Degron-tagged reporters probe membrane topology and enable the specific labelling of membrane-wrapped structures

Abstract

Visualization of specific organelles in tissues over background fluorescence can be challenging, especially when reporters localize to multiple structures. Instead of trying to identify proteins enriched in specific membrane-wrapped structures, we use a selective degradation approach to remove reporters from the cytoplasm or nucleus of C. elegans embryos and mammalian cells. We demonstrate specific labelling of organelles using degron-tagged reporters, including extracellular vesicles, as well as individual neighbouring membranes. These degron-tagged reporters facilitate long-term tracking of released cell debris and cell corpses, even during uptake and phagolysosomal degradation. We further show that degron protection assays can probe the topology of the nuclear envelope and plasma membrane during cell division, giving insight into protein and organelle dynamics. As endogenous and heterologous degrons are used in bacteria, yeast, plants, and animals, degron approaches can enable the specific labelling and tracking of proteins, vesicles, organelles, cell fragments, and cells in many model systems.

Fig. 1

Comparison of degron approaches. a Proteins with a zinc finger 1 degron (ZF1, red) are stable before expression of the ubiquitin ligase adaptor ZIF-1 (blue) in C. elegans embryos. ZIF-1 starts to be expressed in a stereotyped pattern of somatic cells after the two-cell stage, starting with the anterior cells (red + blue = purple). Proteins with a ZF1 tag are degraded in somatic cells, starting with the anterior cells (purple to blue). Cells that do not express ZIF-1, such as the posterior germ cell, do not degrade proteins with a ZF1 tag (red). b The C-terminal phosphodegrons (CTPD) of OMA-1 are inert until phosphorylated and CTPD-tagged proteins (red) are stable, despite the presence of SCF ubiquitin ligases (blue). Phosphorylation of CTPD occurs during the first mitosis in C. elegans embryos, leading to degradation of CTPD-tagged proteins during the first cell division. c Expression of the TIR1 ligase adaptor (blue) is not sufficient to induce robust degradation of proteins tagged with the auxin-inducible degron (AID, red). Addition of auxin family hormones induces degradation of AID-tagged proteins in cells where TIR1 is expressed (purple to blue)

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 PH_PLC1∂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

Fig. 3

The CTPD domain leads to transient degradation and labelling of extracellular vesicles. a An mCherry and CTPD-tagged PH reporter localizes to the plasma membrane in one-cell embryos, but begins to be degraded during mitosis. Weaker fluorescence persists in embryonic cells from the two-cell stage on, but polar bodies remain brightly labelled (arrowhead, n = 42). Scale bar: 10 µm. b After tat-5 knockdown, the mCh::PH::CTPD reporter still degrades, but bright patches of extracellular vesicles are visible in the eggshell, floating around the embryo, and on the embryo surface, in addition to the brightly labelled polar bodies (arrowhead, n = 43). See also Supplementary Movie 2. c Loss of mCh::PH::CTPD fluorescence from the cell surface begins ~3 min before cytokinetic furrow ingression, but degradation tapers off leaving residual mCh::PH::CTPD fluorescence after the two-cell stage. Fluorescence intensity was normalized to the first polar body to correct for photobleaching. Bars represent mean ± s.e.m. (n = 5–12). Source data are provided as a Source Data file. d Phosphorylation of the CTPD degrons leads to recognition of the CTPD by multiple SCF ubiquitin ligases, ubiquitination, and degradation

