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. 2021 Apr 16;296:100675. doi: 10.1016/j.jbc.2021.100675

Figure 1.

Figure 1

Optogenetic recruitment of talin to the plasma membrane leads to activation of integrin αIIbβ3.A, linear depiction of talin domains. In a structural model of “autoinhibited” talin (15), the interactions between rod domain R9 and FERM subdomain F3 (red dashed line) and R12 and F2 (blue dashed line) limit F3 domain access to plasma membrane lipids and integrin β tails. Talin F1 contains a unique amphipathic helix loop that may facilitate talin interaction with the plasma membrane. B, depiction of the CIBN–GFP–CAAX and CRY2–mCherry–talin fusion proteins that were stably expressed in A5 CHO cells. CIBN–GFP–CAAX is constitutively anchored to the plasma membrane. As a result, when the cells are exposed to 450-nm (blue) light, the CIBN moiety should interact with CRY2, resulting in talin recruitment to the plasma membrane. C, a model of CRY2–mCherry–talin using atomic coordinates of each protein (CRY2PHR, mCherry, and talin head domain (THD)). The view highlights CRY2PHR (shown in blue), mCherry (shown in gray), and the F0, F1, F2, and F3 subdomains of talin (shown in green). For simplicity, the talin rod domain is not shown. Membrane translocation of CRY2–mCherry–talin was observed by confocal microscopy (panel D, top) in experiments where cultured cells were first trypsinized, suspended in buffer, and then exposed to pulsed blue light for 30 min with a frequency of 1 s each 75 s. No such membrane recruitment of talin was observed if cells expressed CIBN–GFP–CAAX and mCherry-talin (without CRY2) (panel D, bottom). The scale bar represents 20 μm. E, optogenetic recruitment of talin to the plasma membrane promotes αIIbβ3 activation, as monitored by specific PAC-1 binding quantified by flow cytometry. PAC-1 binding is expressed as the fold increase in binding relative to that observed in the same cells maintained in the dark. In this and subsequent figures, unless specified otherwise, PAC-1 binding was normalized to that observed when cells expressing CIBN–GFP–CAAX and CRY2–mCherry–WT talin were maintained in the dark. Data represent the means ± SEM of five experiments (asterisk, p < 0.05). CHO, Chinese hamster ovary; CRY2, Arabidopsis cryptochrome 2; FERM, 4.1 protein/ezrin/radixin/moesin; PAC-1, activation-dependent anti-αIIbβ3 monoclonal antibody; PHR, photolyase homology region.