strains in this study were jm837 ( leu132  h- ) , jm1262 ( pil1-mcherry::natr h- ) , jm1293 ( fhn1::kanmx6 ura4-d18 leu132 ade6-m210  h ) and jm1461 ( sle1::kanmx6 ura4-d18 leu132 ade6-m210  h ) . expression from plasmid pjm512 ( prep41-pil1-gfp ) was induced by growth in the absence of thiamine for 36  hours at 32c . to observe endogenous pil1-mcherry rods , strain jm1262 was grown on ye4s plates at 32c for 3 d. cells were scraped from the plate and immediately imaged . electron microscopy : for thin sectioning , cells carrying plasmid pjm512 ( prep41-pil1-gfp ) or control plasmid pjm211 ( prep41-gfp ) were grown in the absence of thiamine for 36  hours at 32c , and then prepared and imaged as previously described . for immunolabeling of pil1 bundles , fixations and digestion were the same except for the following changes : initial fixation of 3% pfa/1% gta was used instead of 3% gta/1% pfa . samples were incubated in 0.05  m glycine in nacac buffer for 30  min to remove excess aldehydes . additionally , 1% p - phenylenediamine ( ppd ) was included in each etoh solution . four additional 85% etoh/1% ppd rinses over 1  hour were performed , and then pellets were resuspended in a 2:1 mixture of 85% etoh/1% ppd : lr white medium resin . this suspension was placed on a rotator for 2  hours at room temperature and then resuspended in 1:1 85% etoh/1% ppd : lr white medium resin , rotated for 1  hour , and then kept at 4c overnight . the following day , samples were resuspended in 1:2 85% etoh/ 1% ppd : lr white medium resin rotated for 1  hour at room temperature and then resuspended in 100% lr white medium resin . solution was changed until clear , and then 3 more changes over 6  hours rotating at room temperature were performed before placing at 4c overnight . samples were warmed to room temperature and 4 changes of 100% lr white medium resin were made with 1  hour each change . samples were transferred to gelatin capsules and left for 4  h at room temperature , then polymerized at 50c for 24  h. thin sections ( 7080  nm ) were placed on g300 nickel - coated grids ( electron microscopy sciences ) . grids with sections were washed in pbs for 5  min , put in block solution of pbs + 5% bsa ( sigma - aldrich ) for 15  min , incubated for 1  hour in 1/25th dilution of rabbit anti - gfp , washed 6  times for 2  min , incubated for 30  min in a 1/25th dilution of goat anti rabbit 6  nm gold - conjugated secondary antibody ( 25104 ; electron microscopy sciences ) , then washed 6  times for 2  min with 2 additional washes in pbs and 2 final washes in distilled water . washes and antibody dilutions were in pbs + 0.1% bsa ( sigma - aldrich ) at rt . grids were stored at room temperature and stained with 2% aqueous uranyl acetate for 4  min and reynold 's lead citrate for 10 sec . light microscopy : fluorescence microscopy was performed as previously described using a deltavision imaging system ( applied precision ) . for filipin staining , cells expression from plasmid pjm512 was induced by growth in the absence of thiamine for 20  hours at 32c ; filipin ( sigma f9765 ) was added at a final concentration of 5 g / ml to live cells . this work was funded by national institutes of health grant gm099774 ( to j.b.m . ) and t32-gm008704 ( to r.k . ) .
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 sle1 and fhn1 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 .