Images (16 bit depth) were acquired using an Orca-ER (C4742-80) Cooled CCD digital camera (Hamamatsu Italy, Milan, Italy)

Images (16 bit depth) were acquired using an Orca-ER (C4742-80) Cooled CCD digital camera (Hamamatsu Italy, Milan, Italy). between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of MK-2206 2HCl MK-2206 2HCl the molecular clutch. cell adhesion receptors. These non-integrin adhesion receptors, including syndecans, discoidin domain name receptors and CD44, are MK-2206 2HCl thought to mediate transmission transduction and cytoskeleton coupling by lateral associations with integrins (Schmidt & Friedl, 2010). One such non-integrin adhesion receptor is the urokinase-type plasminogen activator receptor (uPAR) that promotes cell adhesion through its direct interaction with the provisional ECM protein vitronectin (VN) (Wei is usually supported by observations that this expression of crucial genes, required for embryo development, is supported by integrin chimeras lacking the ligand-binding domain name (Martin-Bermudo & Brown, 1999). Furthermore, ligand-binding deficient mutants of v3 are qualified in supporting tumour growth through the formation of an oncogenic complex with SRC kinase (Desgrosellier em et?al /em , 2009). Ligand-independent integrin signalling shares many common features with canonical integrin signalling including the requirement for an active conformation of the integrin, the binding of intracellular scaffolding proteins, as well as force generation on a rigid ECM. What clearly distinguishes the two types of integrin signalling, aside from the requirement for ligand binding, is the role of membrane Mouse monoclonal to 4E-BP1 tension. In canonical integrin signalling, the relaxation of membrane tension does not impair cell distributing but rather increases it (Raucher & Sheetz, 2000). Membrane tension is in fact known to antagonise cell protrusions and to rise during cell distributing and polarisation (Raucher & Sheetz, 2000; Houk em et?al /em , 2012). In the ligand-independent integrin signalling, explained here, the relaxation of membrane tension abrogates cell distributing, while increasing membrane tension MK-2206 2HCl enhances cell distributing. This is possibly explained by the finding that in ligand-independent integrin signalling, the (tense) membrane is usually a critical component of the molecular clutch responsible for force transmission between the extracellular matrix and the cytoskeleton. In canonical ligand-dependent integrin signalling, the membrane is not an integral component of the clutch as integrins directly connect the ECM and the cytoskeleton (observe cartoon in Fig ?Fig88). Consistent with our finding that membrane tension is critical for cell distributing on non-integrin substrates, it has previously been reported that non-ligated 1 integrins are localised at the leading edge during cell protrusion (Galbraith em et?al /em , 2007), coinciding with zones of high membrane tension (Houk em et?al /em , 2012). The biological importance of membrane tension is usually furthermore substantiated by studies showing that membrane tension is required for the polarisation of neutrophils (Houk em et?al /em , 2012) and for efficient cell migration and lamellipodia organisation (Batchelder em et?al /em , 2011). Material and Methods Materials HEK 293 Flp-In T-REx cells, expression vectors pcDNA5/FRT/TO and pOG44, zeocin, blasticidin S HCl and F-12 (Ham) medium were from Invitrogen. Dulbecco’s altered Eagle’s medium (DMEM) was from Lonza. PBS, trypsin, glutamine, penicillin and streptomycin were obtained from EuroClone, while foetal bovine serum (FBS) was from HyClone. Non-tissue culture plates were from Falcon Becton Dickinson. Tetracycline, poly-L-lysine, anti-vinculin antibody (hVIN-1) and MK-2206 2HCl CHO protein-free culture medium were from Sigma. FuGENE 6, fibronectin and hygromycin B were from Roche. Pro-uPA was kindly provided by Dr. Jack Henkin (Abbott Laboratories). Antibodies against total (cat no. 13383) and phosphorylated p130Cas (cat no. 4011), total ERK1/2 (cat. no. 9102) and phosphorylated ERK1/2 (cat. no. 9101) were from Cell Signalling Technology. The talin monoclonal antibody (cat. no. T3287) was from Sigma. Blocking antibodies against v3 (LM609), 51 (P1D6) and v5 (P1F6) integrins were obtained.