TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo

Allison M Lynch; Yuyun Zhu; Bethany G Lucas; Jonathan D Winkelman; Keliya Bai; Sterling CT Martin; Samuel Block; Mark M Slabodnick; Anjon Audhya; Bob Goldstein; Jonathan Pettitt; Margaret L Gardel; Jeff Hardin

doi:10.1016/j.cub.2022.10.045

. Author manuscript; available in PMC: 2023 Dec 5.

Published in final edited form as: Curr Biol. 2022 Nov 15;32(23):5189–5199.e6. doi: 10.1016/j.cub.2022.10.045

TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo

Allison M Lynch ^1,^*, Yuyun Zhu ^1,^*, Bethany G Lucas ⁵, Jonathan D Winkelman ⁶, Keliya Bai ^7,¹¹, Sterling CT Martin ², Samuel Block ^4,¹², Mark M Slabodnick ^8,⁹, Anjon Audhya ⁴, Bob Goldstein ⁹, Jonathan Pettitt ⁷, Margaret L Gardel ^6,¹⁰, Jeff Hardin ^1,^2,^3,^13,^†

PMCID: PMC9729467 NIHMSID: NIHMS1846259 PMID: 36384139

SUMMARY

LIM domain-containing repeat (LCR) proteins are recruited to strained actin filaments within stress fibers in cultured cells ^1–3, but their roles at cell-cell junctions in living organisms have not been extensively studied. Here, we show that the Caenorhabditis elegans LCR proteins TES-1/Tes and ZYX-1/Zyxin are recruited to apical junctions during embryonic elongation, when junctions are under tension; in genetic backgrounds in which embryonic elongation fails, junctional recruitment is severely compromised. The two proteins display complementary patterns of expression: TES-1 is expressed in lateral (seam) epidermal cells, whereas ZYX-1 is expressed in dorsal and ventral epidermal cells. tes-1 and zyx-1 mutant embryos display junctional F-actin defects; loss of either protein strongly enhances morphogenetic defects in hypomorphic mutant backgrounds for cadherin/catenin complex (CCC) components. The LCR regions of TES-1 and ZYX-1 are recruited to stress fiber strain sites (SFSSs) in cultured vertebrate cells. Together, these data establish TES-1 and ZYX-1 as components of a multicellular, tension-sensitive system that stabilizes the junctional actin cytoskeleton during embryonic morphogenesis.

RESULTS AND DISCUSSION

We previously conducted a genome-wide RNAi screen in a sensitized HMP-1/α-catenin background to identify genes that, when knocked down, enhanced the severity of the hmp-1(fe4) phenotype during morphogenesis in C. elegans embryos ⁴, including a gene on chromosome IV (Video S1). Previously named TAG-224 (Temporarily Assigned Gene 224), we renamed the protein TES-1 given its significant homology to vertebrate Tes. ClustalW analysis indicated that TES-1 is approximately 35% identical and 64% similar to human Tes. Pfam analysis showed that both proteins have an N-terminal PET domain followed by three C-terminal LIM domains (Figure 1A).

TES-1 is an F-actin-binding protein that functionally interacts with hmp-1/α-catenin at the C. elegans apical junction

100% of hmp-1(fe4); tes-1(RNAi) embryos arrested during the elongation stage of morphogenesis with junctional actin defects (Figure 1B–E). tes-1(ok1036); hmp-1(fe4) double homozygotes similarly exhibit 93.8% lethality and elongation arrest (n = 516 embryos examined), and tes-1 RNAi enhanced lethality in a hmp-2/β-catenin hypomorph (hmp-2(qm39); Figure S1). tes-1 RNAi exacerbated junctional proximal actin defects in hmp-1(fe4) homozygotes (Figure 1F–H). In 26 % of hmp-1(fe4); tes-1(RNAi) embryos (6 of 23 embryos examined via 4d microscopy) cells leaked out of the ventral midline, compared with 0% of hmp-1(fe4) homozygotes (0 of 22 embryos examined; significantly different, Fisher’s exact test, p = 0.02). Ventral enclosure involves formation of CCC-dependent junctions at the ventral midline ⁵, suggesting that TES-1 is also involved in this process (Figure 1E, arrow). Like vertebrate Tes ^6,7, recombinant TES-1 cosediments with F-actin (Figure 1I) to an extent statistically indistinguishable from HMP-1/α-catenin ⁸ (Figure 1J).

TES-1 localizes to apical junctions in the embryonic epidermis

We constructed an endogenously tagged version of tes-1; mNG::tes-1 embryos, larvae, and adults were phenotypically indistinguishable from wildtype (Figure 2A). In larvae, TES-1 was visible at alae, epidermal structures produced by seam cells; in adults, TES-1 was expressed in vulval tissues (data not shown). In early embryos, mNG::TES-1 was visible in the cytoplasm of epidermal cells; at the 2-fold stage of elongation, mNG::TES-1 puncta began to accumulate at sites of cell-cell contact, expanding and becoming more evenly distributed along cell borders as elongation continued. Strikingly, mNG::TES-1 was maintained at seam-dorsal and seam-ventral, but not seam-seam borders (Figure 2B, arrow).

We next performed knockdown of junctional components in mNG::tes-1 embryos. In hmr-1(RNAi) embryos TES-1::GFP failed to accumulate at junctions (Figure 2C). In contrast, ajm-1(RNAi) did not prevent junctional localization of mNG::TES-1 (Figure 2D); however, TES-1 foci did not spread to form a continuous, intense band as in wildtype, which may reflect the failure of ajm-1(RNAi) embryos to elongate fully.

Endogenously tagged HMP-1/α-catenin::mScarletI and mNG::TES-1 displayed substantial overlap in embryos (Figure 2E; Pearson’s R value above threshold = 0.58, n = 10 junctions), whereas there was little to no overlap with DLG-1/Discs large::dsRed, which localizes basal to the CCC (Figure 2F; R = 0.25, n = 10 junctions; significantly different, p < 0.0001, unpaired Student’s t-test). Partial localization of Tes with the CCC has similarly been reported in cultured vertebrate cells ⁹. Although one study reported that vertebrate α-catenin and Tes can be coimmunoprecipitated ¹⁰, we were unable to replicate this result with C. elegans CCC components in a generalized proteomics screen ¹¹ or in directed coIP experiments (Figure S2A–B), suggesting that the interaction of TES-1 with the C. elegans CCC is indirect. Alternatively, force-dependent interactions between LCR proteins and cell-cell junctions may be transient and weak, as suggested by a recent BioID study of zyxin ¹², and thus difficult to demonstrate using traditional biochemical approaches.

