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Research projects
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PostDoc May 2014- Oct. 2018
Mechanotrasnduction impacts on centrosome dynamics and duplication cycle
Vitiello et al, Nat Com, Jan 2019
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Location: Liphy, UGA, Grenoble, France
Advisor: Martial Balland
Awarded fellowship: ARC (Aide pour la recherche contre le cancer) postdoc grant
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In 2016, Farina et al reported that in vitro purified centrosomes are capable of nucleating actin fibres. Interestingly, actin cables are one of the main components of the mechanosensing cascade, due to their direct interaction with focal adhesions and cell-cell junctions, the main hubs of mechanic signal perception (Hytönen & Wehrle-Haller, 2016). Moreover, actin fibres in combination with the molecular motor myosin II represent the major force generating apparatus of the cell. Contractile forces are required for a great variety of cellular processes such as cell division, cell motility, vesicle trafficking and organelle positioning. This implies that upon mechanosensing stimuli, acto-myosin fibres rearrange in particular configurations, and produce specific force profiles, which will dramatically impact the life of a cell.
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During my postdoc I investigated whether acto-myosin fibres could affect the centrosome duplication cycle. The basic idea of my hypothesis was that acto-myosin cables could control centrosome duplication via regulation of centriole separation. In 2015, Shukla et al suggested that higher centriole-to-centriole distances, which they observed in prophase (up to 300 nm), could act as a block to reduplication (Shukla et al., 2015). Driven by these data, indicating that centriole separation is associated to centrosome duplication, I decided to control actin organization and traction force profiles and monitor the response on centriole separation and centrosome duplication.
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Under the supervision of Dr Martial Balland, I utilized fibronectin micropattens printed on soft hydrogels to impose geometrical constraints and induce precise acto-myosin organization and force profiles (Mandal et al, 2014). Via actin orientation quantification and traction force microscopy (TFM) I was able to characterize a set of geometrical shape inducing low degree of acto-myosin force polarity (Square and Tripod: random organization of actin and traction force axis); and high degree of acto-myosin force polarity (H and T: with one/2 main axis of actin orientation and force).
By imaging HeLa cells stably transfected with Centrin1-GFP (centriolar marker) on different micropatterns, I observed that the degree of acto-myosin force organization controls centrosome separation direction and distance: the more oriented the acto-myosin force axis is the more the centrioles would be able to separate; moreover, the separation direction would follow the direction of the force axis imposed by the geometrical constrain. Accordingly, inhibition of acto-myosin contractility impairs centriole separation.
Alongside, we reported that the degree of acto-myosin force organization regulates the efficiency of centriole duplication: cells with high degree of acto-myosin force polarity (H and T) would perform correct centrosome duplication (resulting in 4 centrin dots) in about 90% of the cases, whereas cells with low degree of acto-myosin force organization would fail replicating the centrosome (>4 centrin dots) in 40% of the cases. Additionally, we showed that acto-myosin forces influence centrosome duplication by tuning PLK4 recruitment at the centrosome to prevent centriole amplification (Fig.1)
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Fig.1 Scheme of postdoc work.
Panel A illustrates the micropatterns (adhesive geometrical surfaces) utilized: they were characterized for actin orientation and traction force via TFM: as shown in the figure, two sets of shape were identified (Square and Tripod with low acto-myosin force organization, and H and T with high acto-myosin force organization). Panel B: by Centrin1-GFP live imaging, I showed that the shapes with high organization of acto-myosin forces separate for wider distances the two centrioles. Panel C: cells with high organization of acto-myosin forces could control centrosome duplication more efficiently. Panel D: model.
