Pier Paolo D'Avino
Defects in cell division often cause abnormal distribution of the genetic material between the two daughter cells. These abnormalities generate genetic instability, which is considered to be a key step in the development of human cancer. Therefore, an understanding of the mechanisms that control the activity of cell cycle regulatory proteins may lead to the identification of therapeutic targets for the treatment of tumour pathologies.
My recent research interests focus on the mechanisms and signalling pathways that control the final act of mitosis, cytokinesis. In a typical animal mitosis, a cleavage furrow forms at the equatorial cortex after anaphase onset and advances inwards to generate two daughter cells. The ingression of this furrow is achieved through the assembly and contraction of actomyosin filaments whose dynamics appears to be regulated by the small GTPase RhoA. I am currently trying to address two major questions: (i) What is the nature of the signal that positions the cleavage plane during cytokinesis and (ii) what are the signaling components downstream of RhoA that control actomyosin filaments dynamics.
The finely orchestrated process of cytokinesis requires the harmonized action of a large set of proteins to regulate three major processes: (1) the determination of the cleavage plane; (2) the assembly and contraction of the actomyosin ring; and (3) the formation and addition of new membrane between the two daughter cells. In order to execute specific functions, these proteins associate in stable or transient multiprotein complexes whose interactions are regulated by enzymatic complexes that mediate spatio-temporally coordinated protein degradation and phosphorylation/dephosphorylation. Thus, a thorough understanding of the mechanisms that control cytokinesis requires the identification of all the components that constitute the plethora of complexes involved in cytokinesis.
This is the aim of the work of the Cell Cycle Proteomics Group, a collaboration involving Vincent Archambault, Marcin Przewloka (Glover group), Ernest Laue, Wei Zhang and Kathryn Lilley (Department of Biochemistry, University of Cambridge). To this goal, we have adapted the TAP (Tandem Affinity Purification) methodology to Drosophila S2 cells.
This method consists of the fusion of proteins of interest with two different affinity purification tags. Cell lines stably expressing these proteins are generated and the isolation of the TAP-tagged bait products and their partners is accomplished through single or double affinity purifications. The purified protein complexes are then resolved by SDS PAGE and identified by mass spectrometry.
We have tested a series of tags and created constitutive and inducible vectors for either amino- or carboxy-terminal tagging. We have used these vectors to tag several proteins known to function in different aspects of cytokinesis. These include microtubule associated proteins (MAPs), molecular motors and their cargoes, kinases, and components of the Rho signalling and membrane trafficking pathways. Purification and characterization of complexes containing these tagged proteins has confirmed previously known interactions and identified novel partners.
Areas of interest:- Cytokinesis
- Signalling pathways
- Cytoskeleton architecture
Dr. Paolo D'Avino
Address:
Department of Genetics,
University of Cambridge,
Downing Street,
Cambridge,
CB2 3EH,
England.
Email:
ppd21@
mole.bio.cam.ac.uk
D'Avino PP, Savoian MS and Glover DM (2005)
Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy.
Journal of Cell Science 118: 1549-1558
D'Avino PP, Savoian MS and Glover DM (2004)
Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac.
Journal of Cell Biology 166: 61-71
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