Fig. 4: StlP oligomerizes and controls membrane fluidity at hyphal tips.
A StlP assembles into oligomers, consistent with other stomatin proteins. The stomatin domain of StlP (StlPSD) was purified and concentrated to 1 mg ml−1. For the SDS-PAGE analysis, 2 and 20 µg StlPSD were loaded. For the Native-PAGE gel, 20 µg StlPSD was used. Protein bands were visualized with Coomassie blue staining. B AlphaFold predictions of an StlP hexamer (left) or decamer (right). The StlP decamer assembles into a 10-fold symmetric ring structure. For the prediction, the amino acid sequence spanning the two N-terminal transmembrane helices (TMHs) at positions 106-126 and 149-168, as well as the stomatin domain at positions 209-311 of StlP were used. Magenta represents an StlP monomer. Deletion of stlP increases membrane fluidity at hyphal tips of S. coelicolor. C Visualization of a fluid membrane region (yellow spots) at hyphal tips. 16 h-old cultures of S. coelicolor M145, the stlP mutant and the complemented mutant strain (∆stlP + pXZ15) were incubated with 100 µM DilC12 and grown for a further 3 h in L-phase broth (LPB) liquid medium prior to imaging. White arrows indicate fluid regions at hyphal tips. Please note that the extruded cell wall-deficient (CWD) cells are highly fluid. BF brightfield. Qualitative (D) and quantitative (E) analysis of membrane fluidity using Laurdan staining. Mycelia from 16 h-old cultures of different strains were collected and labeled with 1 mM Laurdan. Red and blue pixels represent high and low fluidity, respectively (D). BF brightfield (D). The values in the graph (E) indicate the generalized polarization (GP), which ranges from −1 (more fluid) to +1 (less fluid). The number of measured tips was 27 for the wild-type strain (M145), 30 for the stlP mutant and 26 for the complemented mutant. Box plots display the distribution of apical fluorescence measurements. The center line represents the median (50th percentile). The box spans from the first quartile (25th percentile) to the third quartile (75th percentile), representing the interquartile range (IQR). Whiskers extend to the smallest and largest values within 1.5 times the IQR. Data points beyond this range are considered outliers and are shown as individual dots. ***p < 0.001; unpaired two-tailed Student’s t test. F Increased growth temperature restores membrane fluidity of the stlP mutant. Here, strains were grown in LPB liquid medium at 22, 30 or 37 °C for 16 h and the membrane fluidity is indicated as GP value. Approximately 45 tips were measured for each strain. ***p < 0.001 for 22 °C and 30 °C; p = 0.58 for 37 °C; unpaired two-tailed Student’s t test. Scale bar represents 5 μm (C), 10 μm (D, main images) and 0.5 μm (D, insets), respectively.
