Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

Abstract

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

“…7J; Fig. S8D) with a diffusion constant D diff = 0.087 ± 0.001 µm 2 .s -1 (mean ± SEM) that is comparable to that of free diffusing transmembrane proteins (integrins: (Rossier et al, 2012)) or of a lipid-anchored protein bound to the plasma membrane by its PH domain (kindlin:(Orre et al, 2021). Confined SEPT9_i3-mEos3.2 was also diffusing very slowly (Fig.…”

Tau forms dynamic hot spots that are resistant to microtubule perturbations and cholesterol depletion

“…A case in point is the molecular dynamics linked to focal adhesion (FA) zones. For instance, some proteins, such as Rac1 and kindlin, are targeted to the membrane where they can undergo free diffusion before being recruited to and confined at these zones 37,38 . By contrast, proteins such as talin and PIX are directly recruited from the cytoplasm to these zones and become confined 37,38 .…”

“…For instance, some proteins, such as Rac1 and kindlin, are targeted to the membrane where they can undergo free diffusion before being recruited to and confined at these zones 37,38 . By contrast, proteins such as talin and PIX are directly recruited from the cytoplasm to these zones and become confined 37,38 . Why proteins choose different recruitment pathways appears to be dictated by their functions and is still a matter of intense investigation.…”

Talin and kindlin cooperate to control the density of integrin clusters

“…Li et al, 2017; Liu et al, 2011). This domain triggers the localization of kindlin at the membrane and promotes its diffusion, allowing kindlin to reach and activate integrins (Orré et al, 2021).…”

“…Instead, the same study showed that in cells kindlin increases integrin affinity for multivalent but not monovalent extracellular ligands, suggesting a role for kindlin in talin-activated integrin clustering. Interestingly, tracking of single proteins in cells revealed that talin associates with integrins in FAs from a cytoplasmic pool (Rossier et al, 2012), whereas kindlin membrane diffusion allows kindlin to reach FAs and activate integrins (Orré et al, 2021). Furthermore, the association of kindlin with integrins is more labile than that of talin, indicating that these two regulators may also act sequentially or independently of each other.…”