We reasoned that TES-1 could stabilize CCC-dependent junctional proximal actin networks during morphogenesis, and so we compared F-actin in tes-1(ok1036) homozygous embryos wild-type for hmp-1 with fully wild-type embryos (Figure 2G–I). Unlike wild-type embryos (Figure 2G), most tes-1(ok1036) embryos displayed significantly narrower zones of junctional proximal actin (Figure 2H; quantified in Figure 2J), as well as more severe phenotypes, including gaps between circumferential filament bundles (CFBs), CFB collapse, and complete loss of preserved junctional-proximal actin (Figure 2I; quantified in Figure 2K). We conclude that TES-1 stabilizes junctional-proximal actin during morphogenesis.

TES-1 requires its PET and LIM domains

To identify functionally important subdomains of TES-1 we analyzed endogenously tagged tes-1 deletions. Unlike full-length mNG::TES-1 (Figure 3A), mNG::TES-1ΔPET localized along all seam cell borders in the epidermis (Figure 3B). mNG::TES-1ΔLIM1-3 localized along structures that appear to be CFBs (Figure 3C). This result suggests that the latent ability of TES-1 to bind to CFBs is not normally manifest when the N terminus is present, and is similar to vertebrate Tes, which can co-immunoprecipitate actin ⁷ and localize via its N terminus in a non-mechanosensitive manner ^10,13,14. Line scans indicated that when either the PET or LCR domains were deleted, TES-1 still localized to seam-dorsal and seam-ventral junctions (Figure 3D), but embryos showed ectopic TES-1 junctional localization at seam-seam junctions (Figure 3E). Deletion of the PET domain led to an increase in junctional vs. cytoplasmic signal compared to wildtype, while removal of all three LIM domains resulted in the opposite effect (Figure 3F). It is possible that the PET and LCR domains interact, restricting their domain-specific binding affinities, as has been proposed for vertebrate Tes based on biochemical assays⁷. These results indicate that both the LCR and PET domains are required for normal levels and sites of TES-1 junctional recruitment.

We also expressed various TES-1::GFP deletion constructs (Figure S3A) in transgenic embryos. Full-length TES-1∷GFP, TES-1ΔPET::GFP, and TES-1ΔLIM1-3 recapitulated the expression of endogenous knock-ins (Figure S3B–D). TES-1::GFP rescued lethality in tes-1(ok1036)/+; hmp-1(fe4) embryos. tes-1(ok1036)/+; hmp-1(fe4) worms exhibited 80% lethality (n = 20 embryos scored); the addition of extrachromosomal TES-1::GFP reduced lethality to 38% (n = 92 embryos scored). tes-1(ok1036); hmp-1(fe4) worms could develop to adulthood, but only if they expressed tes-1::gfp, indicating the TES-1::GFP is functional. Deletion of LIM1 (Figure S3E) or LIM2 (Figure S3F) both led to sporadic recruitment to epidermal junctions, including some seam-seam junctions, and what appear to be actin-containing structures in epidermal cells. Deletion of LIM3 rendered TES-1::GFP largely cytoplasmic (Figure S3G).

Due to maternal effects and gonadal defects, assessing synergistic lethality of tes-1::gfp deletion constructs in tes-1(ok1036); hmp-1(fe4) homozygous mothers proved challenging. Fertile tes-1(ok1036); hmp-1(fe4) worms harboring tes-1ΔLIM1::GFP could not be obtained; occasional tes-1(ok1036); hmp-1(fe4)/+; tes-1ΔLIM1::GFP embryos were able to grow to adulthood, but were sterile. We therefore tested for the ability of TES-1::GFP fragments to rescue synergistic lethality in tes-1(ok1036); hmp-1(fe4)/+ embryos (Figure S3H). TES-1ΔPET::GFP significantly rescued some embryonic lethality in this genetic background, but progeny displayed germline malformations, protruding vulvae, and sterility. TES-1ΔLIM1-3::GFP, TES-1ΔLIM2::GFP, and TES-1ΔLIM2::GFP were unable to rescue the 39% lethality observed among progeny of tes-1(ok1036); hmp-1(fe4)/+ mothers. Overall, these results indicate that the LIM domains of TES-1 are crucial for tes-1 function during morphogenesis.

The difference in localization pattern of TES-1ΔLIM3::GFP and TES-1ΔLIM1-3::GFP was curious, since the entire LCR region, with appropriate spacing between LIM domains, has been suggested to be crucial for F-actin binding ^3,15. It has been suggested, however, that the LIM1-2 domain of vertebrate Tes can engage in both heterophilic binding to proteins such as zyxin and homodimerization via interaction with the PET domain of Tes ¹⁰. While it is not currently known if homodimeric Tes is sequestered away from cell-cell adhesion sites, deletion of LIM3 could favor such homodimerization. Alternatively, deletion of LIM3 may cause misfolding of the truncated protein.

TES-1 localizes to junctions in a tension-dependent manner

Tes is required for maintenance of stress fibers in cultured vertebrate cells ¹⁶, accumulates at “focal adherens junctions” (spot-like foci of cell-cell adhesion) in human vascular endothelial cells ⁹, and accumulates at stress fibers downstream of Rho signaling ¹⁴. These data suggest that Tes might play tension-dependent roles in stabilizing F-actin networks at adherens junctions during morphogenesis. A coordinated change in the shape of epidermal cells drives elongation of the C. elegans embryo to approximately 4-fold its original length ¹⁷, during which contractile forces result in elevated tension specifically at seam-ventral and seam-dorsal junctions ^5,18–21. Given the localization of TES-1, we sought to test whether it is recruited to junctions in a tension-sensitive manner during embryonic elongation.

Because hmr-1/cadherin, hmp-1/α-catenin, and hmp-2/β-catenin homozygous null mutant embryos fail to progress past the two-fold stage of elongation, we could not assess whether disruption of TES-1::GFP recruitment to junctions is due primarily to physical absence of CCC components or to the pre-elongation death of the embryos. We therefore examined hmp-1(fe4) embryos expressing TES-1::GFP. While some hmp-1(fe4) embryos failed to elongate appreciably, other embryos extended to the 2-fold stage of elongation. TES-1::GFP did not localize to junctions in hmp-1(fe4) embryos that failed to elongate past 1.5-fold (10 of 10 embryos; Figure 3G,J), even in embryos that survived and hatched. However, TES-1::GFP did localize to junctions in the rare hmp-1(fe4) embryos that elongated to at least 2-fold their original length (5 of 5 embryos examined; significantly different; Fisher’s exact test, p = 0.0003; Figure S3I). The correlation between the extent of elongation of fe4 embryos and TES-1::GFP junctional recruitment suggests that TES-1 is recruited to junctions in cells that generate sufficient tension to elongate to the 2-fold stage.

