Asymmetric cell division is the main mode of division of stem cells and plays a central role in generating cell diversity during development. Indeed, during asymmetric cell division of polarized cells, biomolecules are asymmetrically segregated between daughter cells. The position of the mitotic spindle within the cell determines whether the cell will divide symmetrically or asymmetrically. Spindle positioning, in turn, depends on the interaction of astral microtubules (cMTs) with cortical factors. A complex network of proteins, such as non-motor microtubule-associated proteins (plus-end tracking proteins, +TIPs), kinesins, dynein and actin-interacting proteins, are involved in these interactions.
Budding yeast also divides asymmetrically and is used as a model organism to study asymmetric cell division. Spindle positioning in yeast depends on two genetically identified pathways: i) the dynein pathway that includes complexes of the microtubule-dependent motor dynein, and ii) the Kar9 pathway. Kar9 is the yeast functional equivalent of the Adenomatous Polyposis Coli (APC) tumour suppressor, a protein with a central role in spindle positioning from Drosophila to mammals. Dynein, Kar9 and APC homologues are universal mediators of spindle positioning during eukaryotic asymmetric division, but their regulation is poorly understood.

In the laboratory, we explore how cell cycle regulators modulate the temporal activation and dynamics of dynein and Kar9 complexes during spindle positioning in budding yeast. During their transport along microtubules and interactions with cortical proteins, these factors form highly dynamic protein complexes. Cyclin-dependent kinases control both the formation of these complexes and the coordination of their activity with other cytoskeletal events, such as chromosome segregation and cytokinesis. To understand the regulation of Kar9 and dynein complexes we use genetics to study their interactions. Using protein biochemistry, we analyse the Kar9/dynein complex composition and phosphorylation. Finally, we investigate complex dynamics in living yeast cells by time-lapse fluorescence imaging.

Bone is a highly dynamic, mineralized tissue. Indeed, the whole skeleton is renewed in about 10 years. Bone dynamics (homeostasis) is ensured by two cell types: osteoclasts and osteoblasts. Osteoclasts degrade the bone matrix: they solubilize the mineral components and digest proteins, forming Howship’s lacunae at the bone surface that are then filled in by osteoblasts. Therefore, the skeleton dynamic homeostasis to ensure the maintenance of the bone mass depends on the proper coordination of the osteoclast and osteoblast activities.

Osteoclast activity is abnormally high in several physiopathological situations. In these cases, bone loss cannot be compensated by new bone formation and bones become more fragile. Different hormonal disorders are associated with increased osteoclast activity, for instance in post-menopausal women. Very active osteoclasts can be observed also in people with inflammatory diseases, such as rheumatoid arthritis, as well as in people with bone metastases.

Our group studies the molecular mechanisms that regulate osteoclast bone-resorption activity. These giant multinucleated macrophages are the only body cells that can degrade the mineralized extracellular matrix of bone. Specifically, we are interested in the Rho family of GTPase signalling molecules. These molecules regulate the dynamics of actin, which plays a major role in the different aspects of osteoclast biology, particularly during their differentiation and during their bone matrix degradation activity.

Our more recent work has highlighted the essential role of WRCH1/RHOU during osteoclast differentiation. WRCH1/RHOU controls the adherence and migration of osteoclast precursors by interfering with the signalling by the αvß3 integrin or by the receptor of vitronectin, the main integrin in osteoclasts. WRCH1/RHOU is also essential for the fusion of precursors during osteoclast differentiation.
We have also demonstrated that the exchange factor DOCK5, a Rac activator, controls the organization of podosomes in osteoclasts through a mechanism that involves p130Cas/BACR1, downstream of the αvß3 integrin. DOCK5 promotes the organization of the osteoclast podosome belt in order to form the sealing zone, which is the osteoclast-specific adhesion structure that is essential for bone degradation. We identified an inhiibtor of Dock5 that can protect against pathological bone loss in various models of osteolytic diseases. Our results open novel therapeutic possibilities against osteoporosis.

To study bone degradation by osteoclasts and its regulation by the Rho family of GTPases, we use different approaches:
– we study osteoclasts and their bone resorption activity in culture. This allows us to analyse their adhesion structures by fluorescence microscopy and to follow their dynamics by time-lapse microscopy. Particularly, we assess how the Rho GTPase signalling pathways influence osteoclast motility and their bone degradation capacity.

– we also study the bone dynamics in different mouse models. We use mouse lines in which interesting genes have been genetically ablated and also mouse models of bone disorders, such as post-menopausal osteoporosis, rheumatoid arthritis or bone metastases. We carry out histo-morphometric and micro-tomographic analyses in order to understand how the actin dynamics in osteoclasts affects the normal and pathological skeleton homeostasis.