Determinants of endocytic membrane geometry, stability, and scission

Abstract

During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.

Fig. 1.

Endocytic vesicle scission assay. (A) Sla1-GFP dynamics in rvs167Δ and rvs161Δ mutants. Blue, red, and yellow bars represent the percentages of patches that were internalized followed by scission, that were internalized followed by retraction to the plasma membrane (PM), and that did not internalize, respectively. (B) Kymographs of single patches from movies of Sla1-GFP in an rvs167Δ mutant. Three patterns described in A are displayed. Each panel shows three individual patches. (Scale bars: 400 nm.) (C) Colocalization of fluorescent α-factor with internalizing (WT cells) and retracting (rvs167Δ mutant cells) Sla1-RFP patches. The time series shows the position of endocytic patches from movie frames (1 frame per second). Images from 3-s intervals are shown. (Left) White arrowheads represent a released endocytic vesicle in WT cells. (Right) Dashed lines represent the position of Sla1-RFP origin parallel to the PM. (Scale bars: 200 nm.) (D) Kymograph representations of fluorescent α-factor and Sla1-RFP for a single patch in an rvs167Δ mutant from a two-color movie (1 frame per second). (Scale bar: 200 nm.)

Fig. 2.

Importance of actin assembly and Myo5, Las17, and Pan1 NPF activity for membrane scission. (A) Single frames from live-cell movies showing Sla1-GFP localization at a Lat-A concentration that allows actin assembly at endocytic sites. Sla1-GFP dynamics in WT (B) and rvs167Δ mutant (C) cells treated with Lat-A. Results shown are representative of three independent experiments. (D) Sla1-GFP dynamics in strains carrying rvs167Δ combined with site-directed mutations in genes encoding NPF proteins, specifically eliminating their Arp2/3-activating activity. Data shown are the mean ± SD. Three independent experiments were performed. (E) Fluorescence microscopy analysis of LY endocytic uptake. Cells were incubated with LY for 2 h at 25 °C. Means are the fractions of the cells displaying LY accumulation in the vacuole. (Scale bars: A and E, 5 μm.)

Fig. 3.

Synaptojanin Sjl2 contributes to membrane scission. (A) Sla1-RFP dynamics in WT or rvs167Δ cells with or without P_GAL1-GFP-SJL2 cultured in glucose-containing medium to turn off SJL2 expression. Lack of GFP-Sjl2 expression was confirmed by fluorescence microscopy. (B) Effect of GFP-SJL2 overexpression on Sla1-RFP dynamics in WT or rvs167Δ cells with or without P_GAL1-GFP-SJL2. Cells were cultured in galactose-containing medium to induce GFP-SJL2 overexpression. Data shown are the mean ± SD.

Fig. 4.

F-BAR protein Bzz1 contributes to membrane scission synergistically with Rvs proteins and actin assembly. (A) Domain structure of Bzz1 and sequence alignment of F-BAR domains from different proteins. Black boxes indicate identical amino acids (A. A.). Gray boxes indicate similar A. A. The open inverted triangle (R37E) above the sequences indicates the mutated A. A. residue. (B) Sla1-GFP dynamics in a bzz1Δ rvs167Δ las17-WCAΔ triple mutant. Data shown are the mean ± SD. (C) Deletion of BZZ1 exacerbates the growth defect of the las17-WCAΔ rvs167Δ double mutant. After streaking cells, YPD plates were incubated at 25 °C for 2 d. The results shown are representative of results for four independent clones for each genotype. (D) Coimmunoprecipitation (IP) of Bzz1-HA and Bzz1-Myc from extracts of heterozygous diploid cells. Immunoblot analyses using antibodies against Myc (Upper) and HA (Lower) are shown. The results presented are representative of three experiments. (E) Binding of Bzz1 and mutant Bzz1 to liposomes made from PC/PE mix with or without 5 mol% PI(4,5)P₂. (F) Fluorescence image of GFP-tagged Bzz1 and Bzz1 mutants. Means for the lifetime of Bzz1-GFP ± SD were calculated from at least 50 patches from 10 cells. (Scale bar: 5 μm.) (G) Sla1-GFP dynamics in the bzz1 mutant combined with the rvs167Δ mutant. Data shown are the mean ± SD.

Fig. 5.

