Biomedical subjects

Helder Maiato

Publications and source records attributed to Helder Maiato.

12 recordsLinked to original sources

PubMed2006

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function.

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.

Aneuploidy↗

PubMed2006

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells.

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40-50 nm thick. On average, each S2 kinetochore binds 11+/-2 MTs, in contrast to the 4-6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.

Animals↗

PubMed2006

Kinetochores use a novel mechanism for coordinating the dynamics of individual microtubules.

Chromosome alignment during mitosis is frequently accompanied by a dynamic switching between elongation and shortening of kinetochore fibers (K-fibers) that connect kinetochores and spindle poles . In higher eukaryotes, mature K-fibers consist of 10-30 kinetochore microtubules (kMTs) whose plus ends are embedded in the kinetochore . A critical and long-standing question is how the dynamics of individual kMTs within the K-fiber are coordinated . We have addressed this question by using electron tomography to determine the polymerization/depolymerization status of individual kMTs in the K-fibers of PtK1 and Drosophila S2 cells. Surprisingly, we find that the plus ends of two-thirds of kMTs are in a depolymerizing state, even when the K-fiber exhibits net tubulin incorporation at the plus end . Furthermore, almost all individual K-fibers examined had a mixture of kMTs in the polymerizing and depolymerizing states. Therefore, although K-fibers elongate and shrink as a unit, the dynamics of individual kMTs within a K-fiber are not coordinated at any given moment. Our results suggest a novel control mechanism through which attachment to the kinetochore outer plate prevents shrinkage of kMTs. We discuss the ramifications of this new model on the regulation of chromosome movement and the stability of K-fibers.

Animals↗

PubMed2004

Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres.

The motion of a chromosome during mitosis is mediated by a bundle of microtubules, termed a kinetochore fibre (K-fibre), which connects the kinetochore of the chromosome to a spindle pole. Once formed, mature K-fibres maintain a steady state length because the continuous addition of microtubule subunits onto microtubule plus ends at the kinetochore is balanced by their removal at their minus ends within the pole. This condition is known as 'microtubule poleward flux'. Chromosome motion and changes in position are then driven by changes in K-fibre length, which in turn are controlled by changes in the rates at which microtubule subunits are added at the kinetochore and/or removed from the pole. A key to understanding the role of flux in mitosis is to identify the molecular factors that drive it. Here we use Drosophila melanogaster S2 cells expressing alpha-tubulin tagged with green fluorescent protein, RNA interference, laser microsurgery and photobleaching to show that the kinetochore protein MAST/Orbit - the single CLASP orthologue in Drosophila - is an essential component for microtubule subunit incorporation into fluxing K-fibres.

Animals↗

PubMed2004

Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis.

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

Animals↗

PubMed2004

The dynamic kinetochore-microtubule interface.

The kinetochore is a control module that both powers and regulates chromosome segregation in mitosis and meiosis. The kinetochore-microtubule interface is remarkably fluid, with the microtubules growing and shrinking at their point of attachment to the kinetochore. Furthermore, the kinetochore itself is highly dynamic, its makeup changing as cells enter mitosis and as it encounters microtubules. Active kinetochores have yet to be isolated or reconstituted, and so the structure remains enigmatic. Nonetheless, recent advances in genetic, bioinformatic and imaging technology mean we are now beginning to understand how kinetochores assemble, bind to microtubules and release them when the connections made are inappropriate, and also how they influence microtubule behaviour. Recent work has begun to elucidate a pathway of kinetochore assembly in animal cells; the work has revealed that many kinetochore components are highly dynamic and that some cycle between kinetochores and spindle poles along microtubules. Further studies of the kinetochore-microtubule interface are illuminating: (1) the role of the Ndc80 complex and components of the Ran-GTPase system in microtubule attachment, force generation and microtubule-dependent inactivation of kinetochore spindle checkpoint activity; (2) the role of chromosomal passenger proteins in the correction of kinetochore attachment errors; and (3) the function of microtubule plus-end tracking proteins, motor depolymerases and other proteins in kinetochore movement on microtubules and movement coupled to microtubule poleward flux.

Animals↗

PubMed2004

Stuck in division or passing through: what happens when cells cannot satisfy the spindle assembly checkpoint.

Cells that cannot satisfy the spindle assembly checkpoint (SAC) are delayed in mitosis (D-mitosis), a fact that has useful clinical ramifications. However, this delay is seldom permanent, and in the presence of an active SAC most cells ultimately escape mitosis and enter the next G1 as tetraploid cells. This review defines and discusses the various factors that determine how long a cell remains in mitosis when it cannot satisfy the SAC and also discusses the cell's subsequent fate.

Animals↗

PubMed2004

Kinetochore-microtubule interactions during cell division.

Proper segregation of chromosomes during cell division is essential for the maintenance of genetic stability. During this process chromosomes must establish stable functional interactions with microtubules through the kinetochore, a specialized protein structure located on the surface of the centromeric heterochromatin. Stable attachment of kinetochores to a number of microtubules results in the formation of a kinetochore fibre that mediates chromosome movement. How the kinetochore fibre is formed and how chromosome motion is produced and regulated remain major questions in cell biology. Here we look at some of the history of research devoted to the study of kinetochore-microtubule interaction and attempt to identify significant advances in the knowledge of the basic processes. Ultrastructural work has provided substantial insights into the structure of the kinetochore and associated microtubules during different stages of mitosis. Also, recent in-vivo studies have probed deep into the dynamics of kinetochore-attached microtubules suggesting possible models for the way in which kinetochores harness the capacity of microtubules to do work and turn it into chromosome motion. Much of the research in recent years suggests that indeed multiple mechanisms are involved in both formation of the k-fibre and chromosome motion. Thus, rather than moving to a unified theory, it has become apparent that most cell types have the capacity to build the spindle using multiple and probably redundant mechanisms.

Animals↗

PubMed2003

Human CLASP1 is an outer kinetochore component that regulates spindle microtubule dynamics.

One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. Here, we show that CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that we term the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. Our data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior.

Antibodies↗

PubMed2003

Dissecting mitosis by RNAi in Drosophila tissue culture cells.

Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated 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 of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

Journal Article↗

PubMed2002

MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity.

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 early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.

Alleles↗

PubMedDate not supplied

How do kinetochores CLASP dynamic microtubules?

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 that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizes to the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.

Animals↗