Billions of our cells divide every day. This process must be highly regulated and error free to maintain healthy tissues and organs. During cell division, each of the two daughter cells receives one copy of each chromosome. The mechanical forces driving chromosome movement are generated by the mitotic spindle. This structure is composed of thousands of tubular filaments, termed microtubules, which arrange in a bipolar array. How microtubules form and grow to shape the specific geometry of the spindle is not fully understood.
Daniel Gerlich’s group at the IMBA now reports in the Journal of Cell Biology that once a few microtubules have grown out from the two poles of the spindle, most microtubules are generated throughout the spindle body. This results in highly coordinated microtubule growth towards the attachment sites on chromosomes.
Spindles of human cells attach a fiber of 20-40 microtubules to a confined region on each replicated sister chromatid, termed the kinetochore. Each pair of sister kinetochore-fibers binds to centrosomes at opposing spindle poles of the dividing cell, enabling faithful chromosome segregation.
Growing microtubules plus-ends (green) and centromeres (magenta) in a mitotic HeLa cell, imaged by 3D Lattice light-sheet microscopy. Shown are a selected focal plane (left) and a projection of the whole cell volume (right). t=0, nuclear envelope disassembly. Scale bar, 10 µm.
It has been shown that microtubules can be generated at centrosomes, cytoplasmic regions surrounding chromosomes, directly at kinetochores, or on the outer walls of existing microtubules. Yet, to which extent each generation pathway contributes to spindle assembly in human cells has been unclear. To address this, Daniel Gerlich’s group at the IMBA used new live-cell microscopy technology and mathematical modeling to study spindle assembly in dividing human cells.
The current study reveals that only a very small fraction of growing microtubules within the metaphase spindle originates from centrosomes, chromosomes or kinetochores. Instead, Gerlich’s team found that almost 90% of microtubules are generated on the walls of pre-existing microtubules, in a manner dependent on the protein complex Augmin.
Because attachment to kinetochores stabilizes microtubules, the next question was whether this amplification of long-lived template could lead to biased microtubule growth towards kinetochores. For this line of investigation, Gerlich’s team collaborated with groups at the HHMI Janelia Research Campus and UT Southwestern to perform lattice light-sheet microscopy, which enables ultra-fast, three-dimensional imaging of live cells. These analyses revealed that the direction of microtubule growth is not stochastic, but highly biased towards individual kinetochores.
First author Ana David who is a Vienna Biocenter PhD student describes the process as `molecular sorting mechanism’: „You have a very dynamic population of microtubules - those attached to the kinetochore are more stable and so produce more descendants that are just like them: they point in the same direction and reach the same kinetochore.
Original Paper: David et al., “Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth”, Journal of Cell Biology, https://doi.org/10.1083/jcb.201805044