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Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

Jonathan D. Winkelman, Caitlin A. Anderson, Cristian Suarez, David R. Kovar, Margaret L. Gardel

Posted on: 15 April 2020 , updated on: 24 April 2020

Preprint posted on 10 March 2020

Article now published in Proceedings of the National Academy of Sciences at http://dx.doi.org/10.1073/pnas.2004656117

and

Mechanosensing through direct binding of tensed F-actin by LIM domains

Xiaoyu Sun, Donovan Y. Z. Phua, Lucas Axiotakis Jr., Mark A. Smith, Elizabeth Blankman, Rui Gong, Robert C. Cail, Santiago Espinosa de Los Reyes, Mary C. Beckerle, Clare M. Waterman, Gregory M. Alushin

Posted on: , updated on: 24 April 2020

Preprint posted on 7 March 2020

Article now published in Developmental Cell at http://dx.doi.org/10.1016/j.devcel.2020.09.022

Can molecular signatures within cells reveal mechanical stress? How are they recognised? Two preprints present evidence that actin filaments under duress may relay the message by directly recruiting multi-contextual LIM domains.

Selected by Angika Basant

Categories: biochemistry, cell biology

Background:

An immune cell binding its target, a cancer cell migrating through a tissue, a neuronal precursor finding its position in the brain, a dividing cell moving in an epithelial sheet – all have at least one thing in common. They sense and adapt to forces and mechanical cues. Membrane proteins are typically the first-responders to such signals. For example, conformational changes occur in the extracellular domain of the T-cell receptor when its ligand binds (1), and the protein talin undergoes force-dependent stretching at adhesions (2). Are peripheral molecules alone in detecting mechanical changes?

In adhesive cells, actomyosin bundles called stress fibres span the cell, linking cell-matrix adhesion sites. They have been thought of as second messengers for mechanical signals (3). How would they transmit information? Stress fibres undergo breakage when stretched and are subsequently repaired. Some proteins bind strained sites on stress fibres, zyxin being the best characterised among them (4, 5). But curiously, zyxin does not accumulate much with actin when spun down in co-sedimentation assays (6).

Zyxin belongs to a large group of proteins containing a modular protein-binding interface called the LIM domain. This collection of proteins is very diverse, encompassing kinases, transcription factors and cytoskeletal regulators (7).  Zyxin has 3 LIM domains which are required for stress fibre binding. LIM domains typically comprise 50-60 amino acids with 8 highly conserved cysteine and histidine residues at defined intervals allowing coordination of two zinc ions (7). Other zinc-finger-containing domains can bind DNA and lipids but an interaction with actomyosin is not established (8). What do LIM domains recognise on stress fibres? Can any LIM domain bind actomyosin? Some transcription factors and activators contain LIM domains, could they perhaps sense mechanical stress?

Top: GFP-Zyxin localisation upon laser ablation of stress fibres (Winkelman et al) Bottom: Sun et al show localisation of F-tractin (magenta) and a zyxin chimera with FHL3 LIM domains (green)

 

Key findings:

The preprints first address the extent of stress fibre-binding among LIM domains, beyond those of zyxin. Screening LIM domain-containing sequences from various LIM protein sub-families, they test for localisation to stress fibre strain sites in response to natural breakage, laser-ablation or cell stretching. Though not all LIM domains mimic zyxin, several members of paxillin, FHL and testin families show localisation to stressed sites. These LIM domains were interchangeable for function, for instance domains from a transcriptional activator FHL2 could replace those of zyxin. Rather interestingly, LIM modules from fission yeast protein Pxl1 can also do the same. Though the repertoire of LIM proteins in yeast is small, this indicates that mechanosensitivity in LIM domains can be traced back to these unikonts.

In vitro assays demonstrate that LIM domains can bind naked, strained actin; no connecting proteins are required. Labeled actin filaments polymerised on coverslips were strained by adding/activating myosin molecules in the imaging chamber. LIM domains accumulate at specific sites on actin only upon motor activity. Furthermore, binding of LIM domains is not restricted to actin stressed by myosin pulling forces. Winkelman et al show that LIM domains also accumulate at sites of breakage and strain in a burst of polymerising actin. The specific actin conformation involved in this binding could explain why zyxin does not appreciably co-sediment with bulk actin.

