Fig. 2
The ZF1 degron enables labelling of specific cells and vesicles in C. elegans embryos. a–c The PH domain of PLC1∂1 binds to lipids in the plasma membrane and an mCherry-tagged PHPLC1∂1 reporter (mCh::PH) localizes to the plasma membrane and endocytic vesicles (arrow) in 4-, 7-, and 28-cell embryos (n = 19). Scale bar: 10 μm. d–f ZIF-1-driven proteasomal degradation of the degron-tagged mCh::PH::ZF1 reporter starts in anterior blastomere (AB) cells during the four-cell stage (d), leading to the absence of the mCh::PH::ZF1 fluorescence in anterior AB cells at the seven-cell stage (e) and most somatic cells at the 28-cell stage (f, n = 10). The ubiquitin ligase adaptor ZIF-1 is not expressed in the posterior germ line or in anterior polar bodies (arrowhead), resulting in the persistence of mCh::PH::ZF1 in these cells. Arrows indicate labelled intracellular vesicles, which are protected from proteasomal degradation by intervening membranes. See also Supplementary Movie 1. Anterior is left, dorsal is up. g The lipid-binding PH domain tagged with the ZF1 degron is recognized by the ubiquitin ligase adaptor ZIF-1 and ubiquitinated. Polyubiquitination leads to proteasomal degradation of degron-tagged fluorescent reporters (dotted lines). h–j Embryos treated with tat-5 RNAi show increased mCh::PH membrane labelling due to accumulated microvesicles at the 4-, 7-, and 28-cell stage (n = 11). k Increased mCh::PH::ZF1 is visible at the four-cell stage in embryos treated with tat-5 RNAi. l–m Gradual degradation of mCh::PH::ZF1 in somatic cells facilitates visualization of released microvesicles in a 7- and 28-cell embryo (n = 22). Due to degradation of cytosolic mCh::PH::ZF1 reporters at the plasma membrane, even small amounts of released microvesicles are easily visible. See also Supplementary Movie 1 and Supplementary Fig. 2. n Degron-tagged PH domain reporters are released in extracellular vesicles that bud from the plasma membrane before ZIF-1 expression. ZIF-1 expression leads to the ubiquitination and proteasomal degradation of cytosolic ZF1-tagged reporters (dotted lines). ZF1-tagged membrane reporters in released vesicles are not ubiquitinated or degraded and maintain fluorescence
