Final Report Summary - MTOC FUNCTION (Microtubule organizing centers and microtubule nucleation in mitosis)
The centrosome nucleates microtubule polymerisation and contributes to the organisation of microtubules in mitosis, which is important for proper segregation of the chromosomes. Defects in centrosome number and function are frequently observed in cancer cells and centrosome proteins are potential targets for therapeutic intervention. Recent data suggests that some centrosome proteins are also implicated in cellular differentiation, and that several human developmental disorders can be linked to defects in centrosomal components. Despite these observations it is unknown how centrosomes and other microtubule organizing centers (MTOCs) function at a molecular level. In addition to centrosomes, acentrosomal MTOCs also contribute to microtubule nucleation and organisation both during mitosis and during cell differentiation, but the molecular details are largely unknown. This project focuses on the ?-tubulin ring complex (?TuRC), the main nucleator of microtubule polymerisation and key component of MTOCs, with the goal of understanding how centrosomal and acentrosomal MTOCs are formed and regulated, and how they contribute to microtubule nucleation and organisation.
Composition and interactors of the TuRC
Using mass spectrometry we compared the composition of ?TuRCs from non-synchronised and mitotic human cells. Based on our analysis we can now define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic ?TuRCs (Teixidó-Travesa et al., 2010, Mol Biol Cell, 21, 3963-72). We also identified novel regulatory subunits such as Mozart1 and GCP8/Mozart2, which are present in both interphase and mitotic ?TuRCs. GCP8 depletion does not affect ?TuRC assembly, but interferes with ?TuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects suggesting that GCP8 specifically affects the organisation of the interphase microtubule network (Teixidó-Travesa et al., 2010, Mol Biol Cell, 21, 3963-72). We are currently conducting a follow-up study to further characterise this interphase-specific function of GCP8. A similar study has been initiated regarding the mitotic role of Mozart1. The identification and characterisation of these novel TuRC subunits is expected to provide insight into TuRC regulation, which remains poorly understood (Teixidó-Travesa et al., 2012, J Cell Sci, review, in press). Our mass spectrometry analysis of ?TuRC has also allowed us to identify many novel phosphorylation sites in ?TuRC subunits, both in interphase and mitosis, and we are studying the potential regulatory roles of these modifications in ?TuRC structure and function.
In addition to ?TuRC regulation we have also re-investigated the structural roles of the conserved ?TuRC subunits GCP2-GCP6. While it is generally assumed that GCP2-GCP6 are all essential for ?TuRC structure, the data is mainly derived from studies in fungi and Drosophila. Therefore we have conducted a comparative analysis of human GCP2-GCP6 using RNAi in human cells. Surprisingly we found that only GCP6 is essential for ?TuRC assembly and function and that lack of GCP4 or GCP5 only partially impairs ?TuRC function (Teixidó-Travesa et al., manuscript in preparation).
Together these studies provide the first comprehensive analysis describing in detail the structural and regulatory roles of distinct subunits of the human ?TuRC.
Recruitment of the ?TuRC: centrosomal and acentrosomal nucleation sites
?TuRCs are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. Accumulation of ?TuRC at centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. We discovered that Plk1 associates with the ?TuRC subunit GCP-WD in mitosis and that Plk1 activity contributes to phosphorylation of GCP-WD. However, GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit ?-tubulin. Interestingly, we found that Plk1 also controls the recruitment of other proteins implicated in centrosomal ?-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of ?-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates (Haren et al., 2009, PLoS One, 4, e5976).
In addition to centrosomal nucleation we are also studying acentrosomal nucleation including the recently discovered augmin-dependent pathway. During our study we also identified the previously uncharacterised protein CCDC52. Despite the similar localisation pattern CCDC52 has properties that distinguish it from the augmin complex and we have renamed this protein SPICE (spindle and centriole protein). RNAi mediated depletion of SPICE in human cells impairs centriole duplication and causes severe mitotic defects. SPICE depletion compromises spindle architecture, spindle pole integrity and chromosome congression. Our data suggests that SPICE is an important dual-function regulator required for centriole duplication and for proper bipolar spindle formation and chromosome congression in mitosis (Archinti et al., 2010, J Cell Sci, 123, 3039-46).
To gain more insight into the role of non-centrosomal augmin and ?TuRC we have analysed the distribution and the dynamics of both complexes within the mitotic spindle. This work revealed that ?TuRC and augmin cooperate in spindle targeting and that ?TuRC at microtubule minus ends is sorted poleward (Lecland and Lüders, manuscript in preparation). This process involves several molecular motor proteins and contributes to proper spindle assembly and function.
Based on our work and parallel studies by other labs we can assume that we now have a rather complete list of ?TuRC subunits and interactors. In addition we have completed the list of known in vivo ?TuRC phosphorylation sites. We have started characterizing novel interactors and phosphorylation sites and lay grounds for future analyses of ?TuRC regulation.