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PhD thesis Sept 2009- Apr 2014
DLC2 plays a role inmitotic spindle positioning and cell junction crossstalk
Vitiello et al, Nat Com, Dec 2014
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Location: Cell biology, UCL, Institute of Ophthalmology, London, UK
Advisor: Karl Matter
Awarded fellowship: MRC (Medical research council)
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Dividing epithelial cells need to coordinate spindle positioning with shape changes to maintain cell-cell adhesion. Microtubule interactions with the cell cortex regulate mitotic spindle positioning within the plane of division. How the spindle crosstalks with the actin cytoskeleton to ensure faithful mitosis and spindle positioning
has been unclear.
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During my PhD, I demonstrated that the tumour suppressor DLC2, a negative regulator of Cdc42, and the interacting kinesin Kif1B coordinate cell junction maintenance and planar spindle positioning by regulating microtubule growth and crosstalk with the actin cytoskeleton. Loss of DLC2 induces the mislocalization of Kif1B, increased Cdc42 activity and cortical recruitment of the Cdc42 effector mDia3, a microtubule stabilizer and promoter of actin dynamics. Accordingly, DLC2 or Kif1B depletion promotes microtubule stabilization, defective spindle positioning, chromosome misalignment and aneuploidy. The tumour suppressor DLC2 and Kif1B are thus central components of a signalling network that guides spindle positioning, cell-cell adhesion and mitotic fidelity (Fig.2).
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Fig.2 DLC2 is required for junction maintenance and mitotic spindle stability.
(Left panel) Control and DLC2-depleted HCE cells stained for the adherens junction proteins α-catenin (red), α-tubulin and DNA (blue). This figure showes that depletion of DLC2 affects junctions stability and mitotic spindle assembly in epithelial cells. (Right panel) Proposed model for the action of DLC2 and Kif1B in metaphase plate stabilization and spindle positioning. The cartoon shows that to position the spindle and maintain epithelial integrity balanced negative regulation of Cdc42 by DLC2 is required. If DLC2 is inactivated, Cdc42 activity is enhanced and leads to the activation of mDia3, triggering increased microtubule stability and disorganization of cortical actin. The increased microtubule length leads to a loss of normal tension control, distortion of the spindle and, hence, defects in metaphase plate maintenance and cohesion fatigue.
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Master thesis/internship Mar 2007- July 2009
MLK4 mutations accelerate tumour growth in colorectal cancer
Martini et al, Cancer Research, Mar 2013
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Location: Institute for cancer research and treatment (IRCC), Oncogenomics center Candiolo (To), Italy
Advisor : Alberto Bardelli
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Colorectal cancer (CRC) is one of the most common and lethal types of cancer with more than 1.4 million people diagnosed every year, and over 600,000 deaths (Arnold et al., 2017). Unfortunately, the survival chances of CRC patients decreases with the increase of the degree of cancer discovered. It is indeed
necessary to identify markers or mutations that could allow an early detection. In the light of these hopes, Dr. Martini and Prof. Bardelli performed a comprehensive mutational analysis of CRC samples. They identified the mixed lineage kinase 4 (MLK4) protein kinase as a protein frequently mutated in CRC with approximately 50% of the mutations occurring in KRAS- or BRAF-mutant tumours. Dr. Martini showed that previously identified MLK4 mutations in CRC lead to a constitutively active form of the kinase and an increase of the transformation and tumorigenic capacity of RAS-mutated cell lines.
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When I joint the lab of Prof Bardelli, I helped characterizing how MLK4 knockout or knockdown is sufficient to reduce the oncogenic power of KRAS- and BRAF-mutant cancer cells both in vitro and in xenograft models. My strongest contribution to this project focused on dissecting the molecular pathway MLK4 mutations acted on. In particular, we showed that MLK4 directly phosphorylates MEK1 (MAP2K1), causing an acceleration of cell growth. Thus, MLK4 knockout impairs the overactivation of MEK/ERK (MAPK1) signalling, which in turn reduces the tumorigenic power of the knockout cell lines. Overall, these findings suggested that MLK4 inhibitors might represent an efficient target molecule in KRAS- and BRAF mutated CRCs.
