Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions
Preprint posted on 6 August 2020 https://www.biorxiv.org/content/10.1101/2020.08.06.239731v1
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-021-23372-w
Talins and Kindlins: partners in crime with specific modes of transportation.Selected by Amanda Haage
Why This Is Cool – This article is all about providing detailed localization data to the integrin adhesome. The “adhesome” refers to the many proteins that cluster around the cytoplasmic tails of integrins and mediate multiple signaling outcomes and links the complex to the actin cytoskeleton. In 2D cell culture this adhesome is pretty much used interchangeably with focal adhesions (FAs), and previous work has shown that there are distinct layers to FAs extending inward from the membrane (1). Here, the authors utilize cutting edge microscopy techniques to track single proteins, namely integrins, talins, and kindlins, in and out of FAs. By first tracking integrins, they show that they become immobilized in FAs and there are slight differences, where ß3 immobilization seems to be more dependent on Talin, while ß1 requires both Talin and Kindlin. These differences are the start of a line of evidence used to connect differences in diffusion/immobilization of Kindlin with a specific role in FAs. Tracking Kindlin in conjunction with various point mutations shows that unlike Talin (2), Kindlin is in free diffusion along the membrane driven by its unique PH domain. Kindlin binding to integrins mediates its immobilization in FAs and it remains near the membrane and integrin cytoplasmic tails instead of repositioning inward similar to vinculin (3). Ending with function, the PH domain and free diffusion along the membrane of Kindlin seems to be fundamental to promoting integrin activation and cell spreading.
Figure1: Membrane recruitment and diffusion of kindlin‐2 is crucial during cell spreading. Top: Phase contrast images showing the morphological features of cells classified as non‐spread, partially spread, or spread. Bottom: Relative fraction of non‐spread, partially spread, and spread KindKo cells 2 days after re‐expression of kindlin‐2‐WT or mutated variants and 4h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). GFP‐ paxillin: 141 cells; GFP‐paxillin + mEos2‐kindlin‐2‐WT: 312 cells; GFP‐paxillin + mEos2‐kindlin‐2‐ΔPH: 311 cells; GFP‐paxillin + mEos2‐kindlin‐2‐ΔPH‐CAAX: 309 cells; GFP‐paxillin + mEos2‐kindlin‐2‐ QW615/615AA: 284 cells; GFP‐paxillin + mEos2‐kindlin‐2‐WT‐CAAX: 289 cells. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP‐paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP‐paxillin condition: between 31 and 58 cells per experiment).
Why I Selected It – One of the influential reviews in my field that I never forget the name of is “Talins and Kindlins: partners in integrin-mediated adhesion”(4), because in my head I have always read it as “Talins and Kindlins: partners in crime.” Essentially it reviews the well-established theory that both the FERM containing adaptors, Talin and Kindlin, bind to the cytoplasmic tails of integrins and facilitate activation, signaling, and linkage to the cytoskeleton. Talin-1’s role has been investigated more thoroughly (5), but the overlapping roles of these proteins remains an open question. What this study does really well is provide detailed and convincing data for distinct functions in FAs via distinct localizations and trafficking between the two.
Open Questions –
- For the functional experiments, Kindlin knockout cells are used to demonstrate rescue, but could the other results be influenced by the various expression levels induced with transfected constructs?
- Though the differing recruitment of Talin is cited, can you also replicate these important results with your system?
- Here you are focusing on the classical proteins of the field, i.e. ß3/ß1 integrins, Talin-1, Kindlin-2, but how much of this work is applicable to the increased complexity of the adhesome? Say Talin-2, the other Kindlins, and the other integrins.
Related References –
- Distinct Layers in FAs
- Kanchanawong, P. et al. Nanoscale architecture of integrin‐based cell adhesions. Nature 468, 580–584 (2010).
- Talin Recruitment
- Rossier, O. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Cell Biol. 14, 1057–67 (2012).
- Vinculin Organization
- Case, L. B. et al. Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Cell Biol. 17, 880–892 (2015).
- Talins and Kindlins
- Calderwood, D. a, Campbell, I. D. & Critchley, D. R. Talins and kindlins: partners in integrin‐ mediated adhesion. Rev. Mol. Cell Biol. 14, 503–17 (2013).
- Talin the Master Regulator
- Klapholz, B. & Brown, N.H. Talin – the master of integrin adhesions. Cell Sci 130(15): 2435 – 2446.
Posted on: 18 September 2020
doi: https://doi.org/10.1242/prelights.24322Read preprint
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