Pil1 cytoplasmic rods contain bundles of crosslinked tubules

Cytoskeletal polymers are organized into a wide variety of higher-order structures in cells. The yeast BAR domain protein Pil1 self-assembles into tubules in vitro, and forms linear polymers at cortical eisosomes in cells. In the fission yeast S. pombe, over-expressed Pil1 forms thick rods that detach from the plasma membrane. In this study, we used thin-section electron microscopy to determine the ultrastructure of these cytoplasmic Pil1 rods. We found that cytoplasmic rods contained crosslinked Pil1 tubules that displayed regular, hexagonal spacing. These bundles were stained by filipin, a sterol-binding fluorescent dye, suggesting that they contained lipids. Cytoplasmic Pil1 rods were present but less abundant in sle1Δ and fhn1Δ mutant cells. We also found that endogenous Pil1 formed thick rods under saturated growth conditions. Taken together, our findings suggest the presence of cellular mechanisms that assemble Pil1 tubules into higher-order structures.