a great deal of noise. To deal with this problem, and to better localize the subjects under study — which can assume various shapes and positions — the team turned to SPA. In this technique, researchers align a large number of images along reference points in three-dimensional space and then average them into a single, characteristic image. The result is a sharper, more reliable picture of what is going on within the cell.
“Small deviations that can be due to noise are eliminated, and we can localize proteins with high confidence, often with even greater precision than via SIM alone,” says Jaspersen. “We estimate the precision to be in the 10-30 nm resolution range.”
The SPA-SIM technique made up part of a two-color structured illumination microscopy approach that used endogenously expressed fluorescent protein derivatives. Through genetic techniques, the researchers fused the proteins in question to two standard fluorescent proteins, which would show up when the yeast naturally expressed the gene.
According to Unruh, this study represents the first combined use of SPA with SIM, and one of the first dual-color super-resolution SPA papers. Moreover, where other studies concentrated on a few protein pairs, the current study “characterized a significant portion of a large, many-protein complex.”
“The big-picture take away is that even structures that are considered by many to be ‘solved’ — we know what they look like by EM, we know all the parts, we know many of the physical interactions between those parts — have remarkable surprises when one is finally able to study their formation in cells.”
Among those surprises: SPB duplication, once thought to occur in the G1 period of interphase, now appears to begin near the end of mitosis. Moreover, the structures by which SPBs attach to the nuclear envelope do not form at the end of duplication, as once thought, but rather during the duplication process itself.
The study also reported a number of never-before-seen structural features, including “the structure and timing of half-bridge elongation, the composition of the satellite and the formation of the membrane pore.”
But that’s just the beginning. According to Jaspersen, the SPA-SIM technique is applicable to a wide variety of subjects beyond SPB structure.
“This method can be applied to any regular intracellular structure if there is some known reference protein that can be used.”