We have also revealed mechanisms involved in the targeting of ?TuRC to centrosomes and spindle microtubules during mitosis. Another major achievement is the demonstration of a motor-dependent poleward sorting mechanism for spindle-associated ?TuRC that helps organizing microtubule minus ends within the mitotic spindle.
In summary this project provides a better understanding of how the ?TuRC interacts with centrosomal and non-centrosomal MTOCs and identifies regulatory mechanisms that control ?TuRC-dependent organisation of the microtubule network.
Composition and interactors of the TuRC
Using mass spectrometry we compared the composition of ?TuRCs from non-synchronised and mitotic human cells. Based on our analysis we can now define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic ?TuRCs (Teixidó-Travesa et al., 2010, Mol Biol Cell, 21, 3963-72). We also identified novel regulatory subunits such as Mozart1 and GCP8/Mozart2, which are present in both interphase and mitotic ?TuRCs. GCP8 depletion does not affect ?TuRC assembly, but interferes with ?TuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects suggesting that GCP8 specifically affects the organisation of the interphase microtubule network (Teixidó-Travesa et al., 2010, Mol Biol Cell, 21, 3963-72). We are currently conducting a follow-up study to further characterise this interphase-specific function of GCP8. A similar study has been initiated regarding the mitotic role of Mozart1. The identification and characterisation of these novel TuRC subunits is expected to provide insight into TuRC regulation, which remains poorly understood (Teixidó-Travesa et al., 2012, J Cell Sci, review, in press). Our mass spectrometry analysis of ?TuRC has also allowed us to identify many novel phosphorylation sites in ?TuRC subunits, both in interphase and mitosis, and we are studying the potential regulatory roles of these modifications in ?TuRC structure and function.
In addition to ?TuRC regulation we have also re-investigated the structural roles of the conserved ?TuRC subunits GCP2-GCP6. While it is generally assumed that GCP2-GCP6 are all essential for ?TuRC structure, the data is mainly derived from studies in fungi and Drosophila. Therefore we have conducted a comparative analysis of human GCP2-GCP6 using RNAi in human cells. Surprisingly we found that only GCP6 is essential for ?TuRC assembly and function and that lack of GCP4 or GCP5 only partially impairs ?TuRC function (Teixidó-Travesa et al., manuscript in preparation).
Together these studies provide the first comprehensive analysis describing in detail the structural and regulatory roles of distinct subunits of the human ?TuRC.
Recruitment of the ?TuRC: centrosomal and acentrosomal nucleation sites
?TuRCs are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. Accumulation of ?TuRC at centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. We discovered that Plk1 associates with the ?TuRC subunit GCP-WD in mitosis and that Plk1 activity contributes to phosphorylation of GCP-WD. However, GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit ?-tubulin. Interestingly, we found that Plk1 also controls the recruitment of other proteins implicated in centrosomal ?-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of ?-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates (Haren et al., 2009, PLoS One, 4, e5976).
In addition to centrosomal nucleation we are also studying acentrosomal nucleation including the recently discovered augmin-dependent pathway. During our study we also identified the previously uncharacterised protein CCDC52. Despite the similar localisation pattern CCDC52 has properties that distinguish it from the augmin complex and we have renamed this protein SPICE (spindle and centriole protein). RNAi mediated depletion of SPICE in human cells impairs centriole duplication and causes severe mitotic defects. SPICE depletion compromises spindle architecture, spindle pole integrity and chromosome congression. Our data suggests that SPICE is an important dual-function regulator required for centriole duplication and for proper bipolar spindle formation and chromosome congression in mitosis (Archinti et al., 2010, J Cell Sci, 123, 3039-46).
To gain more insight into the role of non-centrosomal augmin and ?TuRC we have analysed the distribution and the dynamics of both complexes within the mitotic spindle. This work revealed that ?TuRC and augmin cooperate in spindle targeting and that ?TuRC at microtubule minus ends is sorted poleward (Lecland and Lüders, manuscript in preparation). This process involves several molecular motor proteins and contributes to proper spindle assembly and function.
Based on our work and parallel studies by other labs we can assume that we now have a rather complete list of ?TuRC subunits and interactors. In addition we have completed the list of known in vivo ?TuRC phosphorylation sites. We have started characterizing novel interactors and phosphorylation sites and lay grounds for future analyses of ?TuRC regulation.
We have also revealed mechanisms involved in the targeting of ?TuRC to centrosomes and spindle microtubules during mitosis. Another major achievement is the demonstration of a motor-dependent poleward sorting mechanism for spindle-associated ?TuRC that helps organizing microtubule minus ends within the mitotic spindle.
In summary this project provides a better understanding of how the ?TuRC interacts with centrosomal and non-centrosomal MTOCs and identifies regulatory mechanisms that control ?TuRC-dependent organisation of the microtubule network.