Fig 1.
Protein recruitment dynamics at yeast CME sites and an automated strategy to generate separate circumferential kymographs for mother and daughter cells from a movie of budded yeast cells.
(A) Previously determined timeline for the presence of a subset of endocytic proteins at CME sites (left) [1]. The timeline can be divided into 2 phases separated by the vertical dotted line: the early phase on the left and the late phase on the right. Pan1 and End3 link the 2 phases [50,51]. The mammalian homologs of the yeast proteins are indicated in parentheses. The Pan1-End3-Sla1 complex contains similar interaction domains to the mammalian endocytic protein intersectin and is proposed to perform a similar function [53]. The length of the early phase is variable (30 s to 2 min), indicated by “//”, while the length of the late phase is much more regular (approximately 30 s). AP-2, Pal1, Pal2, and clathrin exhibit similar dynamics to the Yap1801 and Yap1802 [24]. Actin patch (white dot) localization in budding yeast at different cell cycle stages (right) [27,28]. The cells with small or medium buds are undergoing polarized cell growth. (B–G) Show the workflow we developed to quantify protein lifetimes at CME sites. A detailed description is provided in the Results and Materials and methods sections. The scale bar is 5 μm. CME, clathrin-mediated endocytosis.
Fig 2.
Frequency and dynamics of key endocytic proteins at CME sites in mother vs. daughter cells of budding yeast undergoing polarized growth.
(A) Representative circumferential kymographs of Myo5-GFP in mother and daughter cells of yeast cells undergoing polarized growth. (B, C) Myo5-GFP patch lifetime and frequency in mother vs. daughter cells. The numerical data are presented in S1 Data. (D) Representative circumferential kymographs of indicated GFP-tagged proteins in mother and daughter cells. Note: the kymograph of Pan1 in the mother cell reveals 2 stages, a low abundance stage followed by a high abundance stage, with transition points between the stages indicated by arrows. The kymograph numbers identify different ROIs for the mother and daughter cells. (E, F) Mean with SD and coefficients of variation for the lifetimes of the indicated proteins in mother and daughter cells. The numerical data are presented in S1 Data. The scale bar is 30 s. CME, clathrin-mediated endocytosis; ROI, regions of interest; SD, standard deviation.
Fig 3.
The composition of early-arriving endocytic proteins at CME sites differs between mother and daughter cells of budding yeast undergoing polarized growth.
(A–C and E–H) Two-color circumferential kymograph representations of GFP- or mScarlet-I-tagged proteins in mother and daughter cells. The boxes on the kymographs from daughter cells show the dynamic behavior of the indicated protein pair at CME sites. (D) Ede1-GFP patch lifetime and frequency of appearance in mother vs. daughter cells. The numerical data are presented in S2 Data. (E) Some endocytic sites in mother cells lack detectable Yap1801 and Yap1802. The arrows in the mother cell kymograph show productive CME sites labeled by Myo5 but lacking Yap1801 and Yap1802. (G) Most (87%) Myo5 sites in mother cells lack Yap1802-GFP. CME, clathrin-mediated endocytosis.
Fig 4.
Comparison of CME site initiation in mother vs. daughter cells of yeast undergoing polarized bud growth.
(A) The lifetimes of early- and mid-arriving endocytic proteins are much shorter and less variable in the daughter cells (black lines) than in the mother cells (brown lines). In contrast, Sla1 and Myo5 exhibit similar, regular lifetimes in mother and daughter cells. Yap1801 and Yap1802 signals are noticeably dimmer in mother cells (dotted line), and most CME sites in mother cells lack detectable Yap1802. The Pan1 signal in mother cells can be divided into a low-abundance stage (dotted brown line) followed by a high-abundance stage (black line). The length of Pan1’s high abundance phase in mother cells is similar to Pan1’s lifetime in daughter cells. CME sites (represented by Ede1) initiate approximately 5 times more frequently in daughter cells compared to the mothers, correcting for differences in the surface distance sampled. The different shades of gray color are intended to signify that cargo and anionic phospholipids exhibit asymmetric distribution, with higher levels on the plasma membrane of daughter cells in small to medium budded cells. (B) Summary of protein lifetimes (mean with standard deviation (sec)) at CME sites in mother and daughter cells of yeast undergoing polarized bud growth. ND means not determined. ¶ The true lifetimes should be greater than the indicated values because some events are not completed within the duration of our movies. § Lifetime represents the low-intensity and high-intensity stages of Pan1 combined. The numerical data are presented in S1 Data and S3 Data. CME, clathrin-mediated endocytosis.