Fig. 4

Degron protection assay reveals protein topology. a A degron-tagged clathrin reporter ZF1::mCh::CHC-1 initially localizes to the plasma membrane (arrowhead) and intracellular puncta (n = 9). Scale bar: 10 µm. b After tat-5 knockdown, ZF1::mCh::CHC-1 is enriched at the plasma membrane (arrowhead, n = 14). c After expression of ZIF-1 begins in anterior cells, ZF1::mCh::CHC-1 is degraded throughout the anterior cells in control embryos (n = 19). d Although ZF1::mCh::CHC-1 is still enriched at the plasma membrane in posterior cells (arrowhead) in tat-5 RNAi-treated embryos, ZF1::mCh::CHC-1 is lost from the plasma membrane in anterior cells (arrow, n = 18), indicating that clathrin is accessible to ubiquitin ligases. GFP::PH::ZF1 labels the plasma membrane and extracellular vesicles. e Quantification of clathrin enrichment on a posterior cell contact (EMS:P2) or anterior cell contact (ABa:ABp) compared to the neighbouring cytoplasm at the four- and six-cell stage from two independent experiments. ZF1::mCh::CHC-1 fluorescence was significantly increased at the posterior EMS:P2 cell contact (***p < 0.001 using Student’s t-test, ctrl n = 19, tat-5 RNAi n = 18). No change was observed at anterior cell contacts (p > 0.05). Bars represent mean ± s.e.m. Source data are provided as a Source Data file. f If clathrin were in extracellular vesicles, ZF1::mCh::CHC-1 would be protected from ZIF-1-mediated degradation. However, as clathrin is inside the plasma membrane, ZF1::mCh::CHC-1 is accessible to ZIF-1-mediated degradation

Fig. 5

Degron reporters label phagocytosed cell debris and enable tracking. a–c The histone reporter mCh::H2B labels chromosomes in embryonic nuclei and two polar bodies (n = 13). The first polar body (arrowhead) is trapped in the eggshell (dashed oval). Scale bar: 10 µm. a The second polar body (2PB, arrow) neighbours the anterior blastomere (AB cell) in a two-cell embryo. b The 2PB is engulfed in a phagosome in a 15-cell embryo. Due to H2B fluorescence from surrounding embryonic nuclei, it is hard to track the 2PB. c Automated tracking of the 2PB (yellow) results in crossing tracks from nearby nuclei (white), increasing the potential need for manual correction. d–f Embryos labelled with ZF1-tagged mCh::H2B (n = 12). d The released 2PB neighbours a two-cell embryo. e After engulfment, the 2PB is easily trackable due to degradation of ZF1::mCh::H2B in somatic cell nuclei. See also Supplementary Movie 3. f Automated tracking of the 2PB (yellow) is simplified by removing the label of nearby nuclei (white). g Degron reporters released in cells or other debris prior to expression of the ZIF-1 ubiquitin ligase adaptor are protected from ZF1-mediated degradation. After engulfment in a second layer of membrane, they are still protected from proteasomal degradation. As degron reporters are degraded in the cytosol, only the reporters within the phagosome remain fluorescent, improving the signal-to-noise ratio

Fig. 6

Degron reporters label specific membranes. a The mCh::PH::CTPD reporter brightly labels the plasma membrane of the polar bodies and weakly labels the plasma membrane of embryonic cells at the four-cell stage (n = 22). Scale bar: 10 µm. b After phagocytosis of the second polar body (2PB, arrow), the plasma membrane of the 2PB is distinctly visible. c Several minutes later, the corpse plasma membrane breaks down inside the phagolysosome and the mCh::PH::CTPD reporter disperses throughout the phagolysosome lumen, as demonstrated by line scans through the phagolysosome (insets). See also Supplementary Movie 2. d The mCh::PH::CTPD reporters inside the corpse membrane are protected from degradation before and after phagocytosis. Model of membrane breakdown inside the phagolysosome, demonstrating the shape change from a hollow to a filled sphere

