18 research outputs found
Análise funcional do domínio de ligação aos microtúbulos da proteína MAST
Mestrado em Métodos Biomoleculares AvançadosA Mast/Orbit/CLASP é uma família conservada de proteínas associadas aos
microtúbulos (MAPs) essenciais para a organização e função do fuso mitótico
(Inoue et al., 2000; Lemos et al., 2000; Akhmanova et al., 2001; Maiato et al.,
2002; Maiato et al., 2003a; Mimori-Kiyosue et al., 2005). Estas proteínas
surgem associadas aos microtúbulos, centrossomas e cinetocoros e diversos
estudos sugerem que desempenham um papel importante na regulação das
propriedades dinâmicas dos microtúbulos (Akhmanova et al., 2001; Maiato et
al., 2002; Maiato et al., 2003a; Maiato et al., 2005). As isoformas humanas,
CLASPs, fazem parte de um conjunto de proteínas (+TIPs) que exibem uma
forte acumulação na ponta de crescimento (+) dos microtúbulos em
polimerização (Schuyler and Pellman, 2001). Estas proteínas dissociam-se do
polímero formado o que origina uma localização em forma de cometa na
extremidade do microtúbulo. Neste trabalho mostramos que em Drosophila, a
proteína Mast também é uma +TIP. Adicionalmente, definimos o domínio de
ligação da proteína aos microtubulos e demonstrámos que, in vitro, a Mast se
associa directamente com a tubulina num processo sensível a nucleótidos de
guanina. O GTP favorece a ligação aos heterodímeros de tubulina, mas não
influencia a ligação aos microtubulos. Contrariamente, o GDP inibe fortemente
a ligação da Mast aos microtúbulos e heterodímeros de tubulina. Finalmente,
provamos que a Mast liga e hidrolisa GTP, o que a torna a primeira +TIP com
características de GTPase e sugere um novo mecanismo para a localização
dinâmica das +TIPs. Estes resultados são consistentes com um modelo no
qual a Mast-GTP copolimeriza com os heterodímeros de tubulina ou se
associa directamente à extremidade (+) dos microtúbulos em crescimento.
Após a associação ao microtúbulo dá-se a hidrólise do GTP e consequente
formação de Mast-GDP que causará uma alteração conformacional da
proteína promovendo a sua dissociação do microtúbulo. Este estudo sugere
que uma proteína associada aos microtúbulos pode utilizar a actividade
GTPásica na regulação da sua ligação aos microtúbulos.Mast/Orbit/CLASP is a conserved MAP protein family essential for the
organization and function of mitotic spindle (Inoue et al., 2000; Lemos et al.,
2000; Akhmanova et al., 2001; Maiato et al., 2002; Maiato et al., 2003a;
Mimori-Kiyosue et al., 2005). It accumulates at centrosomes, kinetochores and
microtubule plus-ends where it is thought to regulate their dynamic properties
(Akhmanova et al., 2001; Maiato et al., 2002; Maiato et al., 2003a; Maiato et
al., 2005; Mimori-Kiyosue et al., 2005). CLASPs, the human homologues
(Akhmanova et al., 2001), are members of the microtubule plus-end tracking
protein (+TIP) family (Schuyler and Pellman, 2001). +TIPs show strong
accumulation at the polymerizing end of microtubules, dissociating from the
polymer soon afterwards giving the appearance of a comet-like structure. Here
we show that the Drosophila homologue Mast also displays +TIP behaviour.
Moreover, we defined the microtubule binding domain of Mast and showed that
it associates directly with tubulin in a guanine nucleotide sensitive manner.
