Yuko MIMORI-KIYOSUE (Riken CLST)
“A rotating view of whole-cell 3D live imaging of a mitotic HeLa cell by lattice light-sheet microscopy”
|Title:||Dissecting 3D Dynamics of Mitotic Spindles by Lattice Light-Sheet Microscopy|
|Affiliation:||Unit Leader, Cellular Dynamics Analysis Unit, RIKEN Center for Life Science Technologies|
Whole-cell 3D live imaging of a mitotic HeLa cell by lattice light-sheet microscopy. The movie is replayed while rotating it around the spindle axis. Green: EB1-GFP; magenta: H2B-RFP (chromosome).
|The lattice light-sheet microscope, recently developed in Eric Betzig’s lab (Howard Hughes Medical Institute), enables 3D scanning at subsecond intervals with high spatial resolution (Science 346:1257998, 2014). This technology is an advanced type of light-sheet microscopy, but in contrast to the conventional light sheet created with several-micron-thick Gaussian beams, the lattice light sheet is generated from a massive parallel array of nondiffracting light beams that mutually interfere to create an ultrathin light sheet extending over cellular dimensions. Spreading the excitation light across many foci greatly reduces the intensity at any single focus, and in turn, reduces fluorescence photobleaching and phototoxicity. This strategy allowed us to image whole cells for hundreds of volumes at subsecond intervals.
The mitotic spindle, which comprises hundreds of microtubules, plays an essential role in cell division, ensuring the correct segregation of chromosomes into each daughter cell. Recently, we succeeded in detecting 3D microtubule growth dynamics throughout the cell cytoplasm using lattice light-sheet microscopy in conjunction with microtubule growth marker protein end-binding 1 (EB1), a microtubule plus-end-tracking protein, which was fused to green fluorescent protein (EB1-GFP). Furthermore, we developed a method to analyse spatial variations in microtubule growth dynamics within the mitotic spindle by adapting mathematical computing and geometric representation techniques (J Biomed Opt. 20:101206, 2015). Using these techniques, we analyzed the behavioral change of spindle microtubules in cells depleted of APC tumor suppressor protein, dysfunctioning of which leads to chromosomal instability and carcinogenesis. Our analyses revealed that APC protein has a function in centrosome maturation and maintenance of the spindle checkpoint through regulation of mitotic kinases.
|Biographical Sketch:||Yuko Mimori-Kiyosue received her Ph.D. from the Osaka University Graduate School of Engineering Science, Japan, in March 1995. After working in the field of structural biology with a helium-cooled cryo electron microscope, she joined the ERATO Tsukita Cell Axis Project from 1997 and studied cell biology using GFP technology, which led to the discovery of important cell axis regulators, microtubule plus-end tracking proteins (+TIPs). Currently, she is a unit leader at the RIKEN Center for Life Science Technologies and is pursuing developmental cell biology research employing advanced imaging technologies.|