Fig 1: STRN3 interacts directly with Rac1(A and B) Glutathione S-transferase (GST) pull-down from P5 wild-type (WT) mouse sciatic nerve lysate, comparing GST-Rac1-GDP versus GST-Rac1-GTP(A) Volcano plot showing the Welch difference (log2 fold change) of Rac1 interactors. Proteins in red, such as STRN3 and several well-established Rac1 interactors, are significantly (false discovery rate [FDR] = 0.05) enriched in the active Rac1-GTP fraction compared to the inactive Rac1-GDP fraction. MOB4 was found to be enriched in the Rac1-GTP fraction in a separate biological replicate (data not shown).(B) GST-Rac1-GDP/GTP pull-down of P5 WT sciatic nerve lysate was repeated and analyzed by western blot (WB). STRN3 was observed to elute with the active Rac1-GTP fraction but not the inactive Rac1-GDP fraction.(C) Immunoprecipitation of Rac1 from SC lysates. STRN3 was found to interact with Rac1 in SCs. n = 5 individual coIPs per targeting antibody (anti-Rac1 or IgG isotype control). Unpaired two-tailed t test (t = 3.730, df = 8, p = 0.0058). Error bars indicate SEM. **p < 0.01.
Fig 2: STRN3 is required for SC elongation and lamellipodia formationSCs were isolated from WT and Strn3SCKO littermates at P45–P60.(A and B) SCs isolated from Strn3SCKO mice have absent Strn3 protein expression by western blot (A) and immunohistochemistry (B). Similar results were obtained with a second anti-STRN3 antibody (data not shown). STRN3 was stained in green, and nuclei were labeled with DAPI (blue). Scale bar: 20 μm.(C) Representative immunohistochemistry of mouse SCs, with nuclei labeled with Sox10 (green) and F-actin labeled with phalloidin (red).(D) Loss of STRN3 in SCs does not alter the purity of SC isolation. Unpaired two-tailed t test (t = 0.01239, df = 4, p = 0.9907).(E) Deletion of Strn3 in SCs does not affect the density of SCs adhering to the substrate in vitro. Unpaired two-tailed t test (t = 0.9691, df = 4, p = 0.3874).(F) SCs isolated from Strn3SCKO mice form reduced numbers of radial and total, but not axial, lamellipodia. Axial lamellipodia (white arrowhead) are defined as extending from within a 20° angle of the long axis of the cell, whereas radial lamellipodia (white arrow) protrude from outside of this range. Two-way multiple comparisons ANOVA with Bonferroni post hoc test. F (1,6) genotype = 45.63, p = 0.0005; F (2,6) lamellipodia type = 21.75, p = 0.0018; paxial = 0.5662, pradial = 0.0142, and paxial+radial = 0.0033.(G and H) SCs ablated for Strn3 have reduced cell elongation.(G) Unpaired two-tailed t test (t = 2.978, df = 4, p = 0.0408).(H) Lognormal nonlinear regression, extra sum-of-squares F test; F (3, 90) = 14.47. n = 3 individual mouse SC isolations per genotype. Error bars indicate SEM. *p < 0.05 and **p < 0.01.
Fig 3: Striatin protein levels are developmentally regulated(A) STRN1, STRN3, and STRN4 protein levels from WT mouse sciatic nerves at P3, P5, P20, and P45.(B) Densitometry analysis shows a progressive reduction in STRN1, STRN3, and STRN4 during development in peripheral nerves of WT mice. n = 3–4 animals per age group. One-way multiple comparisons ANOVA with Bonferroni post hoc test. STRN1 (F (3,9) = 7.66, p = 0.0072, pP3–P5 > 0.9999, pP3–P20 > 0.9999, pP3–P45 = 0.0474, pP5–P20 = 0.3379, pP5–P45 = 0.0083, and pP20–P45 = 0.3277). STRN3 (F (3,10) = 8.772, p = 0.0038, pP3–P5 > 0.9999, pP3–P20 = 0.0813, pP3–P45 = 0.0471, pP5–P20 = 0.0196, pP5–P45 = 0.0117, and pP20–P45 > 0.9999). STRN4 (F (3,10) = 113.4, p < 0.0001, pP3–P5 = 0.5436, pP3–P20 < 0.0001, pP3–P45 < 0.0001, pP5–P20 < 0.0001, pP5–P45 < 0.0001, and pP20–P45 = 0.0939).(C) Representative western blots of STRN1, STRN3, and STRN4 from sciatic nerves of WT and Strn3SCKO mice at P10.(D) Densitometry analysis shows an increase in Strn4 at P10 in peripheral nerves of Strn3SCKO mice. n = 3–5 animals per genotype per age group. Two-way multiple comparisons ANOVA with Bonferroni post hoc test. STRN1 (F (4, 15) age = 1.335; p = 0.3026; F (1, 11) genotype = 3.755, p = 0.0787; pP3 = 0.5333, pP5 > 0.9999, pP10 = 0.0896, pP20 > 0.9999, and pP45 > 0.9999). STRN4 (F age (4, 15) = 1.475, p = 0.2591; F (1, 12) genotype = 1.305, p = 0.2755; pP3 > 0.9999, pP5 > 0.9999, pP10 = 0.0190, pP20 > 0.9999, and pP45 > 0.9999).Error bars indicate SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001.
