The detailed force profiles of struts in the OT structure were further assessed during spreading (Supplementary Fig. S4).
This idea is supported by our observation that conventional (cPKC) and novel (nPKC) PKC isoforms are also not required for EPAC1-dependent cell spreading (Supplementary Fig. 6).
The intensities of FHOD1 and Myosin 1E were found to change in unison during the protrusive and contractile phases of lamellipodia spreading (supplementary material Movie 4).
Subsequent to the establishment of integrin KD in hMSC, we investigated for changes in their cell attachment, spreading (Supplementary Figures 2 and 4b) and migration on collagen I.
Tip accumulation of Myosin 1E increased as the lamellipodia moved forward in the contractile spreading phase and peaked at the end of active spreading (supplementary material Movie 1; Fig. 1H,I white arrows).
In addition, the X and Y chromosomes were partially synapsed at the pseudoautosomal region in both Bscl2+/+ and Bscl2−/− pachytene spreads (Supplementary Figure S5), indicating that Bscl2−/− spermatocytes did not present obvious synaptic errors during prophase I.
Treatment with channel blocking drugs, probenecid (Pbn, 1 mM, blocks pannexin channels) and lanthanum (La3+, 200 µM, blocks Cx channels) also had no effect on B cell spreading (supplementary material Fig. S2A D).
As reported previously (Choi et al., 2008), GFP-paxillin was not seen initially at the tip (Fig. 2F initial spreading; supplementary material Fig. S4D; Movie 6), but appeared strongly with the onset of contractile spreading phase (Fig. 2F white dotted arrow; supplementary material Movie 6).
Despite being activated in response to cAMP, inhibition of ERK activity with the MEK inhibitors, 1 μM AZD6244 and 1 μM PD184352, had little effect on EPAC1-dependent cell spreading (Supplementary Fig. 5A and B) indicating that although ERK is a downstream target for cAMP signalling this occurs independently to the changes in cell morphology associated with activation of EPAC1.
The variants appear spread within supplementary material and in tables.
