New 3D imaging technique allows deep insights into subcellular structures

Apr 5, 2021 // Using a new microscope and methods from biophysics and biochemistry, scientists from IRI Life Sciences and the Max Planck Institute for the Physics of Light succeeded in visualizing the density of the spindle and the cell interior

Due to the current pandemic, we all pay attention to maintaining distance - on the way to work, while shopping or walking in the park. The inner life of our cells remains unimpressed by such regulations. There, a dense crowd of molecules prevails. This crowding is very important for the cell, since interactions between molecules are necessary for biochemical reactions to take place.

Of particular interest are structures within the cell that perform critical tasks. For example, the mitotic spindle - a self-organizing molecular machine that performs the function of segregating chromosomes during cell division. But how densely packed is a cell? How does the spindle manage to line up the chromosomes in this crowded environment? And how does one measure it?

How are the molecular and mechanical properties of the spindle related?

Every time a cell divides, the chromosomes must be distributed evenly between the two daughter cells that are formed. This task is completed by the mitotic spindle, which is built from hundreds of molecules that continuously interact to maintain a dynamic steady-state. Despite being made of components that turn over rapidly (within seconds), spindles are able to maintain their overall shape and size for extended time periods (hours).

While the last few decades have helped to identify the molecules essential for spindle function, a major gap remains in the understanding of how the different molecular processes collectively give rise to the spindle’s mesoscale material properties. "There are two main reasons for this", explains Simone Reber, research group leader at IRI Life Sciences. "In complex mixtures, like a densely packed cell, molecular properties of individual components overlap or form entirely new ones. Also, some properties remain unknown simply because it has been impossible to measure them", she adds.

To understand how the spindle forms and how it exerts forces on chromosomes during cell division, it is vital to understand the relationship between the spindle’s individual building blocks and its complex material properties.

Imaging technique ODT sheds new light on the spindle

Traditional imaging methods such as fluorescence microscopy provide us with images that are not only visually stunning but are ideal for identifying the subcellular localization of fluorescently labelled proteins. With the help of such techniques, the metaphase spindle has so far been proposed to be a physically denser phase of the cytoplasm, where proteins required to maintain the structure localize in high concentrations. Looking at an image of a spindle labelled with fluorescent tubulin (the spindle’s primary building block) confirms this notion. But is that actually true?

Left: A new imaging setup allows for correlative fluorescence and quantitative phase imaging. Middle: A Xenopus (clawed frog species) spindle as traditionally observed via fluorescence microscopy. Right: A density map of the same spindle obtained via ODT. @ Abin Biswas

New setup provides qualitative and quantitative information via one optical system

To answer this question, Abin Biswas (who is also a member of the Reber Lab at IRI Life Sciences) and Kyoohyun Kim from the group of Jochen Guck, director at the Max Planck Institute for the Physics of Light in Erlangen, built a new optical system capable of performing correlative confocal fluorescence microscopy and optical diffraction tomography (ODT). ODT has the advantage of providing quantitative 3-D refractive index distributions with micron-level resolution. Combined with fluorescence imaging, the setup provides both qualitative and quantitative information via one optical system. "ODT works much like an optical mini-balance", says Abin, first author of the study. "What was surprising, was that we couldn't see the spindle via ODT at all." This means that the spindle has the same refractive index, and therefore the same density as the surrounding cytoplasm. Thus, the molecules within the spindle are not more densely packed than the rest of the molecules in the cell. The current study also suggests that cellular phase transitions may not necessarily involve a density transition. Therefore, sub-cellular structures such as the spindle are formed by the concentration of essential molecules without affecting local density. These findings toss out previous assumptions about the spindle being a denser cytoplasmic phase and reinforce the need to better characterize biologically relevant mesoscale structures - which remains an exciting topic for future research.

The findings of the study additionally imply that within a complex and crowded environment like the cytoplasm, a simplified view of global density transitions taking place during liquid phase separation may not necessarily hold true. Instead, the composition and spatial distribution of molecules change without impacting the local physical density, as has been suggested in recent reviews. The relationship between molecular specificity and material properties of (phase-separating and transitioning) subcellular compartments within crowded physiologically relevant solvents such as the cyto- or nucleoplasm remains an exciting and open topic for future research.

A green laser (532nm wavelength) is used for imaging and is focused into an optical fiber. © Robert Kircher-Reineke

Light coming out of the fiber is used to illuminate the sample at multiple angles. © Robert Kircher-Reineke

Powerful objectives are used to focus light into a sample and collect the light scattered from it. © Robert Kircher-Reineke

Medical implications

Why is it important to measure the physical properties of the spindle? "We know that defective spindles cannot distribute chromosomes accurately. This can have fatal consequences", Abin explains. Cells with too many or missing chromosomes are found in malignant tumors. If errors occur early in embryonic development, they can result in trisomies, such as Down syndrome. It is therefore not only of interest for basic research but also for clinical medicine to understand the exact mechanisms that determine the material properties of the mitotic spindle.

Original publication

Abin Biswas, Kyoohyun Kim, Gheorghe Cojoc, Jochen Guck and Simone Reber. The Xenopus spindle is as dense as the surrounding cytoplasm. Developmental Cell (2021). DOI: 10.1016/j.devcel.2021.03.013

Contact

For further information and other requests please contact Abin Biswas from the Reber Lab via email abin.biswas(at)iri-lifesciences.de.