Fig 4: Ablation of Strn1 and Strn3 in SCs does not alter Rac1 activity(A) Representative western blots of Rac1 and CDC42 active (GTP) and total (tot) forms from pooled P20 sciatic nerves and brachial plexuses of WT and Strn1/3dSCKO mice.(B and C) Densitometry analysis shows no significant change in Rac1 or CDC42 activity in peripheral nerves of Strn1/3dSCKO mice at P20. n =5 samples per genotype. Samples were pooled from 3–5 animals each. Unpaired two-tailed t test. Rac1 activity (t = 1.649, df = 8, p = 0.1379) and CDC42 activity (t = 1.257, df = 8, p = 0.2443).(D) Representative western blots of PAK1 and NF2 phosphorylated and tot forms from P20 sciatic nerves and brachial plexuses of WT and Strn1/ 3dSCKO mice.(E and F) Densitometry analysis shows no significant change in phosphorylation or tot levels of the Rac1/CDC42 effectors PAK1 or NF2 in peripheral nerves of Strn1/3dSCKO mice at P20. n = 4 animals per genotype. Unpaired two-tailed t test. p-PAK1 (t = 1.484, df = 6, p = 0.1884), PAK1 (t = 0.5307, df = 6, p = 0.6147), p-NF2 (t = 1.160, df = 6, p = 0.2900), and NF2 (t = 0.7988, df = 6, p = 0.4548). Error bars indicate SEM.
Fig 5: Loss of striatin proteins in SCs results in mild to severe developmental defects(A–C) Semithin sections (A and C) and electron micrographs (B) of WT and striatin mutant sciatic nerves at P20 (A–C) and P60 (A). At P20, Strn1/3dSCKO nerves present with immature bundles of unsorted axons, myelinated axons inside unsorted bundles (#), and amyelinated axons (*). Scale bars: 10 mm (A and C) and 2 μm (B).(D–F) At P20, sciatic nerves of Strn1/3dSCKO and Strn3/4dSCKO mice have significantly fewer total myelinated fibers, a lower density of myelinated fibers, and thinner, hypoplastic sciatic nerves. n = 3–6 animals per genotype. Two-way multiple comparisons ANOVA with Bonferroni post hoc test. Total fibers (F (5, 35) model = 32.90; p < 0.0001; F (1, 35) WT versus KO = 76.14, p < 0.0001; pStrn1-SCKO > 0.9999, pStrn3-SCKO > 0.9999, pStrn4-SCKO = 0.4678, pStrn1/3-dSCKO < 0.0001, pStrn1/4-dSCKO > 0.9999, and pStrn3/4-dSCKO < 0.0001). Fiber density (F (5, 35) model = 34.94; p < 0.0001; F (1, 35) WT versus KO = 71.50, p < 0.0001; pStrn1-SCKO > 0.9999, pStrn3-SCKO > 0.9999, pStrn4-SCKO > 0.9999, pStrn1/3-dSCKO < 0.0001, pStrn1/4-dSCKO > 0.9999, and pStrn3/4-dSCKO < 0.0001). Nerve area (F (5, 35) model = 4.602; p = 0.0025; F (1, 35) WT versus KO = 11.41, p = 0.0018; pStrn1-SCKO > 0.9999, pStrn3-SCKO > 0.9999, pStrn4-SCKO > 0.9999, pStrn1/3-dSCKO = 0.0001, pStrn1/4-dSCKO > 0.9999, and pStrn3/4-dSCKO = 0.0117). Error bars indicate SEM. *p < 0.05, ***p < 0.001, and ****p < 0.0001.
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