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Dynein and Mast/Orbit/CLASP have antagonistic roles in regulating kinetochore-m...
Reis, Rita; Feijão, Tália; Gouveia, Susana; Pereira, António J.; Matos, Irina; Sampaio, Paula; Maiato, Helder; Sunkel, Claudio E.Establishment and maintenance of the mitotic spindle requires the balanced activity of microtubule-associated proteins and motors. In this study we have addressed how the microtubule plus-end tracking protein Mast/Orbit/CLASP and cytoplasmic dynein regulate this process in Drosophila melanogaster embryos and S2 cells. We show that Mast accumulates at kinetochores early in mitosis, which is followed by a polewar...
Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator
Lince-Faria, Mariana; Maffini, Stefano; Orr, Bernard; Ding, Yun; Florindo, Cláudia; Sunkel, Claudio E.; Tavares, Álvaro; Johansen, JørgenA putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived...
Dual Role of Topoisomerase II in Centromere Resolution and Aurora B Activity
Coelho, Paula A.; Queiroz-Machado, Joana; Carmo, Alexandre M.; Moutinho-Pereira, Sara; Maiato, Helder; Sunkel, Claudio E.Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomera...
Microtubule-associated proteins and their essential roles during mitosis
Maiato, Helder; Sampaio, Paula; Sunkel, Claudio E.Microtubules play essential roles during mitosis, including chromosome capture, congression, and segregation. In addition, microtubules are also required for successful cytokinesis. At the heart of these processes is the ability of microtubules to do work, a property that derives from their intrinsic dynamic behavior. However, if microtubule dynamics were not properly regulated, it is certain that microtubules ...
How Do Kinetochores CLASP Dynamic Microtubules?
Maiato, Helder; Rieder, Conly L.; Earnshaw, William C.; Sunkel, Claudio E.Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed...
Dissecting mitosis by RNAi in Drosophila tissue culture cells
Maiato, Helder; Sunkel, Claudio E.; Earnshaw, William C.interference (RNAi) in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study ...
Human CLASP1 is an outer kinetochore component that regulates spindle microtubu...
Maiato, Helder; Rieder, Conly L.; Swedlow, Jason R.; Cole, Richard; Sunkel, Claudio E.; Earnshaw, William C.One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto the plus-ends of dynamic microtubules that are actively gaining or losing tubulin subunits. Here we show that the microtubule-associated protein CLASP1 is localized preferentially near the plus-ends of growing microtubules during spindle formation and is also a component of a novel region that we term the outer kinetochore...
MAST/Orbit has a role in microtubule-kinetochore attachment and is essential fo...
Maiato, Helder; Sampaio, Paula; Lemos, Catarina L.; Findlay, John; Mar, Carmena; Earnshaw, William C.; Sunkel, Claudio E.Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing e...
Mast, a conserved microtubule-associated protein required for bipolar mitotic s...
Lemos, Catarina L.; Sampaio, Paula; Maiato, Helder; Costa, Madalena; Omel’yanchuk, Leonid V.; Liberal, Vasco; Sunkel, Claudio E.Through mutational analysis in Drosophila, we have identified the gene multiple asters (mast), that encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organised by an irregular number of MTOCs of abnormal size and shape. The mast gene product is evolutionary conserved since homologues were id...
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