The role of neuronal integrin-mediated adhesions in sensing biochemical and mechanical properties of the extracellular environment

Abstract

In fibroblasts integrin receptors are the primary adhesion receptors and act as the "sensory organs" of cells to sense external signals. My aim was to understand how integrins, together with the adhesion-associated plaque proteins Talin and Vinculin, regulate sensing of extracellular biochemical and mechanical stimuli in neurons. A first set of experiments served to characterise the sensing behaviour of CNS-derived primary cells and cell lines on different extracellular matrix (ECM). Integrin antibodies helped to characterise their adhesion receptor profiles and showed that specific integrin family members were involved in adhesion and axon/neurite outgrowth when plated on ECM proteins of laminin (LN), fibronectin, and collagen. Data showed that integrin-ECM interactions affected axon/neurite outgrowth and pathfinding. Presentation of LN promoted axon/neurite outgrowth and furthermore, presenting it in stripes dramatically enhanced outgrowth speeds showing that not only ECM but how it is presented directs growth. In a further set of experiments I established procedures that enabled me to determine the impact of mechanosensing in axon/neurite outgrowth. The established methodology helped to define point contact areas at the base of filopodia as sites of traction force exertion. Growth cones exerted dynamic stresses of around 10-30 Pa. To examine whether and how growth cones sense mechanical differences in their surrounding environment axon/neurite outgrowth was tracked when cells were plated on polyacrylamide. Data showed that axon/neurite outgrowth lengths and speeds were significantly longer and faster, respectively, on soft (0.4 kPa) than on stiff (8 kPa) substrates. Interestingly, the depletion of vinculin in neurons blocked the ability of growth cones to determine between soft and stiff substrates. Altogether, the interdisciplinary approach in this study helped to gain new insight into how neurons sense biochemical and mechanical properties of the ECM.

Date of Award	1 Aug 2015
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andreas Prokop (Supervisor) & Christoph Ballestrem (Supervisor)