We next introduced the full-length TES-1::GFP into let-502(sb118ts)/Rho kinase worms to reduce actomyosin contractility in the epidermis (Figure 3H; Figure S3K–L). When let-502(sb118ts); tes-1::gfp embryos were imaged at the permissive temperature, TES-1::GFP localized to junctions normally (Figure S3K; quantified in Figure 3J, let-502(sb118ts) ≥ 1.5x). At the restrictive temperature (25°C), however, TES-1::GFP remained entirely cytoplasmic in embryos that failed to elongate (Figure 3H; quantified in Figure 3J, let-502 (sb118) 1.25x). We also attempted the converse experiment by knocking down MEL-11/myosin phosphatase, which is known to result in excessive epidermal contractility ^19,20. However, adhesion complexes underwent changes in morphology that made this experiment difficult to interpret: the initially continuous distribution of junctional TES-1::GFP was progressively lost, as TES-1∷GFP became fragmented and pulled into puncta (Figure 3I). One possibility consistent with this result is that excessive tension leads to collapse of junctional proximal actin around CFB insertion sites, including associated TES-1.

ZYX-1/zyxin localizes to junctions in a tension-dependent manner complementary to TES-1

Studies in vertebrate tissue culture cells indicate similar, but not entirely overlapping, localization of Tes and zyxin at spot adherens junctions ^9,22. Moreover, targeted interaction studies ⁷ and proteomics screens ¹⁰ suggest that the two proteins may physically associate, either directly or as part of a complex. We used an endogenous mNG::ZYX-1a knock-in ²³ (hereafter ZYX-1) to assess zyx-1 expression in C. elegans embryos. ZYX-1 had been reported to localize at muscle attachment sites ^24,25 and sites of cell-cell contact in gastrulating embryos ²³. However, its localization at adherens junctions in the embryonic epidermis had not been reported. ZYX-1 showed strong localization at seam-dorsal and seam-ventral junctions in the epidermis during mid-late elongation. Strikingly, however, ZYX-1 showed a pattern complementary to that of TES-1: whereas mNG::TES-1 showed strong expression in seam cells, ZYX-1 was expressed strongly within non-seam cells (Figure 4A).

Figure 4. — (A) mNG::ZYX-1 is recruited to both dorsal-seam and seam-ventral junctions (white arrow), and it also colocalizes with CFBs after the two-fold stage (yellow arrowhead). (B) In *mNG::zyx-1ΔLIM1-3* embryos ZYX-1 is largely absent from junctions and is not recruited to CFBs. (C) *let-502*(*RNAi)* embryos partially lose junctional localization of mNG::ZYX-1. Scale bars = 10 μm. (D-I) Recruitment of TES-1 LCR::mCherry and ZYX-1 LCR::mCherry to stress fiber strain sites (SFSS) in transfected mouse embryonic fibroblasts. (D) Representative kymographs of laser-induced recruitment of the ZYX-1 LCRmCherry and mouse GFP::Zyxin to SFSS. For a timelapse sequence of the entire cell, see Video S2. White dashed and gray solid lines indicate where fluorescence and distance were measured. Dashed gray vertical line indicates t₀, when strain is first observed. (E) Quantification of GFP and mCherry accumulation over time in the kymograph from (D). (F) Representative kymographs of laser-induced recruitment of TES-1 LCR mCherry and mouse GFP::Zyxin to SFSS. For a timelapse sequence of the entire cell, see Video S3. (G) Quantification of GFP and mCherry accumulation over time in the kymograph from (F). (H-I) Intensity of *C. elegans* ZYX-1 LCR::mCherry (H) and *C. elegans* TES-1 LCR::mCherry (I) relative to full-length mouse GFP::Zyxin present in the same cells. Blue dots in each graph represent mCherry alone relative to GFP::MmZyx. TES-1 LCR::mCherry accumulates markedly (p=0.023, n>10) but to a lesser extent than MmZyx, error bars indicate 95% confidence intervals. (J) mNG::ZYX-1 does not co-localize with mScarletI::TES-1. Inset shows magnification of boxed region. Scale bar = 10 μm. See also Figure S2, S4, and Video S2, S3.

Like mNG::ZYX-1, epidermally expressed transgenic ZYX-1::GFP colocalized with the CCC, and its localization was disrupted by HMP-1 depletion (Figure S4A–C). Since the LCR domain of zyxin is thought to be required for interaction with F-actin ^3,15, we created an endogenously tagged ΔLIM1-3 strain. mNG::ZYX-1ΔLIM1-3 was much more weakly recruited to junctions (Figure 4B; for quantification, see Figure S4E). We found that loss of zyx-1 function enhanced lethality of hmp-1(fe4) homozygotes to 100%. This enhancement could be rescued with a ZYX-1::GFP expressed under the control of an epidermal-specific promotor, suggesting that its key role is in this tissue (Figure S4D). We next stably expressed GFP-tagged, truncated forms of ZYX-1 in epidermal cells. ZYX-1ΔLIM1-3::GFP was unable to rescue (Figure S4D). Intriguingly, however, a construct lacking LIM1 and LIM3 could very weakly rescue when overexpressed in the epidermis, suggesting a more stringent requirement for the middle of the LCR during morphogenesis. hmp-1(fe4); zyx-1(gk190) embryos could not be rescued by epidermal ZYX-1::GFP lacking the N terminus (Figure S4D), indicating a role for the N terminus that is yet to be elucidated. Like mNG::TES-1, mNG::ZYX-1 was much more weakly recruited to seam/non-seam junctions in let-502(RNAi) embryos (Figure 4C; for quantification, see Figure S4E). Junctional F-actin defects in zyx-1(gk190) homozygotes were more subtle than those in tes-1(ok1036) homozygotes (see Figure 2G–K): we did not detect effects on CFBs, but did observe small ruptures in the junctional proximal actin network at seam-dorsal and seam-ventral boundaries in the embryonic epidermis not observable in controls (Figure S4F–H).

Both TES-1 and ZYX-1 can be recruited to strained actin fibers

Mammalian LIM domain proteins are recruited to strained actin fibers via their LIM domain-containing region ^2,3,26. Recruitment of the LCRs of such proteins to stress fiber strain sites (SFSSs) can be induced by laser irradiation in cultured mammalian cells ¹⁵. We tested whether the LCRs of TES-1 and ZYX-1 behave similarly. When transfected into mouse embryonic fibroblasts, ZYX-1(LIM1-3)::mCherry was recruited to SFSSs with kinetics similar to the LCR of full-length, eGFP-tagged M. musculus zyxin (Figure 4D; quantified in Figure 4E,H; for a movie of the entire cell, see Video S2). Compared with full-length M. musculus GFP-zyxin, recruitment of the TES-1 LCR was less pronounced, but significant compared to the mCherry negative control (Figure 4F; quantified in Figure 4G,I; also see Video S3).