What distinguishes LIM domains that bind actin? Sun et al demonstrate that a highly conserved phenylalanine residue in the primary sequence is critical for stress fibre binding. Winkelman et al highlight unique elements of LIM domain organization. Binders tend to have 3 or more LIM domains that are linked in tandem (not in parallel), and inter-domain linkers of ~8 residues are ideal; longer linkers result in poor actin binding.

Finally, Sun et al also show that tensed actin retains transcriptional activator FHL2 in the cytosol (out of the nucleus) providing a possible link between mechanical status of a cell and gene expression.

What I like about these preprints:

Away from the cell surface, there are few molecular details as to how a cell would detect mechanical strain to mount a suitable response. These studies provide mechanistic insight into this problem. In addition, it is great to see two separate studies addressing the same theme, arriving at similar conclusions yet bringing out distinct aspects of the mechanism.

Questions for the authors:

  1. The results obviously point to a structural feature on actin that LIM domains recognise. Are there intermediates known in cofilin-mediated severing that might resemble stressed actin and may point to what LIM domains interact with?
  2. In in vitro assays is it possible to discern the length (number of actin subunits) and thickness (number of bundled filaments) that favour LIM domain binding?
  3. Would you expect “non-binders” from say the Lhx family of transcription factors to still bind F-actin weakly, which may play a role in their nuclear function?

References:

  1. Blumenthal and Burkhardt, Journal of Cell Biology, (2020)
  2. Hu et al., Protein Science (2017)
  3. Martino et al., Frontiers in Physiology (2018)
  4. Smith et al., Developmental Cell (2010)
  5. Smith et al., PLoS ONE (2013)
  6. Crawford et al., Journal of Cell Biology (1992)
  7. Kadrmas and Beckerle, Nature Reviews Molecular Cell Biology (2004)
  8. Laity et al., Current Opinion in Structural Biology (2001)

 

doi: https://doi.org/10.1242/prelights.18645

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Author's response

Jonathan Winkelman shared about Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

1. The results obviously point to a structural feature on actin that LIM domains recognize. Are there intermediates known in cofilin-mediated severing that might resemble stressed actin and may point to what LIM domains interact with?

That is an interesting question! Since cofilin severs actin filaments and LIM domains bind to a filament conformation that appears before a filament breaks, the idea that LIM would bind to a structure similar to one induced by cofilin is intriguing. I can think of at least one hypothetical structure that cofilin binding is thought to produce that LIM could bind:

Cooperative cofilin binding results in a softer, floppier, F-actin region. Soft (cofilin-bound) regions next to stiffer (unbound) regions on the actin filament result in soft/stiff boundaries where stresses accumulate and become highly strained. Stress at the boundary may result in the breaking of filament stabilizing contacts between the D-loop and the neighboring actin subunit, weakening the filament and exposing the D-loop. This D-loop is thought to be hydrophobic and as the Alushin lab showed, a major residue thought to be important for binding actin in LIM domains is hydrophobic. So, binding to a strain-induced, exposed D-loop in an actin filament is a hypothetical target for LIM.

We observe LIM binding to actin filament networks at what we are calling “T-junctions”. For example, a single filament may enter into a two-filament bundle and when the network is pulled on by Myosin, filament strain is likely localized to this junction and we observe frequent filament breaking events there. LIM domains could be interacting with any number of features that may arise on these filaments prior to breaking, including an exposed D-loop, a change in the helical twist of the filament due to twist-bend coupling. We hypothesize that tandemly connected LIM domains may act as a ruler that measures and binds when subunits move far enough apart or twist in a particular way.

2. In in vitro assays is it possible to discern the length (number of actin subunits) and thickness (number of bundled filaments) that favour LIM domain binding?