Fig 5.
Polarized enrichment of Yap1801, Yap1802, and Syp1 at the daughter cell plasma membrane is independent of Ede1.
(A) Schematic diagrams of domain structures in Yap1802, Ede1, and Syp1. ANTH domain, AP180 N-terminal homology domain; NPF, Asn-Pro-Phe motif; CBM, clathrin box motif; EH, Eps15 homology; PQ-rich, proline and glutamine-rich motif; UBA, Ubiquitin-Associated domain; F-BAR, FCH-bin-amphiphysin-Rvs domain. (B, C) MIP from 3 min movies taken from indicated yeast strains. The arrows point to the enriched localization of the GFP-labeled proteins in the daughter cells of yeast with small- to medium-sized daughters. The average ratio (daughter vs. mother) of GFP signal intensity along the cortex of polarized cells is indicated in the upper left or upper right corner of images in (B) or (C), respectively. The numerical data are presented in S4 Data. The scale bar is 5 μm. MIP, Maximum Intensity Projection.
Fig 6.
Cytoplasmic Ede1 puncta appear in mutants lacking various early-arriving endocytic proteins.
Maximum Z-projections of Ede1 localization in the indicated null mutants. The number in the upper right corner in each panel indicates the percentage of cells that contain cytoplasmic Ede1 puncta. n equals the number of cells analyzed. Arrows indicate representative cytoplasmic puncta in mutants lacking the indicated early-arriving endocytic protein(s). “*” indicates that Ede1 cortical patches are still visible at cell cortex. The scale bar is 5 μm.
Fig 7.
EH and Syp1 binding domains of Ede1 are required for its cortical localization.
(A) Schematic diagram of Ede1 domain structure. (B–G) Maximum Z-projections of indicated GFP-tagged ede1 mutant proteins in yeast cells. The numbers in the upper right corner of the images in (B) and (C) indicate the percentage of cells that contain cytoplasmic puncta. n equals the number of cells analyzed. (E) A merged image of the GFP channel (D) and corresponding brightfield view. Note: the cell outlines are no longer visible in the GFP channel (D) due to the GFP signal concentrating in the cytoplasmic puncta. “*” indicates cells in which cortical patches are still visible in ede1 mutants. The scale bar is 5 μm.
Fig 8.
Syp1 and Yap1802 function in concert to guide CME site initiation through Ede1 recruitment.
(A) In the absence of rapamycin, Yap1802-GFP-FRB and Pil1-TagBFP2-FKBP12 exclusively localize either to daughter cells or mother cells, respectively. Ede1-mScarlet-I patches in mother cells do not colocalize with Pil1-TagBFP2-FKBP12. (B) After treatment with 10 μm rapamycin for 10 min, Yap1802-GFP-FRB (and presumably Syp1-FRB) efficiently relocates to Pil1-TagBFP2-FKBP12 puncta in the mother cell through the FKBP12-rapamycin-FRB interaction, triggering Ede1-mScarlet-I to form patches on Pil1-TagBFP2-FKBP12 puncta in the mother cell. (C, D) Ede1-mScarlet-I localization after either Yap1802-GFP-FRB or Syp1-GFP-FRB alone was targeted to Pil1-TagBFP2-FKBP12 puncta. All data shown are Maximum Z-projections generated from 3-color Z-stacks generated from Airyscan fixed cell imaging. The numerical data are presented in S5 Data. The scale bar is 1 μm. CME, clathrin-mediated endocytosis.
Fig 9.
The molecular mechanism of CME site initiation.
(A) A model in which the Yap180s and Syp1 play major roles in CME site establishment through their interaction with lipids, cargo, and Ede1. The Yap180s and Syp1 are recruited to the plasma membrane (gray line) through interactions with anionic phospholipids and possibly cargos on the plasma membrane. Ede1 forms a complex with Syp1 in the cytoplasm, and this complex is recruited to the plasma membrane while the Ede1-Yap180s interactions occur at the plasma membrane. Mammalian homologs of indicated yeast proteins are shown in gray. Dotted arrow lines: interaction at the plasma membrane. Two-way solid arrow line: interaction in cytoplasm. (B) A model for polarized CME site initiation in budding yeast. Anionic phospholipids and cargo molecules are enriched at the plasma membrane of daughter cells, where they promote recruitment of the Yap180s and Syp1. Yap180s and Syp1 ensure that Ede1-driven CME site formation occurs where cargo levels are high and acidic phospholipids are abundant. CME, clathrin-mediated endocytosis.