ZYX-1/zyxin and TES-1/Tes act largely independently during elongation

We next assessed the interdependence of TES-1 and ZYX-1 in the epidermis during embryonic elongation. Endogenously tagged TES-1 and ZYX-1 appeared to abut one another across cell-cell junctions (Figure 4J); they did not colocalize quantitatively at junctions (Pearson’s R above threshold = 0.0, 13 junctions measured). We saw no change in localization of mNG::TES-1 to specific boundaries at the 3–4-fold stage in zyx-1(gk190) or zyx-1 null (cp419) ²³ homozygotes (Figure S4I–K), nor did we see mislocalization of mNG::ZYX-1 in tes-1(ok1036) homozygotes (Figure S4L–M). We did not see any obvious enhancement of lethality in tes-1; zyx-1 double loss-of-function embryos, but occasional tes-1(syb5622); zyx-1(cp419) animals showed minor body morphology defects that became less severe during larval molts (3 out of 30 embryos). Finally, based on previous studies of vertebrate homologues ^7,27, we assessed the physical interaction of TES-1 and ZYX-1. While we were able to coIP TES-1 and ZYX-1 (Figure S2C–D), we were only able to detect a very weak, substoichiometric interaction between TES-1 and ZYX-1 via pulldown of bacterially expressed proteins (Figure S2D).

In summary, our results suggest that two LCR proteins – ZYX-1 in non-seam cells and TES-1 in seam cells – act largely independently to bolster cadherin-dependent connections to the junctional-proximal F-actin network during embryonic elongation. A similar division of labor between these two cell types has been elegantly demonstrated previously in the case of non-muscle myosin and other proteins in a series of investigations ^21,28–30. Our results are consistent with experiments in vertebrates, which show that while depletion of zyxin can reduce the amount of Tes at focal adhesions ⁷, Tes can still localize independently of zyxin ²⁷. Our results further suggest that loss of one of these LCR proteins in an otherwise wild-type background in C. elegans is insufficient to decrease tension below the threshold required for recruitment of the other in the complementary group of epidermal cells.

The TES-1 LCR showed less avid recruitment to SFSSs than the ZYX-1 LCR when expressed heterologously. A previous study in tissue culture cells suggested that a crucial phenylalanine (F66) is found in the LIM domains of proteins that show mechanosensitive recruitment to SFSSs ³. Notably, zyxin has the F66 feature, but Tes does not ^3,15. There may be assay dependence regarding this requirement, however, as F66 is not required for recruitment of isolated LCR domains to SFSSs ¹⁵. Moreover, Tes has recently been shown to be activated by Rho signaling ¹⁴; since Rho activity is upregulated in seam cells during embryonic elongation in C. elegans ^28,31, activation of TES-1 in these cells could result in less functional difference in activity of TES-1 and ZYX-1 in vivo. Whether ZYX-1 and TES-1 play subtly different roles at the subcellular level is an interesting avenue for future investigation.

Elongating epidermal cells in the C. elegans embryo are likely to be subject to “self-injury”, as they must remodel their junctional-proximal actin networks to undergo dramatic changes in shape. Our previous experiments indicated that UNC-94/tropomodulin is recruited to junctions under tension, where it presumably protects minus ends of F-actin filaments from subunit loss³². Our current results are consistent with a model in which actomyosin-mediated tension generated in elongating embryos leads to strain-dependent recruitment of TES-1 and ZYX-1 to these same junctions during elongation, stabilizing strained junctional actin filaments against the rigors of mechanical stress during morphogenesis.

STAR METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Jeff Hardin (jdhardin@wisc.edu).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact upon request.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

C. elegans strains were maintained on standard nematode growth medium plates seeded with OP50 E. coli ³³ at either 15°C (temperature sensitive strains) or 20°C (all other strains). Bristol N2 was used as wildtype. Details of strains used in this study can be found in the key resources table.

Key Resource Table.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti-GFP	Invitrogen	Cat#A11120
Rabbit anti-GFP	Invitrogen	Cat#A11122
Rabbit polycolonal anti-HMP-1	Callaci et al.¹¹	N/A
Rabbit polycolonal anti-HMR-1	Callaci et al.¹¹	N/A
Mouse monoclonal anti-ZYX-1	Lecroisey et al.⁵⁴	N/A
Mouse monoclonal anti-AJM-1, MH27	Hardin lab ascites	N/A
Goat anti-rabbit IgG Texas Red	Invitrogen	Cat#T-2767
Goat anti-rabbit IgG FITC	Invitrogen	Cat#31635
Goat anti-mouse IgG Texas Red	Abcam	Cat#ab6787
anti-mouse FITC	Sigma-Aldrich	Lot#SLBZ0072
IRDye® 680RD Goat anti-Rabbit IgG Secondary Antibody	Li-COR	Cat#926-68071
IRDye® 680RD Donkey anti-Mouse IgG Secondary Antibody	Li-COR	Cat#926-68072
IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody	Li-COR	Cat#926-32211
IRDye® 800CW Donkey anti-Mouse IgG Secondary Antibody	Li-COR	Cat#926-32212
Bacterial and virus strains
Escherichia coli OP50	CGC	N/A
Escherichia coli BL21-Gold (DE3)	Sigma-Aldrich	Cat#69450-M
Escherichia coli clone for C. elegans CDS B0496.8 (tes-1)	Ahringer library; Kamath et al.⁴¹	N/A
Escherichia coli clone for C. elegans CDS C10H11.9 (let-502)	Ahringer library; Kamath et al.⁴¹	N/A
Escherichia coli clone for C. elegans CDS C06C3.1 (mel-11)	Ahringer library; Kamath et al.⁴¹	N/A
Chemicals, peptides, and recombinant proteins
Alexa Fluor 488 Phalloidin	Invitrogen	Cat#A12379
Alexa Fluor 660 Phalloidin	Invitrogen	Cat#A22285
ProTEV Plus	Promega	Cat#V6102
polymerized chicken F-actin	Cytoskeleton, Inc	Cat#AS99-B
T7 Megascript kit	ThermoFisher	Cat#AM1334
T3 Megascript kit	ThermoFisher	Cat#AM1338
Phusion DNA polymerase	ThermoFisher	Cat#F630S
Experimental models: Cell lines
NIH 3T3 fibroblasts	American Type Culture Collection	CRL-1658
Mouse: Embryonic fibroblasts (MEFs)	Gibco	Cat#A34181
Experimental models: Organisms/strains
C. elegans N2: wildtype	CGC	N2
C. elegans HR1157: let-502(sb118ts)I	Mains Lab	HR1157
C. elegans LP810: zyx-1(cp415[mNG::zyx-1a])II	Goldstein Lab	LP810
C. elegans LP831: zyx-1Δ(cp419[Pmyo-2>GFP])II	Goldstein Lab	LP831
C. elegans ML1651: mcIs46 [dlg-1::RFP + unc-119(+)]	Labouesse Lab	ML1651
C. elegans MQ468: hmp-2(qm39)I	Hekimi Lab	MQ468
C. elegans PE532: xnIs96[pJN455(hmr-1p::hmr-1::GFP::unc-54 3’UTR) + unc-119(+)]	Pettitt Lab	PE532
C. elegans PE633: feEx324[zyx-1::mCherry rol-6(su1006)]	Pettitt Lab	PE633
C. elegans PE636: feEx327[zyx-1::gfp Pmyo-2::dTomato]	Pettitt Lab	PE636
C. elegans PE644: zyx-1(gk190)II; feEx327[zyx-1::gfp myo-2p::dTomato]	Pettitt Lab	PE644
C. elegans PE647: zyx-1(gk190)II; hmp-1(fe4)/nT1V, feEx328[zyx-1D376-603::gfp myo-2p::dTomato]	Pettitt Lab	PE647
C. elegans PE649: zyx-1(gk190)II; hmp-1(fe4)/nT1V; feEx329[zyx-1D479-603::gfp myo-2p::dTomato]	Pettitt Lab	PE649
C. elegans PE650: zyx-1(gk190)II; hmp-1(fe4)/nT1V; feEx330[zyx-1D526-603::gfp myo-2p::dTomato]	Pettitt Lab	PE650
C. elegans PE651: zyx-1(gk190)II; hmp-1(fe4)/nT1V; feEx331[zyx-1D166-200::gfp myo-2p::dTomato]	Pettitt Lab	PE651
C. elegans PE671: mcIs46[dlg-1::RFP + unc-119(+)]; feEx327[zyx-1::gfp myo-2p::dTomato]	Pettitt Lab	PE671
C. elegans PE97: hmp-1(fe4)V	Pettitt Lab	PE97
C. elegans PHX5560: zyx-1(syb5560[mNG::zyx-1a, deltaLIM1-3])II	This paper	PHX5560
C. elegans PHX5622: tes-1(syb5622[mNG::FLAG::tes-1, deltaLIM1-3])IV	This paper	PHX5622
C. elegans PHX5627: tes-1(syb5622[mNG::FLAG::tes-1, deltaPET])IV	This paper	PHX5627
C. elegans SU1042: tes-1(jc71[mNeonGreen::tes-1])IV; zyx-1(gk190)II	This paper	SU1042
C. elegans SU1043: tes-1(jc71[mNeonGreen::tes-1])IV; mcEX40[plin-26::vab-10::mcherry; myo-2::gfp])IV	This paper	SU1043
C. elegans SU1044: tes-1(jc71[mNeonGreen::tes-1])IV; curIs[plin-26::lifeact::mcherry::unc-54 3'UTR; unc-119(+)]	This paper	SU1044
C. elegans SU1058: tes-1(jc71[mNG::tes-1])IV; zyx-1Δ(cp419[Pmyo-2>GFP])II	This paper	SU1058
C. elegans SU1072: tes-1(jc71[mNG::FLAG::tes-1])IV; hmp-1(jc58[hmp-1::mScarlet-I+LoxP511])V	This paper	SU1072
C. elegans SU1073: zyx-1Δ(cp419[Pmyo-2>GFP])II; tes-1(ok1036)IV	This paper	SU1073
C. elegans SU1085: tes-1(jc110[mScarlet-I::FLAG::tes-1+LoxP511])IV	This paper	SU1085
C. elegans SU1087: zyx-1(cp415[mNG::zyx-1a])II; curIs[plin-26::lifeact::mcherry::unc-54 3'UTR; unc-119(+)]	This paper	SU1087
C. elegans SU1088: zyx-1(syb5560[mNG::zyx-1a, deltaLIM1-3])II; curIs[plin-26::lifeact::mcherry::unc-54 3'UTR; unc-119(+)]	This paper	SU1088
C. elegans SU1090: tes-1(jc110[mScarlet-I::FLAG::tes-1+LoxP511])IV; zyx-1(syb5560[mNG::zyx-1a, deltaLIM1-3])II	This paper	SU1090
C. elegans SU1091: tes-1(jc110[mScarlet-I::FLAG::tes-1+LoxP511])IV; zyx-1(cp415[mNG::zyx-1a])II	This paper	SU1091
C. elegans SU1094: zyx-1(cp415[mNG::zyx-1a])II; tes-1(ok1036)IV	This paper	SU1094
C. elegans SU1100: zyx-1(gk190)II; curIs[plin-26::lifeact::mcherry::unc-54 3'UTR; unc-119(+)]	This paper	SU1100
C. elegans SU1101: tes-1(syb5622[mNG::FLAG::tes-1, deltaLIM1-3])IV; curIs[plin-26::lifeact::mcherry::unc-54 3'UTR; unc-119(+)]	This paper	SU1101
C. elegans SU1107: zyx-1Δ(cp419[Pmyo-2>GFP])II; tes-1(syb5622[mNG::FLAG::tes-1, deltaLIM1-3])IV	This paper	SU1107
C. elegans SU496: N2; jcEx159 [5kbptes-1::tes-1::gfp; pRF4; F35D3]	This paper	SU496
C. elegans SU708: N2; jcEx229[pRF4; Ptes-1::tes-1deltaPET::gfp F2-8; F35D3]	This paper	SU708
C. elegans SU709: N2; jcEx230[pRF4; Ptes-1::tes-1deltaPET::gfp F2-6; F35D3]	This paper	SU709
C. elegans SU710: N2; jcEx231[pRF4; Ptes-1::tes-1deltaLIM1::gfp; F35D3]	This paper	SU710
C. elegans SU713: N2; jcEx234[pRF4; Ptes-1::tes-1deltaLIM2::gfp F2-7; F35D3]	This paper	SU713
C. elegans SU714: N2; jcEx235[pRF4; Ptes-1::tes-1deltaLIM3::gfp; F35D3]	This paper	SU714
C. elegans SU715: N2; jcEx236[pRF4; Ptes-1::tes-1deltaLIM1-3::gfp; F35D3]	This paper	SU715
C. elegans SU896: hmp-1(jc58[hmp-1::mScarlet-I + Lox511])V	This paper	SU896
C. elegans SU931: curIs[plin-26::lifeact::mcherry::unc-54 3'UTR; unc-119(+)]	This paper	SU931
C. elegans SU955: tes-1(jc71[mNG::FLAG::tes-1])IV	This paper	SU955
C. elegans VC299: zyx-1(gk190)II	Moerman Lab	VC299
C. elegans VC696: tes-1(ok1036)IV	Moerman Lab	VC696
Oligonucleotides
tes-1 N-terminal 5’ Homology arm Forward Primer: GGCTGCTCTTCgTGGtttcttacctattttaaaatgacacctgcc	IDT	N/A
tes-1 N-terminal 5’ Homology arm Reverse Primer: GGGTGCTCTTCgCATCATtactgaaattaattggcatttaacgct	IDT	N/A
tes-1 N-terminal 3’ Homology arm Forward Primer: GGCTGCTCTTCgACGACCGACGTCACGTCTCCCGTTGTtGAC	IDT	N/A
tes-1 N-terminal 3’ Homology arm Reverse Primer: GGGTGCTCTTCgTACGTCTGGAAGTGGTGCCCACGCATAC	IDT	N/A
tes-1 N-terminal sgRNA: GCACGGCTTCTCGTCCACAA	IDT	N/A
tes-1 2kb promoter amplify Forward Primer: GCGTCGACGAGTTTTTGTCAAGAGTAAGAC	IDT	N/A
tes-1 2kb promoter amplify Reverse Primer: GCCCCGGGATCAACTGATCATCCGGATTCG	IDT	N/A
tes-1 5kb promoter amplify Forward Primer: GCCTGCAGGAAGACAACGCTTGTCAAGAAT	IDT	N/A
tes-1 5kb promoter amplify Reverse Primer: GCGTCGACATTTTGCCCTCGAAATGCAATAC	IDT	N/A
Recombinant DNA
cDNA yk662b10 (hmr-1)	NEXTDB, Kohara Lab	https://nematode.nig.ac.jp/doc/readme.php
cDNA yk285a2 (ajm-1)	NEXTDB, Kohara Lab	https://nematode.nig.ac.jp/doc/readme.php
cDNA yk1054c06 (zyx-1)	NEXTDB, Kohara Lab	https://nematode.nig.ac.jp/doc/readme.php
Plasmid: pPD95_75	Addgene	Addgene_1494
F35D3	Whitfield et al.³⁷	N/A
pRF4 [rol-6(su1006) transgenic marker]	Mello et al.³⁸	N/A
Cbr-unc-119(+)	Maduro et al.³⁹	N/A
Pmyo-2::dTomato	Korswagen Lab	N/A
Plasmid: Ptes-1(2kb)::tes-1::gfp	This paper	pAML224
Plasmid: Ptes-1(5kb)::tes-1::gfp	This paper	pAML224v2
Plasmid: SUMO-His-hmp-1	Callaci et al., ¹¹	N/A
Software and algorithms
Fiji	ImageJ	https://imagej.nih.gov/ij/
GraphPad Prism v.9.0	GraphPad	https://www.graphpad.com/scientific-software/prism/
Adobe illustrator	Adobe	https://www.adobe.com
Micromanager	Micromanager	https://micro-manager.org/
Fusion	Andor	https://andor.oxinst.com/downloads/view/fusion-release-2.3
Imaris	Imaris	https://imaris.oxinst.com/
QuickTime movie plugins for ImageJ	Hardin Lab	https://worms.zoology.wisc.edu/research/4d/4d.html
JACoP Plugins	ImageJ	https://imagej.nih.gov/ij/plugins/track/jacop.html

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NIH 3T3 fibroblasts (American Type Culture Collection, Manassas, VA) and mouse embryonic fibroblasts (MEFs) were cultured in DMEM media (Mediatech, Herndon, VA) and supplemented with 10% fetal bovine serum (HyClone; ThermoFisher Scientific, Hampton, NH), 2 mM L-glutamine (Invitrogen, Caarlsbad, CA) and penicillin–streptomycin (Invitrogen).

METHOD DETAILS

Molecular cloning

A ~5kb genomic sequence containing 2kb promoter and entire genomic region of tes-1 was PCR amplified using Phusion polymerase (ThermoFisher). The primers used were: 5’ GCGTCGACGAGTTTTTGTCAAGAGTAAGAC and 3’ GCCCCGGGATCAACTGATCATCCGGATTCG. The PCR product was digested with SalI and SmaI and ligated into a similarly digested Fire lab vector pPD95.75, which contains the GFP sequence. A frameshift was repaired via PCR to generate a Ptes-1(2kb)::tes-1::gfp construct (pAML224). To generate Ptes-1(5kb)::tes-1::gfp, additional promoter sequence was PCR amplified using Phusion polymerase. The primers used were: 5’ GCCTGCAGGAAGACAACGCTTGTCAAGAAT and 3’ GCGTCGACATTTTGCCCTCGAAATGCAATAC. The PCR product and pAML224 were digested using PstI and SalI and ligated together to generate pAML224v2. The identity of pAML224v2 was confirmed via sequencing. Domain deletions were performed using circle PCR as described previously ⁴.

CRISPR

mNG::TES-1 worms were generated via plasmid-based CRISPR/Cas9 ³⁴ using repair templates cloned using SapTrap cloning ³⁵. All domain deletions mutations (PHX strains) were generated by SunyBiotech (Fujian, China). Guides, homology arms primers, and single-stranded repair templates for all CRISPR/Cas9 editing can be found in the key resources table.

Microinjection

DNA was microinjected into worms as described previously ³⁶. Briefly, injection mixes consisting of 5ng/μl of transgenic tes-1 DNA constructs, 20 ng/μl of junk DNA (F35D3³⁷) and 75 ng/μl of pRF4 (rol-6(su1006) transgenic marker DNA)³⁸ were microinjected into both gonads of hermaphrodites. Progeny were screened for the presence of rol-6(su1006), and stable lines were established by passaging of worms. For zyx-1 transgenics, purified zyx-1 deletion construct DNA (100ng/ml) was mixed with coinjection markers pRF4 (200ng/ml), Cbr-unc-119(+) (30ng/ml)³⁹, and Pmyo-2::dTomato (5ng/ml) (courtesy Rik Korswagen, Utrecht Univ.) diluted in sterile water. At least two stable lines from each injected transgene were. used to analyze expression patterns.

Injection RNA interference was performed as described previously ⁴⁰. dsRNA was generated using ThermoFisher T7 and/or T3 Megascript kits; templates included Ahringer library ⁴¹ clones C10H11.9 (let-502) and C06C3.1 (mel-11), and Kohara clones yk662b10 (hmr-1), yk285a2 (ajm-1), and yk1054c06 (zyx-1) (NEXTDB, http://nematode.lab.nig.ac.jp/).

Antibody and Phalloidin Staining

Immunostaining was performed using freeze-cracking ⁴². Staining was performed as described previously ⁴³. Embryos were mounted onto poly-L-lysine-coated ring slides and incubated with primary antibodies in PBST and 5% non-fat dry milk overnight at 4°C. Embryos were then incubated with secondary antibodies in PBST and 5% non-fat dry milk for approximately three hours at room temperature. The following primary antibodies were used: 1:1000 mouse-anti-GFP (Invitrogen), 1:1000 rabbit-anti-GFP, 1:4000 polyclonal rabbit-anti-HMP-1, 1:4000 polyclonal rabbit-anti-HMR-1 and 1:200 monoclonal mouse-anti-AJM-1 (MH27). The following secondary antibodies were used: 1:50 anti-rabbit IgG Texas Red, 1:50 anti-rabbit FITC, 1:50 anti-mouse Texas Red and 1:50 anti-mouse FITC.