GTP favours the binding of Mast to tubulin heterodimers but does not influence
binding to microtubules, while GDP strongly inhibits the binding of Mast to
microtubules. More importantly, we show that Mast can bind and hydrolyse
GTP demonstrating that it is the first +TIP with this feature and hence
suggesting a new mechanism for +TIP behaviour. These results are fully
consistent with a model in which Mast-GTP copolymerizes with tubulin
heterodimers at the growing microtubule plus end. Mast is then released from
the polymer due to hydrolysis of the bound GTP, causing a conformational
change of the protein that promotes its release from the microtubule lattice. Our
data provides evidence that a microtubule associated protein could use its
GTPase activity to regulate its ability to bind microtubules.FCTPOCTI/BCI/49176/200
CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis
The deposited article is a post-print version (author's manuscript from PMC and available in PMC 2017 May 9).This publication hasn't any creative commons license associated.This deposit is composed by the main article and the supplementary materials are present in the publisher's page in the following link: https://www.sciencedirect.com/science/article/pii/S0960982216303001?via%3Dihub#sec4Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.ERC grant: (ERC-2010-StG-261344); FCT grants: (FCT Investigator, EXPL/BIM-ONC/0830/2013, PTDC/SAU-BD/105616/2008); EMBO installation grant.info:eu-repo/semantics/publishedVersio
SLAIN2 links microtubule plus end–tracking proteins and controls microtubule growth in interphase
SLAIN2’s interactions with multiple different microtubule plus end–tracking proteins stimulate processive microtubule polymerization and ensure proper microtubule organization
Mammalian end binding proteins control persistent microtubule growth
© 2009 Komarova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0. The definitive version was published in Journal of Cell Biology 184 (2009): 691-706, doi:10.1083/jcb.200807179.End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.This work was supported by the Netherlands Organization for Scientifi
c Research grants to A.A., by Funda ç ã o para a Ci ê ncia e a Tecnologia
fellowship to S.M. Gouveia, by a FEBS fellowship to R.M. Buey, by the National
Institutes of Health grant GM25062 to G.G. Borisy and by the Swiss
National Science Foundation through grant 3100A0-109423 and by the
National Center of Competence in Research Structural Biology program to
M.O. Steinmetz
PLK4 is a microtubule-associated protein that self assembles promoting de novo MTOC formation
The deposited article version is the Epub Ahead of Print version of the article (the "Accepted Manuscript"), posted online 20th September 2018, provided by Company of Biologists. It has peer-review.The deposited article version contains attached the supplementary materials within the pdf.The centrosome is an important microtubule-organizing centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical to recruit several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation (Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into condensates that recruit α/β-tubulin. In Xenopus extracts, PLK4 assemblies additionally recruit PLK4's substrate, STIL, and the microtubule nucleator, γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similarly to centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and α/β-tubulin, leading to MTOC formation.We are thankful to Anna Akhmanova, Raquel Oliveira and Jeffrey B.Woodruff for reading and discussing the manuscript. We are also thankful to Catarina Nabais for the GFP control construct and Vladimir Joukov for the Xenopus Cep192 antibody. S.M.G was funded by an EMBO Long term fellowship ALTF 1088-2009, a Marie curie Intra-European fellowship (#253373) and a FCT postdoctoral fellowship. The collaboration with J.L. laboratory in the USA was supported by a The Company of Biologists travel grant. S.Z is funded by ERC grant ERC-COG-683258. Research in JL lab was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. M.B-D. Laboratory is supported by an ERC grant ERC-COG-683258 and FCT Investigator to MBD.info:eu-repo/semantics/acceptedVersio
In Vitro Reconstitution of the Functional Interplay between MCAK and EB3 at Microtubule Plus Ends
The kinesin-13 family member mitotic centromere-associated kinesin (MCAK) is a potent microtubule depolymerase [1-4]. Paradoxically, in cells it accumulates at the growing, rather than the shortening, microtubule plus ends. This plus-end tracking behavior requires the interaction between MCAK and members of the end-binding protein (EB) family [5-8], but the effect of EBs on the microtubule-destabilizing activity of MCAK and the functional significance of MCAK accumulation at the growing microtubule tips have so far remained elusive. Here, we dissect the functional interplay between MCAK and EB3 by reconstituting EB3-dependent MCAK activity on dynamic microtubules in vitro. Whereas MCAK alone efficiently blocks microtubule assembly, the addition of EB3 restores robust microtubule growth, an effect that is not dependent on the binding of MCAK to EB3. At the same time, EB3 targets MCAK to growing microtubule ends by increasing its association rate with microtubule tips, a process that requires direct interaction between the two proteins. This EB3-dependent microtubule plus-end accumulation does not affect the velocity of microtubule growth or shortening but enhances the capacity of MCAK to induce catastrophes. The combination of MCAK and EB3 thus promotes rapid switching between microtubule growth and shortening, which can be important for remodeling of the microtubule cytoskeleton
Mammalian end binding proteins control persistent microtubule growth
End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners