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Journal of cell science 133(19), jcs239202 (1-15) - (2020). doi:10.1242/jcs.239202, Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the �� integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn$^{2+}$. Here we show that kindlin-1 can replace Mn2+ to mediate ��3 integrin clustering induced by the talin head, but not that induced by the F2���F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ��3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of ��- and ��3-integrins, in order to activate the ��3 integrin heterodimer, further detailing the mechanism by which the talin���kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ��3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer., Published by Company of Biologists Limited, Cambridge