Phalloidin staining of mutant and wild-type embryos was used to visualize actin in fixed embryos ⁵. Embryos were mounted on poly-L-lysine-coated ring slides and fixed using the following: 4% paraformaldehyde, 0.1 mg/mL lysolecithin, 48 mM Pipes pH 6.8, 25 mM Hepes pH 6.8, 2 mM MgCl₂, and 10 mM EGTA for 20 minutes at room temperature. 1:20 Phalloidin-488 was incubated with embryos at room temperature for 90 minutes. Images of stained embryos were acquired as described below.

For co-immunostaining and phalloidin staining, embryos were gathered in a 1.5 mL Eppendorf tube and permeabilized with a solution of 4% paraformaldehyde, 10% Triton-X-100, 48 mM Pipes pH 6.8, 25 mM Hepes pH 6.8, 2 mM MgCl₂ and 10mM EGTA for 20 minutes at room temperature. Embryos were incubated overnight in PBST+5% dry milk+1:1000 rabbit-anti-GFP at 4C on a nutator. Secondary antibodies (1:10 Phalloidin-660 and 1:50 anti-rabbit FITC) were incubated for 2 hours at room temperature. Images of stained embryos were acquired as described below.

DIC Imaging

Four dimensional DIC movies were gathered on either a Nikon Optiphot-2 connected to a QImaging camera or an Olympus BX5 connected to a Scion camera. Mounts were made as previously described ⁴⁴. QuickTime movie plugins for ImageJ (https://worms.zoology.wisc.edu/research/4d/4d.html) were used to compress and view movies.

Confocal Microscopy

Spinning-disc confocal images of tes-1 transgenics were acquired with a Z-slice spacing of 0.2μm for imaging of actin, 0.3 μm for embryos stained for both GFP and actin, and 0.5μm for all other imaging using either Perkin Elmer Ultraview or Micromanager software ^45,46 and a Nikon Eclipse E600 microscope connected to a Yokogawa CSU10 spinning disk scanhead and a Hamamatsu ORCA-ER charge-coupled device (CCD) camera. Junctional/cytoplasmic signal measurements were performed as described previously ⁴⁷. Fisher’s exact test calculations were performed online at https://www.socscistatistics.com/tests/fisher/default2.aspx or using GraphPad Prism v. 9.0 software (GraphPad Software, San Diego, California USA, www.graphpad.com). The extension of Fisher’s exact test to a 4 × 2 contingency table⁴⁸ was performed online at http://vassarstats.net/fisher2x4.html. Other statistical analyses were performed using GraphPad Prism. For zyx-1 transgenics, imaging was carried out using a Zeiss LSM 710 laser scanning confocal microscope equipped with 10x and 63x oil lenses. For endogenous knock-ins, imaging was performed using a Dragonfly 500 spinning disc confocal microscope (Andor Corp., Belfast, Ireland), mounted on a Leica DMi8 microscope, equipped with a Zyla camera and controlled by Fusion software (Andor Corp.). Images were collected using 0.18 μm slices with a 100×/1.3 NA oil immersion Leica objective at 20°C.

Colocalization Analysis

Colocalization analysis was performed in Fiji using Just Another Colocalization Plugin (JACoP; https://imagej.nih.gov/ij/plugins/track/jacop.html) ⁴⁹. 5 focal planes from >10 junctional segments were combined into single stacks for each genotype. Maximum intensity Z projections were obtained, and automated Costes thresholding within JACoP was visually confirmed in each case. Significant difference in Pearson’s R for colocalizations was assessed using the online Z calculator available at http://vassarstats.net/rdiff.html

Protein Expression and Purification

GST- and SUMO-His-tagged proteins were expressed in BL21-Gold (DE3) Escherichia coli cells and purified as described ^50,51. Cells were induced with 0.1mM IPTG at 18°C for 16 hours. Wash and elution buffers were as follows: GST wash (1X PBS, 500mM NaCl, 0.1% Tween-20, and 1mM DTT), GST elution (50mM Tris pH 8.0, 0.3% glutathione, 150mM NaCl), His wash (50mM Na-Phosphate pH 8.0, 300mM NaCl, 0.1% Tween-20, 10mM Imidazole), and His elution (250mM Imidazole, 100mM NaCl, 10% glycerol, 50mM Hepes pH 7.6). For actin-pelleting assays, the GST tag was cleaved from GST-TES-1 using ProTEV Plus (Promega), according to manufacturer’s instructions.

Actin-Pelleting Assays

Actin co-sedimentation assays were performed as described previously ⁵⁰. Briefly, 5μM purified, cleaved proteins (quantified via a Bradford Assay) were incubated at room temperature for one hour with 0 or 5μM polymerized chicken F-actin (Cytoskeleton, Inc.). BSA was used a negative control, and SUMO-His-HMP-1 ¹¹ was used as a positive control. Samples were then centrifuged at 100,000 rpm for 20 min at 4°C in a TLA-120.1 rotor using a Beckman Optima tabletop ultracentrifuge. Samples were run on 12% SDS-PAGE gels, stained with Coomassie Brilliant Blue, and bands were quantified using ImageJ.

Co-immunoprecipitations and Western Blots

C. elegans expressing TES-1::GFP were grown in liquid culture as previously described ⁵². Co-immunoprecipitations were completed as in ³². Western blots were performed as described previously ⁵³, using rabbit anti-GFP, rabbit anti-HMP-1 ¹¹ and mouse anti-ZYX-1 ⁵⁴ primary antibodies and Li-COR IRDye® secondary antibodies to detect proteins.

Stress fiber strain site assay

A tes-1 LCR::mCherry construct was designed and expressed using the procedures described in detail by Winkelman et al. ². Briefly, a synthetic gBlock DNA encoding a mammalian codon-optimized version of the LIM1-3 domain of TES-1 was ordered from IDT (Coralville, Iowa) and cloned into a CMV-driven expression vector that fused the C-terminus of LCR(Tes) to mCherry, and used to transfect zyxin ^−/− mouse embryo fibroblast cells (MEFs) rescued with stably integrated GFP-zyxin. Transfected MEFs were imaged on an inverted Nikon Ti-E microscope (Nikon, Melville, NY) with a Yokogawa CSU-X confocal scanhead and Zyla 4.2 sCMOS Camera (Andor, Belfast, UK). A 405 nm laser coupled to a Mosaic digital micromirror device (Andor) was used to locally damage stress fibers. Kymography of TES-1(LIM1-3)::GFP was performed using ImageJ as described in ².

QUANTIFICATION AND STATISTICAL ANALYSIS

Graphs were generated using GraphPad Prism. Unpaired Student’s T-test or ANOVA was used to determine statistically significant differences between groups. Statistical test parameters, outcomes and reporting on number of samples used in each experiment are indicated in figure legends.

