Supplemental Material

This article contains the following supporting material:

• Figure 1 - Subcellular localization of CAV-1-GFP in oocytes. Transgenic animals expressing CAV-1-GFP under pie-1 promoter control (green) were stained with anti-RME-2 (A-B), anti-EEA-1 (C-D) and anti-RME-1 (E-F) antibodies (red). In the early stage of oogenesis, some CAV-1-GFP colocalized with RME-2, but the two proteins diverged in their steady-state localization as oocytes grew (A-B). CAV-1-GFP localized to membrane structures that were not labeled with anti-EEA-1 or anti-RME-1 antibodies (C-F). (G) CAV-1 bodies are not labeled by Nile red. Scale bars represent 10μm.

• Figure 2 - The subcellular localization of RME-2-GFP in oocytes was relatively unaffected by knockdown of arf-1 or agef-1. RME-2-GFP was localized normally to the plasma membrane and endosomes of control (A), arf-1 (RNAi) (B), arf-1(ok796) (C) and agef-1 (RNAi) (D) worms. Scale bars represent 10μm.

• Movie 1 - Dynamics of CAV-1-GFP in the gonad. Individual images were acquired once every 20 seconds using spinning disc confocal microscopy. Note the flow of CAV-1-GFP labeled puncta into a newly forming oocyte, and the appearance of CAV-1 bodies soon thereafter.

• Movie 2a - Dynamics of GFP-CAV-1 in the ovulating oocyte and early zygote. (a) Individual images were acquired once every 20 seconds in intact animals using spinning disc confocal microscopy. CAV-1 bodies move actively during ovulation. Note the apparent fusion of the CAV-1 bodies with the plasma membrane quickly followed by endocytosis and degradation.

• Movie 2b - Dynamics of GFP-CAV-1 in the ovulating oocyte and early zygote. (b) Dynamics of GFP-CAV-1 in the early zygote. This time-lapse movie shows endocytosis of GFP-CAV-1 in more detail. Images were taken every 15 seconds.

• Movie 3 - Dynamics of CAV-1-GFP in the arf-1(ok796) gonad. Individual images were acquired once every 20 seconds using spinning disc confocal microscopy. Note the flow of CAV-1-GFP puncta from the rachis into a newly forming oocyte, and the coalescence of signal forming the abnormal Golgi associated structures.

• Movie 4 - Movement of CAV-1-GFP in the agef-1 (RNAi) gonad. Individual images were acquired once every 20 seconds using spinning disc confocal microscopy. Note the flow of CAV-1-GFP puncta in a newly forming oocyte and the lack of coalescence of signal to form larger structures.