An intrinsic code for spindle position and orientation

Mitotic spindle positioning by cortical pulling forces defines the cell division axis and location, which is critical for proper cell division and development. Although recent work has identified developmental and extrinsic cues that regulate spindle orientation, the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle pole and chromosome-derived signals that regulate cytoplasmic dynein localization.

HFSP Long-Term Fellow Tomomi Kiyomitsu and Young Investigator Grant holder Iain Cheeseman
authored on Thu, 08 March 2012

During development in multi-cellular organisms, cells expand the pool of similar cells through symmetric cell division, but create cell-type diversity through asymmetric cell division. The type of cell division is determined in part by the positioning and orientation of the spindle. Both types of cell division are critical for tissue homeostasis in multicellular organisms. Recently, several developmental signals required for asymmetric cell division have been identified.  However, the signals required for symmetric cell division remain unclear.

Figure: Diagram showing a model for spindle pole- (red) and chromosome- (blue) derived signals regulating cortical dynein localisation (yellow). Spindle pole-derived PlK1 signals contribute to center the spindle by negatively regulating cortical dynein localisation when the spindle is off-center (left) while chromosome-derived RAN-GTP gradient defines or maintains the cell division axis by excluding NuMA and LGN (green) upstream cortical receptors for dynein from the central cell cortex in symmetrically dividing cells.

Regardless of the type of cell division, cortical pulling forces generated on astral microtubules define the position and orientation of the spindle. The conserved microtubule-based minus-end directed motor, cytoplasmic dynein(hereafter called dynein), generates a pulling force at the cell cortex using its motor activity. Dynein consists of a catalytic heavy chain and several non-catalytic subunits. Several adaptor proteins such as dynactin complex bind to the non-catalytic subunits of dynein and help target dynein to specific subcellular sites. To properly regulate spindle positioning and orientation, dynein must be properly recruited to defined locations in time and space. Several reports indicate that the conserved cortical protein complex NuMA-LGN-Gai, which is called Mud-Pins-Gain Drosophila and LIN-5-GPR-1/2-Gain C. elegans, functions as a cortical dynein-dynactin receptor. However, whether the NuMA-LGN-Gai complex physically interacts with dynein-dynactin and how their interaction is regulated in human cells was unclear.

Recently, we demonstrated that the NuMA-LGN-Gai complex functions as the cortical dynein-dynactin receptor in symmetrically dividing human cells. We also found that the interaction between dynein-dynactin and the NuMA-LGN-Gai complex is negatively regulated by the spindle pole localized Plk1 kinase both in vivo and in vitro. This mechanism causes the asymmetric cortical localization of dynein when the spindle is off-center, and generates asymmetric pulling forces to correct the mis-positioning of the metaphase spindle. Because Plk1 regulates cortical dynein localization in a distance-dependent manner, we proposed that this mechanism contributes to position the spindle in the middle of the cell.

We also found a second intrinsic mechanism that controls spindle orientation in symmetrically dividing HeLa cells. To maintain the spindle orientation axis, the force generator, dynein, must be restricted to lateral cell cortex. We found that the chromosome-derived RanGTP gradient excludes NuMA and LGN, upstream factors for dynein, from the central cortex thus restricting dynein to the lateral cell cortex. Ran is a small GTPase and is converted from RanGDP to RanGTP by the RanGEF, RCC1. The presence of RCC1 on chromosomes locally generates RanGTP leading to the production of Importin-RanGTP complexes and releasing nuclear localization signal (NLS) containing proteins in the vicinity of the chromosomes. This mechanism functions for nuclear transport during interphase and spindle assembly during mitosis. Importantly, NuMA has an NLS and is an established target of RanGTP. We showed that the NuMA NLS sequence contributes at least in part to exclude NuMA and LGN from the central cell cortex downstream of RanGTP. Because the same cortical force generating machinery is used for spindle orientation in both symmetric and asymmetric cell division, this work will contribute to understanding the common and regulatory mechanisms of spindle orientation. 

Reference

Chromosome and spindle pole-derived signals generate an intrinsic code for spindle position and orientation. Tomomi Kiyomitsu and Iain M. Cheeseman. Nat Cell Biol. 2012 published online Feb 12. doi: 10.1038/ncb2440, Nat Cell Biol. 2012 vol.14, March, p311-318, (cover).

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