Seeing how the building blocks of cells align over time is important for understanding how cells are built, move, grow and divide. Recently, researchers from Japan collaborated with imaging scientists at the Marine Biological Laboratory (MBL) to develop a new cellular probe, POLArIS, that allows real-time imaging of molecular orientations in live cells.
The study, led by researchers at Tokyo Medical and Dental University (TMDU), was published in Proceedings of the National Academy of Sciences.
The technology is based on a simple principle: If a molecule of interest is rigidly connected with a fluorescent tag such as Green Fluorescent Protein (GFP), the polarized fluorescence from the tag reports the orientation of the molecule.
“In previous approaches for monitoring the orientation of proteins of interest, researchers had to develop effective, constrained GFP tagging methods that might be different for each protein,” says one of the lead authors, Ayana Sugizaki. “POLArIS uses an antibody-like binder that is rigidly connected with GFP, allowing both specific and versatile constrained labeling,” adds another lead author, Keisuke Sato.
POLArIS can be designed to discern the alignment of any biological molecules of interest. It can be expressed in specific cell types and organelles, and will be useful for studying architectural dynamics of molecular assemblies in a broad range of cell cultures, tissues, and whole organisms.
“From the point of view of fluorescence polarization imaging, POLArIS has significant advantages because of its genetically encoded nature,” says Tomomi Tani, senior researcher at the National Institute of Advanced Industrial Science and Technology (AIST) in Japan, who joined this project when he was an associate scientist at the MBL.
By using the probe for actin — proteins that form filaments that help cells divide, muscles contract, etc.– in dividing starfish embryonic cells, the team uncovered transient emergence and dissolution of highly ordered F-actin architecture that they named FLARE structure (see movie, above).
The FLARE structure, they found, is involved in orienting the plane of cell divisions. The mechanisms that determine the cell division plane are key for controlling many aspects of development, and yet remain a crucial mystery. This discovery of radially aligned actin architectures sheds light on one of the most fundamental unanswered questions of cell biology.
“We found that the FLARE structure extends up to the cell cortex in association with the astral microtubules,” says corresponding author, Sumio Terada, who had frequently visited the MBL from Tokyo, together with his colleagues in TMDU. “The astral microtubules are responsible for connecting the spindle to the cell cortex and orientating it correctly, controlling the plane of cell divisions.”
Shalin Mehta, another MBL imaging scientist who has since moved to Chan Zuckerberg Biohub, also worked on POLArIS at MBL.
Time-lapse movies of polarized fluorescence imaging of dividing starfish egg expressing POLArIS for F-actin, that specifically binds to actin filaments in a rotationally constrained manner. Magenta parts in the left movie indicate horizontally aligned actin filaments and the green parts for those of actin filaments that are vertically aligned. Right, the same movie with enhanced contrast. Credit: Keisuke Sato and Sumio Terada. More info on POLArIS: Sugizaki et al, PNAS 2021, DOI: 10.1073/pnas.2019071118