Integrin binding dynamics modulate ligand-specific mechanosensing in mammary gland fibroblasts

Abstract

The link between the modulation of integrin activity and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are mechanically distinct from previously studied cell types. In particular, MSFs generate high forces at a low matrix stiffness, equivalent to the soft mammary gland tissue, supported by maximal focal adhesion maturation, strong actin stress fiber formation, and myosin phosphorylation.We describe that loss of the cytosolic integrin inhibitor, SHARPIN, triggers impeded spreading of MSFs specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modelling, we find that reduced expression of the collagen-binding integrin α11β1 in SHARPIN-deficient MSFs leads to faster force-induced unbinding of adhesions from collagen. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells, and increases actin retrograde flow. Our results unveil a collagen-specific mechanosensing mechanism and suggest a key function for integrin activity regulation and integrin α11β1 in MSF mechanotransduction.

Fluctuation-Based Super-Resolution Traction Force Microscopy

“…To allow for functionalization, TFM gels were incubated for 30 min at RT with an activation solution [0.2 mg/mL of Sulfo-SANPAH (Thermo Fisher Scientific, 22589), 2 mg/mL N -(3-(dimethylamino)­propyl)- N ′-ethylcarbo­diimide hydrochloride (EDC) (Sigma-Aldrich, 03450) diluted in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine­ethanesulfonic acid; Sigma-Aldrich, H0887)] under gentle agitation . The glass-bottom dishes containing the gels were then irradiated, without their plastic lids, with ultraviolet (UV) light for 10 min using a UV-chamber (Jelight Company Inc., UVO CLEANER, 342–220).…”

“…To allow for functionalization, TFM gels were incubated for 30 min at RT with an activation solution [0.2 mg/mL of Sulfo-SANPAH (Thermo Fisher Scientific, 22589), 2 mg/mL N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) (Sigma-Aldrich, 03450) diluted in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Sigma-Aldrich, H0887)] under gentle agitation. 49 The glass-bottom dishes containing the gels were then irradiated, without their plastic lids, with ultraviolet (UV) light for 10 min using a UVchamber (Jelight Company Inc., UVO CLEANER, 342−220). Gels were then washed three times with PBS and coated with The structured illumination microscope (SIM) used was DeltaVision OMX v4 (GE Healthcare Life Sciences) fitted with a 60x Plan-Apochromat objective lens, 1.42 NA (immersion oil RI of 1.516) used in SIM illumination mode (five phases × three rotations).…”