Study of the mechanisms by which podosomes recognize nanometric topographies
PhD committee
- Dr. Laura PICAS ESCOFET - Rapporteur - Institut de recherche en infectiologie de Montpellier (IRIM)
- Dr. Pierre-Henri PUECH - Rapporteur - Laboratoire adhésion et inflammation (LAI) Marseille
- Dr. Violaine MOREAU - Rapporteur - Institut de recherche en oncologie de Bordeaux (BRIC)
- Dr. Christophe VIEU - Examiner - Institut national des sciences appliquées de Toulouse (INSA)
- Dr. Renaud POINCLOUX - PhD supervisor - Institut de pharmacologie et biologie structurale (IPBS)
- Dr. Christophe THIBAULT - PhD co-supervisor - Institut national des sciences appliquées de Toulouse (INSA)
Summary
Macrophages are innate immune cells present in all body tissues. They play a fundamental role in host defence, but their massive infiltration of pathological tissues can, in certain contexts such as chronic inflammation or cancer, promote disease progression. Although cell migration has been widely studied on two-dimensional substrates, the extracellular matrix in vivo exhibits complex architectures, ranging from the nanometric relief of the collagen fibre network to micrometric-sized three-dimensional structures such as interstitial channels. While it is known that these structured environments influence cellular behaviour, the mechanisms by which cells detect geometries at such small scales remain poorly understood.
Macrophages form adhesion structures, called podosomes, to probe their environment. The aim of this thesis is to better understand how podosomes perceive and interpret the topographical features of their environment.
As a first step, a method for manufacturing linear topographies etched directly into glass was optimised. Topographies with controlled dimensions ranging from 2 to 300 nanometres in height, compatible with fluorescence microscopy imaging, were successfully produced.
Secondly, characterisation of macrophage responses to these topographies revealed cell elongation and podosome alignment along the patterns, as well as organisational modulation and accumulation of some of their components.
By quantifying these parameters, we estimated detection threshold of topographies between 10 and 20 nanometres in height. Finally, to explore the molecular mechanisms involved in this detection, macrophages were genetically modified using the CRISPR/Cas9 tool. Knock-out studies targeting integrins heterodimers revealed the necessary role of integrin αMβ2 (Mac-1) in the formation and organisation of podosomes on these structured substrates. Moreover, knock-outs generated for several BAR domain proteins, known for their ability to form and detect membrane curvature, began to reveal the involvement of these proteins in the recognition of nanoscale topographies.