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1.  A Primary Microcephaly Protein Complex forms a ring around parental centrioles 
Nature genetics  2011;43(11):1147-1153.
Autosomal recessive primary microcephaly (MCPH) is characterised by a significant reduction in prenatal human brain growth, without alteration of cerebral architecture. The genetic aetiology of MCPH is bi-allelic mutations in genes coding for a subset of centrosomal proteins1-10. While at least three of these proteins have been implicated in centrosome duplication11, the nature of centrosome dysfunction that underlies the neurodevelopmental defect in MCPH is unclear. Here we report a homozygous MCPH-causing mutation in the human CEP63 gene. CEP63 forms a complex with another MCPH protein, CEP152, a conserved centrosome duplication factor12-15. Together, they are essential for maintaining normal centrosome numbers in cells. Using super-resolution microscopy we find that CEP63 and CEP152 co-localise in a discrete ring around the proximal end of the parental centriole, a pattern specifically disrupted in CEP63-deficient patient-derived cells. This work suggests that the CEP152-CEP63 ring-like structure ensures normal neurodevelopment and its impairment particularly affects human cerebral cortex growth.
doi:10.1038/ng.971
PMCID: PMC3299569  PMID: 21983783
centrosome; mitotic spindle; centrosome duplication; microcephaly; brain
2.  A direct look at RNAi screens 
doi:10.1038/msb.2012.14
PMCID: PMC3397413  PMID: 22531120
3.  CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response 
The Journal of Cell Biology  2010;189(1):23-39.
Two domains of centrosomal protein CDK5RAP2, CNN1 and CNN2, link centrosomes to mitotic spindle poles. CNN1 lacking centrosomes are unable to recruit pericentriolar matrix components that mediate attachment to spindle poles.
The centrosomal protein, CDK5RAP2, is mutated in primary microcephaly, a neurodevelopmental disorder characterized by reduced brain size. The Drosophila melanogaster homologue of CDK5RAP2, centrosomin (Cnn), maintains the pericentriolar matrix (PCM) around centrioles during mitosis. In this study, we demonstrate a similar role for CDK5RAP2 in vertebrate cells. By disrupting two evolutionarily conserved domains of CDK5RAP2, CNN1 and CNN2, in the avian B cell line DT40, we find that both domains are essential for linking centrosomes to mitotic spindle poles. Although structurally intact, centrosomes lacking the CNN1 domain fail to recruit specific PCM components that mediate attachment to spindle poles. Furthermore, we show that the CNN1 domain enforces cohesion between parental centrioles during interphase and promotes efficient DNA damage–induced G2 cell cycle arrest. Because mitotic spindle positioning, asymmetric centrosome inheritance, and DNA damage signaling have all been implicated in cell fate determination during neurogenesis, our findings provide novel insight into how impaired CDK5RAP2 function could cause premature depletion of neural stem cells and thereby microcephaly.
doi:10.1083/jcb.200912163
PMCID: PMC2854379  PMID: 20368616
4.  MCAK-Independent Functions of ch-Tog/XMAP215 in Microtubule Plus-End Dynamics▿ †  
Molecular and Cellular Biology  2008;28(23):7199-7211.
The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.
doi:10.1128/MCB.01040-08
PMCID: PMC2593372  PMID: 18809577
5.  The F-Box DNA Helicase Fbh1 Prevents Rhp51-Dependent Recombination without Mediator Proteins 
Molecular and Cellular Biology  2005;25(18):8084-8096.
A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1− mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.
doi:10.1128/MCB.25.18.8084-8096.2005
PMCID: PMC1234329  PMID: 16135800

Results 1-5 (5)