Fig. 7

Degron reporters reveal nuclear envelope topology during cell division. a During interphase, the nuclear envelope is intact, hindering ZIF-1 binding to ZF1-tagged reporters inside the nuclear envelope. b During mitosis, the nuclear envelope is remodelled into the endoplasmic reticulum, allowing binding of ZIF-1 to ZF1-tagged reporters, which leads to ubiquitination and rapid proteasomal degradation. c Prior to ZIF-1 expression, mKate2::ZF1::LMN-1 is similarly bright in the nuclear matrix of the anterior AB cell (arrow) and the posterior cell (n = 14). Scale bar: 10 µm. d After the onset of ZIF-1 expression in anterior daughter cells (ABx), mKate2::ZF1::LMN-1 starts to lose fluorescence in anterior cells (arrows). e, f During mitosis, the nuclear envelope is disassembled, leading to rapid proteasomal degradation of mKate2::ZF1::LMN-1. DIC images show nuclear morphology during cell cycle progression. g–j mKate2::ZF1::LMN-1 is maintained in anterior cells after zif-1 knockdown (n = 11). DIC images show nuclear morphology during cell cycle progression. See also Supplementary Movie 4. k Quantification of mKate2::ZF1::LMN-1 fluorescence in the dorsal ABp nucleus compared to the ventral EMS nucleus with (n = 5–11) and without zif-1 RNAi treatment (n = 5–14). Bars represent mean ± s.e.m. l Normalizing mKate2::ZF1::LMN-1 to zif-1 RNAi-treated embryos demonstrates that fluorescence drops 4–6 times faster during nuclear envelope breakdown (NEBD) in anterior cells (yellow boxes, AB: 16% per min, ABx: 11% per min), when ZIF-1 has full access to mKate2::ZF1::LMN-1. Slow decline in the intensity of mKate2::ZF1::LMN-1 is also visible during interphase (3% per min). NEBD was defined as the first time point the nuclear lamina collapsed in the mother cell until the lamina became round again in daughter cells. Time is given in relation to AB furrow ingression. Source data are provided as a Source Data file. m, n In the absence of ZIF-1, ZF1-tagged reporters are stably expressed, because they are not ubiquitinated or degraded during interphase or mitosis

Fig. 8

Localization of the ubiquitin ligase adaptor spatially controls degradation. a Representative time-lapse images of a HeLa cell transiently expressing a nuclear export signal-tagged ubiquitin ligase adaptor (NES-TIR1) and a Venus- and mAID-tagged lamin LMNA. 0.5 mM NAA was added at t = 0 to induce auxin-dependent degradation. During interphase, Venus-mAID-LMNA in the nuclear lamina is protected from ubiquitination by cytosolic NES-TIR1 and fluorescence persists in the presence of NAA. Scale bar: 30 µm. See also Supplementary Movie 5. b Model of mAID-LMNA protection from cytosolic NES-TIR1 during interphase. c The same cell is shown entering mitosis, which is visible by cell rounding in the brightfield (BF) overlay. After nuclear envelope breakdown (NEBD, t = 0), Venus-mAID-LMNA is accessible by cytosolic NES-TIR1 and fluorescence rapidly disappears. See also Supplementary Movie 6. d mAID-tagged reporters in the nucleus are accessible to cytosolic NES-TIR1 after NEBD occurs during mitosis. e Interphase HeLa cell stably expressing a nuclear localization signal-tagged TIR1 (NLS-TIR1) and NES-TIR1 from a single mRNA in addition to Venus-mAID-LMNA. After treatment with 0.5 mM NAA (t = 0), Venus-mAID-LMNA fluorescence rapidly disappears during interphase. See also Supplementary Movie 7. f Model of mAID-LMNA accessibility to nuclear NLS-TIR1 during interphase. g Quantification of Venus-mAID-LMNA fluorescence before and after NEBD (t = 0) in the presence of cytosolic NES-TIR1 and NAA. Values were normalized to NEBD. h Quantification of Venus-mAID-LMNA fluorescence in interphase cells in the presence of cytosolic NES-TIR1, nuclear NLS-TIR1, and NAA. Values were normalized to t = 0 (NAA addition). i Comparison of Venus-mAID-LMNA degradation velocities from single cells. The degradation velocity was determined for 45 min before NEBD (interphase) and 45 min after NEBD (mitosis) for NES-TIR1-expressing cells from three independent experiments or during the first 45 min after NAA addition for cells expressing both NLS-TIR1 and NES-TIR1 from two independent experiments. Bars indicate the mean of the indicated number of cells. Significance according to unpaired multi-comparison Kruskal–Wallis test with Dunn’s statistical hypothesis testing (****p < 0.0001; ns, p > 0.9999). Source data are provided as a Source Data file