I. Number of actin subunits: With the resolution on our confocal microscopes, we can’t observe the exact number of actin filament subunits that favor LIM binding, but we do observe binding of single molecules of the LIM containing regions (LCR) of zyxin or yeast pxl1. Our working model suggests that a only few strained subunits of the actin filament are sufficient to recruit LIM.

II. Number of filaments in a bundle: We don’t think bundling is necessary although we can’t completely rule it out. We see binding during symmetry breaking in motile beads and we don’t envision these filaments being bundled. Additionally, we observe most binding events at T-junctions that are thinner (less bundled) because those tend to break more readily.

3. Would you expect “non-binders” from say the Lhx family of transcription factors to still bind F-actin weakly, which may play a role in their nuclear function?

I love that idea, but at this point I think that we just don’t know. I do suspect actin filament binding to be an ancient function of the LIM domain which was tinkered with by evolution to produce strain sensitive variants. As for the other “non-binding” LIM families, I think we don’t know if some of harbor some undiscovered actin binding that regulates their function. We didn’t observe any binding to actin networks within the LIM domains from Lhx in cells, but it’s always a little harder to interpret a negative result.

and

Xiaoyu Sun and Greg Alushin shared about Mechanosensing through direct binding of tensed F-actin by LIM domains

The results obviously point to a structural feature on actin that LIM domains
recognise. Are there intermediates known in cofilin-mediated severing that might
resemble stressed actin and may point to what LIM domains interact with?

1. We anticipate that mechanoresponder LIM domains directly bind a strained state of F-actin that is specifically induced by force in the longitudinal direction. This could involve a specific protomer conformation, like the exposure of a cryptic site (e.g., the D-loop) that is involved in the bond formation between adjacent longitudinal protomers in intact F-actin, the formation of a specific filament superstructure in the presence of force that changes the spacing between subunits, or, most likely, both.

The formation of LIM patches along individual filaments suggests this conformation can co-exist with a standard actin conformation within the same filament, which is reminiscent of the formation of cofilin patches. The collaborative work led by De La Cruz and Sindelar has recently shown that the actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments1; this twist disrupts the longitudinal actin-actin bond at the pointed-end boundary while introduces strain in the barbed-end boundary2. We speculate that there could be a shared structural feature between the cofilin-induced actin conformation at the segment boundary and the strained actin conformation recognized by LIM domains.

However, we are open-minded about what precisely LIM proteins recognize. We are currently pursuing cryo-EM studies of F-actin in the presence of myosin-generated forces utilizing a similar reconstitution system as described in our paper, which will hopefully provide a definitive answer soon.

In in vitro assays is it possible to discern the length (number of actin subunits) and
thickness (number of bundled filaments) that favour LIM domain binding?

2. Since there is no crosslinker in our in vitro assay, we believe the majority of the actin filaments remain single filaments. The length of the FHL3 binding patches as measured by TIRF are usually 1-2 m, which in the case of standard F-actin with an approximately 2.5 nm axial spacing between protomers would contain hundreds to ~1,000 subunits. The diffraction limit of TIRF microscopy does impact the accuracy of these length measurements, however, and we don’t know if the subunit spacing changes in the patches. We thus believe cryo-EM will ultimately be required to give an accurate picture of the number of actin subunits in LIM domain-binding regions. What is currently clear is that it is many actin subunits, not just one or two.

Would you expect “non-binders” from say the Lhx family of transcription factors to
still bind F-actin weakly, which may play a role in their nuclear function?

3. All the mechanoresponsive LIM proteins we identified in the screen possess at least three LIM domains in tandem. Point mutations at a site conserved in each LIM domain of these proteins demonstrated that all LIM domains in a tandem array contribute cumulatively to mechanoaccumulation. The LHX-family proteins have two LIM domains. We therefore speculate that its capacity to bind tensed actin remain low.

References
1. Huehn, A. et al. The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments. J. Biol. Chem. 293, 5377–5383 (2018).
2. Huehn, A. R. et al. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments. PNAS 117, 1478–1484 (2020).

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