Advance to GO: Regulation of mitotic progression by the spindle checkpoint kinase MPS1

TY - THES

T1 - Advance to GO: Regulation of mitotic progression by the spindle checkpoint kinase MPS1

AU - Pachis, Spyridon T

PY - 2019/9/24

Y1 - 2019/9/24

N2 - Due to its extreme importance in maintaining a stable genome, chromosome segregation is highly regulated and complex process which is driven by the mitotic spindle. The spindle is comprised of microtubules whose role is to make contacts with chromosomes, capture them and eventually drag duplicated chromosomes (or sister chromatids) to opposite poles of the cell, ensuring the inheritance of a full set of chromosomes by the two daughter cells. The contact points for microtubules on chromosomes are called kinetochores. These are large protein scaffolds comprised of multiple complexes that are built on top centromeric DNA just before mitosis begins. Microtubule-kinetochore contacts can occur in many different configurations, but only a certain kind of attachments (amphitelic) can lead to the equal distribution of chromosomes. For this reason, cells have evolved systems that monitor the attachment status of each sister chromatid kinetochore, and can intervene and destabilize erroneous attachments when they are detected, allowing cells to attempt the formation of the right kind of attachments anew. These are the spindle assembly checkpoint (SAC) and error correction pathways respectively. Monopolar spindle 1 (MPS1) is a key upstream player in SAC signaling which, in the presence of unattached kinetochores, is responsible for the hierarchical recruitment of downstream SAC components which eventually form a diffusible inhibitor of mitotic progression. When microtubules form stable attachments to kinetochores, MPS1 becomes displaced, SAC signaling is interrupted and mitotic progression is able to take place. Aurora B, the catalytically active component of the error correction pathway, phosphorylates proteins in the outer kinetochore, and causes the destabilization of microtubule attachments. Amphitelic attachments lead to kinetochore transformations and the simultaneous recruitment of phosphatases that antagonize Aurora B activity, so that the outer kinetochore Aurora B targets can no longer be phosphorylated and the attachments will be stabilized. The SAC and error correction communicate with each other with many proteins playing a role in both pathways and regulatory feedback loops keep activity of the two systems interwound. This ensures that mitotic progression only takes place when all chromosomes are properly attached to the mitotic spindle. The work presented in this thesis aims to provide novel insights into spindle assembly checkpoint (SAC) signaling through the regulation of the kinetochore localization of a critical SAC kinase, MPS1. MPS1 is responsible for mounting a robust SAC signal in the presence of unattached kinetochores and is one of the major targets of the SAC silencing mechanisms, making it one of the guardians of chromosome segregation fidelity. Understanding in greater detail how MPS1 localizes to kinetochores and how it is removed upon microtubule attachment is therefore of great interest. Panels of MPS1 and other kinetochore proteins such as HEC1 and KNL1 were used in a knockdown-addback strategy that allowed structure-function studies via quantitative immunofluorescence microscopy and time lapse imaging and additionally in in vitro biochemical approaches.

AB - Due to its extreme importance in maintaining a stable genome, chromosome segregation is highly regulated and complex process which is driven by the mitotic spindle. The spindle is comprised of microtubules whose role is to make contacts with chromosomes, capture them and eventually drag duplicated chromosomes (or sister chromatids) to opposite poles of the cell, ensuring the inheritance of a full set of chromosomes by the two daughter cells. The contact points for microtubules on chromosomes are called kinetochores. These are large protein scaffolds comprised of multiple complexes that are built on top centromeric DNA just before mitosis begins. Microtubule-kinetochore contacts can occur in many different configurations, but only a certain kind of attachments (amphitelic) can lead to the equal distribution of chromosomes. For this reason, cells have evolved systems that monitor the attachment status of each sister chromatid kinetochore, and can intervene and destabilize erroneous attachments when they are detected, allowing cells to attempt the formation of the right kind of attachments anew. These are the spindle assembly checkpoint (SAC) and error correction pathways respectively. Monopolar spindle 1 (MPS1) is a key upstream player in SAC signaling which, in the presence of unattached kinetochores, is responsible for the hierarchical recruitment of downstream SAC components which eventually form a diffusible inhibitor of mitotic progression. When microtubules form stable attachments to kinetochores, MPS1 becomes displaced, SAC signaling is interrupted and mitotic progression is able to take place. Aurora B, the catalytically active component of the error correction pathway, phosphorylates proteins in the outer kinetochore, and causes the destabilization of microtubule attachments. Amphitelic attachments lead to kinetochore transformations and the simultaneous recruitment of phosphatases that antagonize Aurora B activity, so that the outer kinetochore Aurora B targets can no longer be phosphorylated and the attachments will be stabilized. The SAC and error correction communicate with each other with many proteins playing a role in both pathways and regulatory feedback loops keep activity of the two systems interwound. This ensures that mitotic progression only takes place when all chromosomes are properly attached to the mitotic spindle. The work presented in this thesis aims to provide novel insights into spindle assembly checkpoint (SAC) signaling through the regulation of the kinetochore localization of a critical SAC kinase, MPS1. MPS1 is responsible for mounting a robust SAC signal in the presence of unattached kinetochores and is one of the major targets of the SAC silencing mechanisms, making it one of the guardians of chromosome segregation fidelity. Understanding in greater detail how MPS1 localizes to kinetochores and how it is removed upon microtubule attachment is therefore of great interest. Panels of MPS1 and other kinetochore proteins such as HEC1 and KNL1 were used in a knockdown-addback strategy that allowed structure-function studies via quantitative immunofluorescence microscopy and time lapse imaging and additionally in in vitro biochemical approaches.

KW - mitosis

KW - spindle assembly checkpoint

KW - MPS1

KW - kinetochore

KW - SAC

KW - chromosome segregation

KW - cell division

M3 - PhD thesis

SN - 978-94-6375-449-1

PB - Utrecht University

ER -