Figure 1. The peripheral ER forms a network of interconnected tubules. Fluorescence microscopy of wild-type cells expressing either Sec63-GFP or ss-GFP-HDEL. Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 mm. The ER Is Dynamic We wondered if the yeast cortical ER is dynamic as it is in higher eukaryotes. Cells expressing Sec63-GFP were visu- alized in the fluorescence microscope at three-minute in- tervals (Fig. 2 A). The peripheral ER in the daughter cell (above the mother cell) is highly dynamic and is com- pletely rearranged in three minutes. To facilitate compari- son between the frames, stars have been placed at identi- cal locations in the daughter cell in each image. The structures near the stars are qualitatively quite different at each time point. The ER in the mother cell was somewhat more stable than in the daughter cell, with some regions showing little change over the course of the experiment. The star in the mother cell indicates a region that remains relatively unchanged during the course of the experiment. This qualitative difference in the extent of ER structural alteration in daughter and mother cells was seen consis- tently in many experiments (not shown). Video microscopy of mammalian cells revealed three types of movement of the ER: tubule branching, ring clo- sure (the contraction of a loop of ER to a single tubule), and tubule sliding (the movement of a junction of ER tu- bules along a tubule; Lee and Chen, 1988). Similar move- ments occur in S. cerevisiae. Fig. 2 B shows time-lapse mi- croscopy of a portion of a mother cell with pictures taken every five seconds. An example of ring closure occurs be- tween 5 and 20 s (Fig. 2, arrow a points to ring). An in- stance of tubule branching occurs between 25 and 30 s (Fig. 2, arrow b points to the new tube). We find that tu- bule branching and ring closure occur at approximately the same rate as in mammals (Lee and Chen, 1988). Therefore, ER tubule movements appear to occur simi- larly in yeast and mammalian cells. Role of Microtubules and Actin Cytoskeleton in ER Structure To examine the role of microtubules in ER formation, we incubated yeast in medium containing nocodazole. After two hours, microtubules could not be detected by immuno- fluorescence with antitubulin antibodies (not shown; Ja- cobs et al., 1988). Neither the structure of the ER (Fig. 3 A) nor ER dynamics (not shown) were affected by the dis- ruption of microtubules. These results indicate that, unlike the situation in mammals, microtubules play no role in the maintenance of ER structure. We next tested the role of actin filaments and patches in the formation and maintenance of the ER. We first as- sessed the alignment of tubules of the peripheral ER with cortical actin filaments in fixed cells (Fig. 3 B). Although fixation slightly distorts the ER, there are clearly a few regions where ER tubules and actin filaments align (indicated by arrows). However, most ER tubules do not align with any actin filament. Next, we treated yeast cells with latrunculin-A, which results in the disrup- tion of actin structures within minutes, as seen by staining with rhodamine-phalloidin (not shown; Ayscough et al., 1997). However, even after growth for two hours in me- dium with latrunculin-A, the ER structure was not dis- rupted, although there appeared to be some minor prolif- eration of ER tubules (Fig. 3 A). Strikingly, minutes after addition of latrunculin-A, the dynamics of the ER was greatly reduced (Fig. 2, compare C with A, daughter cell on top in both). To facilitate comparison between the frames, stars were placed at identical locations in the daughter cell in each frame. To quantitatively compare the changes of ER structure seen in Fig. 2, A and C, we de- termined the pixel values at different time points in the The Journal of Cell Biology, Volume 150, 2000 464