Fig. 9

Degron reporters reveal membrane topology during abscission. a–f Time-lapse images of embryos expressing mCh::PH to label the plasma membrane and NMY-2::GFP::ZF1 to label non-muscle myosin in the cytokinetic ring. Scale bar: 10 µm. a In a control embryo, NMY-2::GFP::ZF1 between the anterior daughter cells (arrow) is protected from ZF1-mediated degradation due to its release outside cells in the midbody remnant after abscission. b NMY-2::GFP::ZF1 in the phagocytosed midbody remnant is protected from proteasomal degradation by engulfing membranes (n = 19). c In embryos depleted of the ESCRT-I subunit TSG-101, NMY-2::GFP::ZF1 localizes to the intercellular bridge normally. d NMY-2::GFP::ZF1 is degraded after tsg-101 knockdown, indicating that abscission is incomplete and NMY-2::GFP::ZF1 is accessible to the degradation machinery. Engulfment of the AB midbody is also delayed, likely due to incomplete abscission (n = 7). e, f Embryos depleted of the septin UNC-59 show rapid degradation of NMY-2::GFP::ZF1 from the intercellular bridge and delayed engulfment of the midbody remnant due to defects in abscission (n = 8). g Fluorescence intensity of the NMY-2::GFP::ZF1 reporter on the intercellular bridge between anterior daughter cells (AB midbody) drops significantly for 2 min after the onset of ZF1 degradation in the cytoplasm of control embryos (blue, n = 11, p < 0.05 using Student’s t-test with Bonferroni correction), showing that NMY-2 is able to diffuse out of the bridge. Fluorescence then persists, showing that formation of a diffusion barrier, symmetric abscission, and engulfment protect the degron-tagged reporter from degradation. In contrast, NMY-2::GFP::ZF1 fluorescence continues to drop in tsg-101 RNAi-treated embryos (red, n = 5, p < 0.05 compared to control after 8 min) and unc-59 mutants (green, n = 6, p < 0.05 compared to control after 3.5 min), showing that the ZF1 degron technique is sensitive to detecting small defects in abscission. Bars represent mean ± s.e.m. Source data are provided as a Source Data file. h In control embryos, degron reporters in the intercellular bridge are protected from proteasomal degradation due to release after abscission and encapsulation in a phagosome. i In abscission mutants, degron reporters are accessible to the cytosol, leading to their removal by proteasomal degradation

Fig. 10

A ZF1 degron reporter in the ER is protected from ZIF-1/ECS-mediated degradation. a mCherry-tagged SP12 localizes to the endoplasmic reticulum (ER) in anterior (somatic, arrow) and posterior (germ line, arrowhead) cells (n = 102). Scale bar: 10 µm. b An RFP- and ZF1 degron-tagged KDEL reporter similarly localizes to the ER in both anterior (somatic, arrow) and posterior (germ line, arrowhead) cells, despite expression of the ZIF-1 ligase adaptor in anterior cells (n = 88). c There was no significant difference in the ratio between the fluorescence of anterior AB lineage cells and of posterior germ line or germ line sister cells at the indicated stages (p > 0.05, Student’s t-test with Bonferroni correction, n = 18–25 mCh::SP12, n = 24–28 ss::tagRFP-T::ZF1::KDEL). Bars represent mean ± s.e.m. Source data are provided as a Source Data file. d The ER membrane protects luminal proteins from ubiquitination by cytosolic ligase complexes