Journal: bioRxiv

Article Title: Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem

doi: 10.1101/504894

Figure Lengend Snippet: (A) Airyscan LSM Image MIP projections of 3T3 fibroblasts expressing both THD-EGFP (green) and mRby2-Paxillin (red). Bottom panel shows the merged image and the paxillin rich ROIs (red lines) with quantitative colocalization of both THD to paxillin in peripherally labelled paxillin rich focal adhesions. The images clearly show a diminishment of co-localized signal of THD to paxillin between the WT control and THD(R35E) mutant, which is further decreased in the THD(R118E) mutant. Quantitation results are displayed in (B) . The number of analyzed adhesions is indicated for each condition. (C) A model of talin1 F1 domain in the cytosol with a positively charged unstructured loop (green) and membrane-associated Rap1b-GTP by its C-terminal geranyl-geranyl (G-G) moiety (orange). Negatively charged phospholipids PI(4,5)P 2 are colored in red. On proximity of the plasma membrane, the low-affinity talin1 F1 Ras-associating (RA) domain probes for Rap1 and the F1 loop seeks negatively charged phospholipids. On contact with Rap1 and negatively charged phospholipids, the F1 RA domain interacts with Rap1 and the F1 loop helical state is favored resulting in cluster of positive charges on one side of the helix. View of THD (green) as seen from the membrane is displayed in the top right panel. Bottom panel shows view of THD as seen from the side. The views highlight the position of the THD F0, F1, F2 and F3 subdomains and the Rap1b (orange) bound to the F0 and F1 subdomains. Both Rap1b C-terminal geranyl-geranyl (G-G) moieties are pointing towards the membrane, as for the F1 loop, the F2 membrane orientation patch (MOP), the F3 association patch (FAP), and the position of the F3 β-integrin. The regions known to interact with the negatively charged phospholipids are shown in blue; the F1 fly-casting loop is shown as a helix, the F2 membrane orientation patch (MOP), and the F3 association patch (FAP). The β-integrin transmembrane and cytoplasmic tail is shown in red bound to the F3 subdomain. Two Rap1b (orange) molecules are shown with their C-terminal geranyl-geranyl (G-G) moiety inserted in the membrane. One Rap1b is bound to the F0 and one to the F1 subdomain. This model summarize the multiple known interactions of THD at the plasma membrane; the negatively charged PI(4,5)P 2 , Rap1 and integrin β-tails.

Article Snippet: Each image consisted of z-stacks of multiple frames that were first processed from a raw Airyscan image to a final integrated, corrected and deconvolved image that was then flattened as a maximum intensity projection using the ZEN software (Zeiss Inc.).

Techniques: Expressing, Control, Mutagenesis, Quantitation Assay, Membrane, Clinical Proteomics