Figure 4. Cholesterol binding is essential for STOML3 to regulate mechanotransduction in sensory neurons.

(a) Alignment of the deduced amino-acid sequence of mouse STOML3 with its homologues including stomatin, MEC-2 podocin and STOML1. The conserved proline is indicted with a solid circle. (b) Representative blots showing that P40S mutation abolishes the distribution of STOML3 in lipid rafts. CHO cells expressing Myc-tagged wild-type STOML3 or P40S mutant were lysed and submitted to sucrose-density-gradient centrifugation. Eight fractions were collected from the top of the gradient (1–8, top to bottom). STOML3 was abundantly detected in both raft fractions (top 2–3) and non-raft fractions (5–8), while STOML3-P40S was mainly detected in non-raft fractions (5–8). STOML3 appeared as a triplet near the predicted molecular weight. The P40S mutation altered the relative intensity of the three bands. See also Supplementary Fig. 5. (c) Quantitative comparison of percentage of raft-associated fractions (fractions 1–3) relative to total amount of STOML3 or STOML3-P40 protein (n=5 for each, unpaired t-test, P<0.01). (d) Stacked histograms showing the proportions of different mechano-gated currents observed in STOML3^−/− neurons treated without or with MβCD (χ²-test, P>0.05). MβCD treatment did not significantly reduce the mechanosensitivity of STOML3^−/− sensory neurons. NS, not significant. (e) Quantitative comparison of the peak amplitude of the RA currents recorded from STOML3^−/− neurons treated without or with MβCD. The amplitude of RA current in STOML3^−/− neurons was not altered by MβCD treatment (unpaired t-test, P>0.05). (f) Left: representative images of EGFP (top), STOML3-EGFP (middle) and STOML3-P40S-EGFP (bottom) expression in transfected STOML3^−/− sensory neurons. Note the punctate distribution of STOML3-EGFP along neurites disappeared in STOML3-P40S-EGFP-transfected STOML3^−/− sensory neurons. Scale bar, 10 μm; right: stacked histograms showing the proportions of different mechano-gated currents observed in STOML3^−/− neurons transfected with EGFP, STOML3-EGFP or STOML3-P40S-EGFP cDNA. Note STOML3-P40S-EGFP transfection failed to restore the mechanosensitivity in STOML3^−/− sensory neurons (χ²-test, EGFP versus STOML3-EGFP, P<0.05; STOML3-EGFP versus STOML3-P40S-EGFP, P<0.05; EGFP versus STOML3-P40S-EGFP, P>0.05). The number of neurons recorded is indicated in parentheses in each panel. *P<0.05; **P<0.01; Error bars indicate s.e.m.