Numerical centrosome aberrations underlie certain developmental abnormalities and may promote can... more Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215-HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome-spindle pole interface by the CEP215-HSET complex could promote survival of cancer cells containing supernumerary centrosomes.
The transition from G1 into DNA replication (S phase) is an emergent behavior resulting from dyna... more The transition from G1 into DNA replication (S phase) is an emergent behavior resulting from dynamic and complex interactions between cyclin-dependent kinases (Cdks), Cdk inhibitors (CKIs), and the anaphase-promoting complex/cyclosome (APC/C). Understanding the cellular decision to commit to S phase requires a quantitative description of these interactions. We apply quantitative imaging of single human cells to track the expression of G1/S regulators and use these data to parametrize a stochastic mathematical model of the G1/S transition. We show that a rapid, proteolytic, double-negative feedback loop between Cdk2:Cyclin and the Cdk inhibitor p27(Kip1) drives a switch-like entry into S phase. Furthermore, our model predicts that increasing Emi1 levels throughout S phase are critical in maintaining irreversibility of the G1/S transition, which we validate using Emi1 knockdown and live imaging of G1/S reporters. This work provides insight into the general design principles of the signaling networks governing the temporally abrupt transitions between cell-cycle phases.
How multiple spindle assembly pathways are integrated to drive bipolar spindle assembly is poorly... more How multiple spindle assembly pathways are integrated to drive bipolar spindle assembly is poorly understood. We performed an image-based double RNAi screen to identify genes encoding Microtubule-Associated Proteins (MAPs) that interact with the highly conserved ch-TOG gene to regulate bipolar spindle assembly in human cells. We identified a ch-TOG centred network of genetic interactions which promotes ensures centrosome-mediated microtubule polymerisation, leading to the incorporation of microtubules polymerised by all pathways into a bipolar structure. Our genetic screen also reveals that ch-TOG maintains a dynamic microtubule population, in part, through modulating HSET activity. ch-TOG ensures that spindle assembly is robust to perturbation but sufficiently dynamic such that spindles can explore a diverse shape space in search of structures that can align chromosomes.
The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a huma... more The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a human protein that localises to the centrosome. At the start of this thesis work, the function of CDK5RAP2 was uncharacterised. Significantly, cdk5rap2 is one of several centrosomal ...
Methods in molecular biology (Clifton, NJ), Jan 1, 2009
Reverse genetic methods, such as homologous gene targeting, have greatly contributed to our under... more Reverse genetic methods, such as homologous gene targeting, have greatly contributed to our understanding of molecular pathways in mitosis, especially in yeast. The chicken B-lymphocyte line, DT40, represents a unique example among vertebrate somatic cells where homologous gene targeting occurs at very high frequency. DT40 cells therefore provide a useful and accessible somatic genetic system for wide-ranging biochemical and cell biological assays. In this chapter, we describe the main principles of homologous gene targeting, the concept of targeting construct design and the detailed experimental protocol of how to achieve successful knockouts. We also mention methods for conditional disruption of essential genes and conclude with specific procedures for the study of mitosis in DT40 cells.
Numerical centrosome aberrations underlie certain developmental abnormalities and may promote can... more Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215-HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome-spindle pole interface by the CEP215-HSET complex could promote survival of cancer cells containing supernumerary centrosomes.
The transition from G1 into DNA replication (S phase) is an emergent behavior resulting from dyna... more The transition from G1 into DNA replication (S phase) is an emergent behavior resulting from dynamic and complex interactions between cyclin-dependent kinases (Cdks), Cdk inhibitors (CKIs), and the anaphase-promoting complex/cyclosome (APC/C). Understanding the cellular decision to commit to S phase requires a quantitative description of these interactions. We apply quantitative imaging of single human cells to track the expression of G1/S regulators and use these data to parametrize a stochastic mathematical model of the G1/S transition. We show that a rapid, proteolytic, double-negative feedback loop between Cdk2:Cyclin and the Cdk inhibitor p27(Kip1) drives a switch-like entry into S phase. Furthermore, our model predicts that increasing Emi1 levels throughout S phase are critical in maintaining irreversibility of the G1/S transition, which we validate using Emi1 knockdown and live imaging of G1/S reporters. This work provides insight into the general design principles of the signaling networks governing the temporally abrupt transitions between cell-cycle phases.
How multiple spindle assembly pathways are integrated to drive bipolar spindle assembly is poorly... more How multiple spindle assembly pathways are integrated to drive bipolar spindle assembly is poorly understood. We performed an image-based double RNAi screen to identify genes encoding Microtubule-Associated Proteins (MAPs) that interact with the highly conserved ch-TOG gene to regulate bipolar spindle assembly in human cells. We identified a ch-TOG centred network of genetic interactions which promotes ensures centrosome-mediated microtubule polymerisation, leading to the incorporation of microtubules polymerised by all pathways into a bipolar structure. Our genetic screen also reveals that ch-TOG maintains a dynamic microtubule population, in part, through modulating HSET activity. ch-TOG ensures that spindle assembly is robust to perturbation but sufficiently dynamic such that spindles can explore a diverse shape space in search of structures that can align chromosomes.
The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a huma... more The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a human protein that localises to the centrosome. At the start of this thesis work, the function of CDK5RAP2 was uncharacterised. Significantly, cdk5rap2 is one of several centrosomal ...
Methods in molecular biology (Clifton, NJ), Jan 1, 2009
Reverse genetic methods, such as homologous gene targeting, have greatly contributed to our under... more Reverse genetic methods, such as homologous gene targeting, have greatly contributed to our understanding of molecular pathways in mitosis, especially in yeast. The chicken B-lymphocyte line, DT40, represents a unique example among vertebrate somatic cells where homologous gene targeting occurs at very high frequency. DT40 cells therefore provide a useful and accessible somatic genetic system for wide-ranging biochemical and cell biological assays. In this chapter, we describe the main principles of homologous gene targeting, the concept of targeting construct design and the detailed experimental protocol of how to achieve successful knockouts. We also mention methods for conditional disruption of essential genes and conclude with specific procedures for the study of mitosis in DT40 cells.
Uploads