Stressed Actin Binding by the Prickle2 LIM Domains and its Regulation in the Full-Length Protein

Figures

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  Figure 1:. Pk2-LIM Domains Localize to Linear Actin Filaments A: Schematic of full-length Pk2, and Pk2-LIM Domain Containing Region (Pk2-LCR). B: Cartoon model of full-length Pk2 and actin localization in planar polarized cells of the Xenopus laevis dorsal mesoderm. C: Cartoon model of Vangl2-Pk2 complexes at the plasma membrane associated with actin. Fluorescence microscopy of dorsal mesoderm expressing fluorescent fusion constructs (D-F) (Scale bar, 5μm). White asterisks indicate anterior puncta. White arrows indicate linear objects. D: Full-length Pk2 localizes into bright puncta alongside actin E: Pk2-LCR localizes to linear structure along actin filaments F: Coexpression of full-length Pk2 and Pk2-LCR highlights diYerential localization of LIM domains.

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  Figure 2:. Localization of Pk2-LIM Domains is Sensitive to Myosin Contractility A: Cartoon model of Pk2-LCR recruitment to linear actin filaments mechanically stressed by contractile myosin-II. Fluorescence microscopy of dorsal mesoderm expressing fluorescent fusion Pk2-LCR and Lifeact at 0 and 15 min following DMSO or 50μM Blebbistatin treatment (B-E) (Scale bar, 10μm). B-C: Bright Pk2-LCR positive objects remain visible 15 min after DMSO treatment. D-E: Bright Pk2-LCR positive objects disappear 15 min after 50μM Blebbistatin treatment. F: Quantification of number of Pk2-LCR positive objects over 15 min time course.

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  Figure 3:. Pk2-LIM Domains Localize to Sites of Stressed Actin via a Conserved Mechanism A: Schematic of Pk2-LCR-WT, Pk2-LCR-F/Y>A mutant, and Zyx-LCR-WT. Fluorescence microscopy of dorsal mesoderm expressing fluorescent fusion constructs and Lifeact (B-D) (Scale bar, 10μm). White arrows indicate linear LCR-positive objects. B: Zyx-LCR-WT localizes to linear actin filaments. C: Pk2-LCR-WT localizes to linear actin filaments. D: Pk2-LCR-F/Y>A mutant exhibits a diYuse cytosolic signal lacking localization to actin filaments TIRF microcopy of dorsal mesoderm expressing fluorescent fusion constructs and Lifeact (E-F) (Scale bar, 5μm) E: Pk2-LCR-WT localizes to Zyx-LCR-WT labeled sites on actin filaments F: Pk2-LCR-F/Y>A does not clearly localize to Zyx-LCR-WT labeled sites on actin filaments. G: Quantification of Pk2-LCR WT or Pk2-LCR-F/Y>A mutant signal relative to Zyx-LCR-WT.

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  Figure 4:. Recruitment of Pk2-LCR is Modulated by the Pk2-PET Domain A: Alphafold3 prediction of Pk2-PET domain (magenta) interaction with Pk2-LIM1 (lime green) and Pk2-LIM2 (forest green). Residues within PET predicted to interact with Pk2-LIM12 highlighted in orange B: Schematic of Pk2-LCR and Pk2-PET-LCR Fluorescence microscopy of dorsal mesoderm expressing fluorescent fusion constructs (C-E) (Scale bar, 10μm). White asterisks indicate anterior puncta. White arrows indicate linear objects. C: Pk2-LCR localizes to linear actin filaments D: Pk2-PET-LCR localizes to linear actin filaments E: Coexpression of Pk2-PET-LCR and full-length Pk2 reveals recruitment of Pk2-PET-LCR into full-length Pk2 puncta TIRF microcopy of dorsal mesoderm expressing fluorescent fusion constructs (F-G,I-L) (Scale bar, 5μm). White asterisks indicate bright puncta. F: Pk2-LCR localizes to Zyx-LCR-labeled regions G: Pk2-PET-LCR localizes to Zyx-LCR-labeled regions as well as bright puncta that do not correspond to a similar local enrichment of Zyx-LCR H: Quantification of Pk2-LCR or Pk2-PET-LCR signal relative to Zyx-LCR.

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  Figure 5:. Recruitment of Pk2-LCR is Modulated by the Pk2-Cterminal Region A: Schematic of Pk2-LCR and Pk2-LCR-Cterm Fluorescence microscopy of dorsal mesoderm expressing fluorescent fusion constructs (B-E) (Scale bar, 10μm). White asterisks indicate bright puncta. White arrows indicate linear objects. B: Pk2-LCR localizes to linear actin filaments C: Pk2-LCR-Cterm localizes to bright puncta alongside actin D: Coexpression of Pk2-LCR-Cterm and full-length Pk2 reveals recruitment of Pk2-LCR-Cterm into full-length Pk2 puncta TIRF microcopy of dorsal mesoderm expressing fluorescent fusion constructs (E-F) (Scale bar, 5μm). White asterisks indicate bright puncta. E: Pk2-LCR localizes to Zyx-LCR labeled sites of stressed actin F: LCR-Cterm localizes to bright puncta that do not correspond with a similar puncta of Zyx-LCR G: Quantification of Pk2-LCR or Pk2-LCR-Cterm signal relative to Zyx-LCR

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  Figure 6:. Pk2 Patient Alleles Exhibit Altered Recruitment to Stressed Actin A: Schematic of Pk2-LCR-WT, Pk2-LCR-Y230C, and Pk2-Y290C TIRF microcopy of dorsal mesoderm expressing fluorescent fusion constructs (B-D) (Scale bar, 5μm). B: Pk2-LCR-WT localizes to Zyx-LCR-labeled regions C: Pk2-LCR-Y230C localizes to Zyx-LCR-labeled regions D: Pk2-LCR-Y290C exhibits weak localization to Zyx-LCR-labeled regions E: Quantification of Pk2-LCR-WT, Pk2-LCR-Y230C or Pk2-LCR-Y290C signal relative to Zyx-LCR