Supplementary Material

2. Video S1. tes-1(RNAi) enhances the severity of morphogenetic defects in hmp-1(fe4) embryos, Related to Figure 1.

Time lapse movie comparing hmp-1(fe4) homozygous and hmp-1(fe4); tes-1(RNAi) embryos. The latter fail consistently during early elongation, and all develop the Humpback phenotype. Time is shown in hours:minutes.

Download video file^{(12.2MB, mp4)}

3. Video S2. Laser-induced recruitment of the ZYX-1 LCR::mCherry and mouse GFP::Zyxin to SFSS, Related to Figure 4.

Time lapse movie showing laser induction of a stress fiber strain site (SFSS) in a representative zyxin ^−/− mouse embryo fibroblast (MEF) rescued with stably integrated M. musculus GFP-zyxin and transiently transfected with a construct encoding ZYX-1 LCR::mCherry related to Figure 4D. White box show where light was targeted, and white arrows denote developing SFSS. Time is shown in minutes:sec.

Download video file^{(8MB, mp4)}

4. Video S3. Laser-induced recruitment of the TES-1 LCR::mCherry and mouse GFP::Zyxin to SFSS, Related to Figure 4.

Time lapse movie showing laser induction of a stress fiber strain site (SFSS) in a representative zyxin ^−/− mouse embryo fibroblast (MEF) rescued with stably integrated M. musculus GFP-zyxin and transiently transfected with a construct encoding TES-1 LCR::mCherry related to Figure 4F. White box show where light was targeted, and white arrows denote developing SFSS. Time is shown in minutes:sec.

Download video file^{(18.7MB, mp4)}

NIHMS1846259-supplement-5.pdf^{(9.9MB, pdf)}

Cell-cell junctions are vulnerable to damage due to high tension generated during dramatic morphogenetic changes. Lynch et al. show that the LIM domain-containing repeat proteins TES-1/Tes and ZYX-1/Zyxin are components of a multicellular, tension-sensitive system that stabilizes the junctional actin cytoskeleton during embryonic morphogenesis.

TES-1 and ZYX-1 promote the integrity of actin networks during elongation
The LIM domains of TES-1 and ZYX-1 are required for normal function and localization
TES-1 and ZYX-1 are recruited to apical junctions in a tension-dependent manner
Both TES-1 and ZYX-1 can be recruited to strained actin fibers

ACKNOWLEDGEMENTS

cDNA clones for hmr-1, ajm-1, zyx-1, zoo-1, hmp-1, and tes-1 (yk collection) were provided by Yuji Kohara (National Institute of Genetics). A.L., Y.Z., B.L., S.M., and J.H. were supported by NIH grants R01GM058038 and MIRA R35GM145312 awarded to J.H.; B.M. was supported by a Gilliam Fellowship from the Howard Hughes Medical Institute, and by an Advanced Opportunities Fellowship and a COVID-19 dissertation completion fellowship from the University of Wisconsin-Madison; S.B. and A.A. were supported by NIH grant R35GM134865 awarded to A.A.; J.W. was supported by NIH grant F32GM122372 and by NIH grant R01GM104032 and Army Research Office Multidisciplinary University Research Initiative W911NF1410403 awarded to M.G.; B.G. and M.M.S. were supported by NIH MIRA R35GM134838 awarded to B.G. and NIH F32GM119348 awarded to M.M.S. Some strains were provided by the Caenorhabditis Genetics Center (CGC; https://cbs.umn.edu/cgc/home), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Abbreviations used in this study:

AJ: adherens junction
CCC: cadherin-catenin complex
CFB: circumferential filament bundle
DIC: differential interference contrast
LIM: Lin-l1, Isl-1, Mec-3
PET: Prickle, Espinas, Testin
CR: cysteine-rich
SFSS: stress fiber strain site

Footnotes

Declaration of Interests

The authors declare no competing or financial interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

2. Video S1. tes-1(RNAi) enhances the severity of morphogenetic defects in hmp-1(fe4) embryos, Related to Figure 1.

Download video file^{(12.2MB, mp4)}

3. Video S2. Laser-induced recruitment of the ZYX-1 LCR::mCherry and mouse GFP::Zyxin to SFSS, Related to Figure 4.

Download video file^{(8MB, mp4)}

4. Video S3. Laser-induced recruitment of the TES-1 LCR::mCherry and mouse GFP::Zyxin to SFSS, Related to Figure 4.

Time lapse movie showing laser induction of a stress fiber strain site (SFSS) in a representative zyxin ^−/− mouse embryo fibroblast (MEF) rescued with stably integrated M. musculus GFP-zyxin and transiently transfected with a construct encoding TES-1 LCR::mCherry related to Figure 4F. White box show where light was targeted, and white arrows denote developing SFSS. Time is shown in minutes:sec.

Download video file^{(18.7MB, mp4)}

NIHMS1846259-supplement-5.pdf^{(9.9MB, pdf)}

Data Availability Statement

All data reported in this paper will be shared by the lead contact upon request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo

Allison M Lynch

Yuyun Zhu

Bethany G Lucas

Jonathan D Winkelman

Keliya Bai

Sterling CT Martin

Samuel Block

Mark M Slabodnick

Anjon Audhya

Bob Goldstein

Jonathan Pettitt

Margaret L Gardel

Jeff Hardin

SUMMARY

RESULTS AND DISCUSSION

Figure 1. TES-1 loss enhances phenotypes in hypomorphic CCC backgrounds.

TES-1 is an F-actin-binding protein that functionally interacts with hmp-1/α-catenin at the C. elegans apical junction

TES-1 localizes to apical junctions in the embryonic epidermis

Figure 2. TES-1 localizes to sites of cell-cell attachment during embryonic elongation.

TES-1 requires its PET and LIM domains

Figure 3. TES-1 localization requires its PET and LCR domains.

TES-1 localizes to junctions in a tension-dependent manner

ZYX-1/zyxin localizes to junctions in a tension-dependent manner complementary to TES-1

Figure 4. ZYX-1 is also recruited to junctions during elongation and both ZYX-1 and TES-1 are recruited to strained actin filaments.

Both TES-1 and ZYX-1 can be recruited to strained actin fibers

ZYX-1/zyxin and TES-1/Tes act largely independently during elongation

STAR METHODS

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Key Resource Table.

METHOD DETAILS

Molecular cloning

CRISPR

Microinjection

Antibody and Phalloidin Staining

DIC Imaging

Confocal Microscopy

Colocalization Analysis

Protein Expression and Purification

Actin-Pelleting Assays

Co-immunoprecipitations and Western Blots

Stress fiber strain site assay

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

ACKNOWLEDGEMENTS

Abbreviations used in this study:

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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