Effect of mechanical stiffness on axonal βII-spectrin in three dimensions.

GF MARTÍNEZ; N UNSAIN; A RICHERI

Santa Cruz

Congreso; IUBMB EMBO Workshop Emerging Concepts of the Neuronal Cytoskeleton. Santa Cruz, Chile.; 2023

EMBO

Introduction: β-spectrin forms a sub-membranous skeleton responsible for maintaining cell adaptationto changing environments and axon stability in response to mechanical stress. Recently, wedemonstrated that substrate stiffness, a mechanical property, negatively impacts axon elongation in athree-dimensional culture (3D environment). The reduced elongation was accompanied by a strongimpact on axonal F-actin cytoskeleton. Almost no F‐actin rich growth cones were recognized, andF‐actin staining was strongly reduced in the axonal compartment. However, the impact of thismechanical property of the stiff 3D environment on other components of the axonal cytoskeletonremains unclear. Aims: To evaluate the impact of a 3D stiff environment on βII-spectrin in two axonalcompartments: axon and Growth Cone (GC). Methods: Superior cervical ganglion explants fromneonatal (4-6 days old) rats were cultured in soft (G´= 18.5 Pa) and stiff (G´= 22.7 Pa) Collagen-3Dmatrices. At 2 days after seeding, 3D-cultures were processed to evaluate: a) the pattern of expressionof βII-spectrin; b) βII-spectrin levels of fluorescence intensity through immunocytochemistry andquantitative fluorescence microscopy. Results: Sympathetic axons grown in a 3D stiff environmentshowed higher spectrin levels than those grown in soft 3D matrices. Interestingly, some axons showedregularly spaced βII-spectrin-immunoreactivity along them in both culture conditions (soft and stiff).Also, preliminary observations indicate that, in soft matrices, βII-spectrin accumulates in axonal tips(near filopodia/GC). Conclusion: Our results suggest that ECM stiffness increases axonal βII-spectrin,which could be indicating an adaptive behavior triggered by mechanical interactions of sympatheticaxons with a stiff 3D environment.