Bzz1 associates with and stabilizes the base of endocytic membrane invaginations. (A) Dynamic localization of Rvs167-RFP relative to Bzz1-GFP in living cells. The time series shows the composition of individual patches from two-color movies (1 frame per second). White dashed lines in merged images represent the plasma membrane (PM). (Scale bar: 300 nm.) (B) Kymograph representation of Rvs167-GFP and Bzz1-RFP in a single patch from a two-color movie. Kymographs are oriented with the cell exterior at the top. (Scale bar: 400 nm.) (C) Displacement of Sla1-GFP from its starting position on retraction to the PM in rvs167Δ bbc1Δ and bzz1Δ rvs167Δ bbc1Δ mutants. The time series shows the position of individual patches from single-color movies (1 frame per second). Arrowheads represent starting positions. (Scale bar: 200 nm.) (D) Tracking of individual Sla1-GFP patches in rvs167Δ bbc1Δ and bzz1Δ rvs167Δ bbc1Δ mutants. The positions of the centers of patches were identified in each frame of a movie (1 frame per second) from the medial focal plane of a cell. Consecutive positions from the start (green) to the end (red) were connected by lines. Patch traces are oriented so that the cell surface is up (dashed line) and the cell interior is down. The time difference between each position along the track is 1 s. (Scale bars: 200 nm.) (E) Histogram shows the distribution of distances between the appearance and disappearance sites for retracting Sla1 patches in the rvs167Δ bbc1Δ, bzz1Δ rvs167Δ bbc1Δ, and bzz1-R37E rvs167Δ bbc1Δ mutants. (F) TIRF microscopy analysis of Las17-GFP dynamics on the PM in living cells. The single frame was taken during bright-field (BF) and GFP fluorescence single-color imaging. A kymograph representation of Las17-GFP in live-cell movies (1 frame per second) in WT cells (Upper) and bzz1Δ (Lower) mutants is shown. White dashed lines indicate the regions used for kymographs. (Scale bar: 2 μm.) (G) Tracking of two individual Las17-GFP patches from TIRF images. Positions of the centers of patches were determined in each frame of a movie (1 frame per second). Consecutive positions from the start (green) to the end (red) are connected by lines. (Scale bar: 100 nm.) (H) Histogram shows the distribution of distances between appearance and disappearance sites for Las17-GFP imaged using TIRF microscopy. (I) Kymograph representation from TIRF imaging of Las17-GFP sliding along the PM in rvs167Δ, bzz1Δ rvs167Δ, rvs167Δ bbc1Δ, and bzz1Δ rvs167Δ bbc1Δ mutants from movies (1 frame per second). (Left) Images are the first frames of each movie. Kymographs were obtained from a single patch for each mutant. White dashed lines represent the PM. (Scale bar: 400 nm.) (J) Kymograph representations of Bzz1-GFP and Sla1-RFP of a single patch in an rvs167Δ mutant from a two-color movie of a medial focal plane in wide-field microscopy (1 frame per second). (Scale bar: 400 nm.)

Fig. 6.

Ultrastructural analysis of BAR and F-BAR domain protein roles in endocytic invagination geometry. (A) Deeply invaginated endocytic membranes (>40-nm length) were identified and measured. Representative electron micrographs of ultrathin sections demonstrating plasma membrane invaginations in WT cells and various mutants are shown. (Scale bar: 50 nm.) (B) Scheme used to measure the diameters of the endocytic membrane tubules and buds. Blue and green lines represent the positions used to measure tubule and bud diameters, respectively. Graphs represent tubule diameter (Lower) or bud diameter (Upper) vs. invagination length.

Fig. 7.

Model for coordinated actions of BAR, F-BAR proteins, and actin assembly during invagination and scission (description is provided in the main text).

Fig. P1.

We propose that Bzz1 F-BAR protein is recruited to endocytic sites when the membrane has a relatively low curvature and assembles a rigid invagination base (step 1). Bzz1 binds to another protein, Las17, and we propose that these proteins form a stable base that resists actin assembly, enabling efficient actin assembly-driven endocytic tubule extension and constriction, which, in turn, generate higher membrane curvature (step 2). The BAR-domain proteins are then recruited to these more highly curved membrane tubules, working cooperatively with actin assembly forces to create deeply invaginated endocytic membranes, resulting in further tubule constriction (step 3) and, ultimately, in scission (step 4).