Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

Nucleation and organization of microtubules by gamma-tubulin complexes – gTuRC

Nucleation and organization of microtubules by gamma-tubulin complexes

The goal of this project is to understand how microtubule nucleation from gamma-TuRC multiprotein complexes can be activated, and how gamma-TuRCs are anchored to the centrosome, and whether these two aspects can be functionally separated. We want to test the role of accessory proteins that may participate in triggering microtubule nucleation by supporting conformational changes within the gamma-TuRC, and that may play a role in anchoring gamma-TuRCs to microtubule-organizing centres.

1) Activation of microtubule nucleation by gamma-TuRC-binding proteins; 2) Conformational changes within GCP proteins ; 3) Cellular role of ninein

1) Activation of microtubule nucleation by gamma-TuRC-binding proteins <br />a) A variety of proteins that are known to bind to gamma-TuRCs will be tested for their potential to activate microtubule nucleation in vitro. The effects of these proteins on conformational changes within the gamma-TuRC, as part of the activation process, will be tested. Microscopical methods will be used for analysis, to follow microtubule nucleation. <br />b) A gamma-TuRC-binding protein that has previously been implicated in activating gamma-TuRC-dependent nucleation of microtubules possesses a consensus sequence that is conserved among many species and that is sufficient for activation. A synthetic peptide of this sequence will be used to study its potential interaction with individual, purified recombinant gamma-TuRC proteins by NMR, to conclude on the molecular mechanism of activation. <br /><br />2) Conformational changes within GCP proteins (partner 2): <br />We will clone and purify recombinant domains of the gamma-TuRC proteins GCP2 and GCP3 and solve their atomic structures by X-ray crystallography. This will enable us to conclude on important structural and conformational differences between GCPs 2 and 3. This will provide crucial information to understanding the mechanisms of microtubule nucleation, which appears to be activated (at least in part) by conformational changes within GCP3. <br /><br />3) Cellular role of ninein in nucleation and anchorage of microtubules: <br />We will study the role of ninein and other centrosomal proteins in binding gamma-TuRCs, using cells without ninein. This experimental system will enable us to study the function of discrete ninein protein domains on a background that is “null” for endogenous ninein. Moreover, this will allow us to evaluate the contribution of other proteins in binding gamma-TuRCs to the centrosome, in the absence of ninein.

The project involves biochemical purification of recombinant proteins, and study of their interaction using biophysical methods, including NMR, fluorescence resonance energy transfer, dynamic light scattering, size exclusion chromatography-multiple angle laser light scattering, differential scanning fluorimetry, X-ray crystallography, and fluorescence microscopy. The study of purified proteins will be accompanied by analysis at a cellular level, involving cell culture and transfection.

We have purified native gamma-tubulin complexes from a cell line expressing tagged gamma-tubulin. We have characterized these complexes in a series of crosslinking studies to determine protein interactions between specific GCP constituents. Moreover, we have expressed and purified various recombinant fusion proteins of previously characterized gamma-TuRC interactors from bacteria. Part of these proteins turned out to be highly insoluble unless solubilised under denaturing conditions, and unsuited for biophysical interaction studies. Others could be purified in a soluble form under non-denaturing conditions, allowing us to pursue our planned project. To study the interactions with GCP proteins, and to address the problem of conformational changes within the gamma-TuRC during its activation, we have purified the main GCP proteins, GCPs 2 and 3. Expression of the full-length constructs of GCPs 2 and 3 failed to yield functional protein. We attempted the purification of the Grip2 domains of both proteins because they contain the gamma-tubulin binding site. We faced again the difficulty of poor yield of soluble protein. A variety of temperatures were chosen for bacterial growth and induction of protein expression. Also, special tags were added to increase the yield of soluble recombinant protein. Our approaches still require further optimization. Other protein domains (Grip 1 and the extreme amino-terminal domains) were cloned into expression vectors, and expression studies have been initiated.
To complement our biochemical/biophysical studies by cell biological characterization of the gamma-TuRC function, we have studied GCP interactions within gamma-TuRCs at the centrosome of living cells, using FRET/FLIM analysis. Moreover, we have studied the role of a potential gamma-TuRC anchoring protein at the centrosome, ninein. The cellular roles of ninein are currently being studied.

In the next phase of the project, we will use the tools generated so far (plasmid constructs, purified proteins, cell lines) for biophysical and cell biological studies. The goal will be to characterize the molecular mechanisms of microtubule nucleation by gamma-TuRCs, and to understand the specific cellular function of individual interacting proteins in the activation process.

N/A

Multiprotein complexes of gamma-tubulin are essential in all eukaryotes for the formation of an intact microtubule network and for correct microtubule dynamics. In most organisms, these multiprotein complexes exist in the form of small, open rings called “gamma-Tubulin Ring Complexes”, or “gamma-TuRCs”. Gamma-TuRCs exist largely in a soluble, inactive pool in the cytoplasm, and in a smaller active pool that is bound to the centrosome or to other microtubule-organizing centres. In many cells, the potential of microtubule nucleation by the gamma-TuRC is thus correlated with its binding to the centrosome. A variety of centrosomal proteins have been previously characterized as gamma-TuRC-binding proteins. Among these, the protein Cdk5rap2 has recently been described as an activator of the gamma-TuRC, containing an amino-terminal domain, CM1 that is sufficient for activation. Most interestingly, this CM1 domain appears to activate gamma-TuRCs independent of their location, i.e. even in the cytoplasm, in a soluble form. This offers the interesting perspective of uncoupling the binding and the activation of the gamma-TuRC. In this project, we will characterize the mechanisms by which Cdk5rap2 may activate the gamma-TuRC, and to what extent other gamma-TuRC-binding proteins may exert any similar activating function. Since gamma-TuRC activation is thought to involve conformational changes within the gamma-TuRC, in particular by one of its structural components, GCP3, we will test the various gamma-TuRC subunits for their potential to bind to CM1, and we will test whether the conformation of GCP3 is altered by CM1 binding. This analysis will involve biophysical methods such as NMR and fluorescence resonance energy transfer (FRET). To understand the potential alterations in conformation at a structural level, we will determine the crystal structures of the “flexible” GCP3 and of the more “rigid” GCP2, using X-ray crystallography of the different domains of these proteins, and using molecular replacement and modelling with our previously published structure of the related gamma-TuRC subunit GCP4. We will further characterize the mechanism of gamma-TuRC-anchoring to the centrosome, by investigating the role of the protein ninein. Using cells from a ninein-knockout mouse, we will characterize the various protein domains of ninein that have previously been implicated in microtubule nucleation and anchoring, on a “ninein-null” background. Moreover, we will test the contribution of other centrosomal proteins that may interact with ninein and that may contribute to the activation and binding of the gamma-TuRC.

Project coordination

Andreas MERDES (Centre de Biologie du Développement) – andreas.merdes@univ-tlse3.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Université Toulouse III Centre de Biologie du Développement
CNRS Institut de Pharmacologie et de Biologie Structurale
CNRS Institut de Pharmacologie et de Biologie Strucutrale

Help of the ANR 350,000 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

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