Ultrastructural dynamics of proteins involved in endocytic budding

Abstract

Fluorescence live-cell imaging has temporally resolved the conserved choreography of more than 30 proteins involved in clathrin and actin-mediated endocytic budding from the plasma membrane. However, the resolution of these studies is insufficient to unveil how the endocytic machinery actually drives membrane deformation in vivo. In this study, we use quantitative immuno-EM to introduce the temporal dimension to the ultrastructural analysis of membrane budding and define changes in the topography of the lipid bilayer coupled to the dynamics of endocytic proteins with unprecedented spatiotemporal resolution. Using this approach, we frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and show that constriction of the invaginations correlates with translocation of membrane-sculpting proteins. Furthermore, we show that initial bending of the plasma membrane is independent of actin and clathrin polymerization and precedes building of an actin cap branched by the Arp2/3 complex. Finally, our data indicate that constriction and additional elongation of the endocytic profiles require the mechanochemical activity of the myosins-I. Altogether, this work provides major insights into the molecular mechanisms driving membrane deformation in a cellular context.

Fig. 1.

The invagination length can be used as a parameter to define the age of the endocytic profiles. (A) Proposed cortical dynamics of endocytic proteins included in the current statistical analysis based on or inferred from live-cell imaging (ref. and references therein). The proteins are grouped in modules (4, 7) represented in different colors. The timing of clathrin recruitment with respect to the early module is not well-defined (–10). The exact time at which the Arp2/3 complex is recruited with respect to other endocytic proteins is controversial (13, 17). Yeast proteins and their respective mammalian homologs are shown in bold and regular types, respectively. Time 0 corresponds to scission (18). The area highlighted in gray shows the broadest time window analyzed in this study. The red bar indicates when initial curvature may appear. (B) Representative immuno-EM micrographs of ultrathin sections from yeast showing endocytic invaginations of different sizes labeled with immunogolds against the HA tag versions of the indicated proteins. (Scale bar: 100 nm.) (C) Diagram illustrating the parameters used to analyze the position of the immunogolds. The levels of the PM and the invagination tip (IT) are indicated. (D) Relative frequency of immunogolds labeling the indicated proteins binned according to the IL. The number of immunogolds recorded for each protein (n_g) was 120. SI Appendix, Fig. S1B shows the GLM analysis of the IL. Proteins labeled with an asterisk were analyzed in ref. and are included as references.

Fig. 2.

Ultrastructure of the yeast endocytic profiles after interfering with actin and clathrin. (A and D) Immuno-EM micrographs showing representative PM invaginations labeled with immunogolds against Sla1-HA (α-Sla1-HA) or Clathrin (α-Chc1) on (A) WT cells treated with 200 μM LatA or DMSO or (D) chc1Δ cells and the isogenic WT (CHC1). (B and E) Scatter graphs representing the GDPM (gold distance to the plasma membrane) of 120 immunogolds against Sla1-HA vs. the corresponding IL (parameter definition in Fig. 1C) for each of the indicated strains and the experimental conditions described in A and D, respectively. The x axis corresponds to the level of the basal PM. The red dotted line indicates the position of the IT. The gray lines converging at the origin define constant GRP values. Blue and red circles indicate the control strains and the LatA-treated or mutant strains, respectively. (C and F) Histograms showing the relative frequency of Sla1-HA immunogolds from the scatter graphs shown in B and E, respectively, binned according to the length of the corresponding invaginations. Student t test P values comparing the length of Sla1-HA–labeled invaginations or the positional parameters of the corresponding immunogolds are described in SI Appendix, Table S1.

Fig. 3.

Localization of immunogolds labeling endocytic proteins on PM invaginations of increasing length. Scatter graphs showing the GDPM vs. the corresponding IL parameters (parameter definition in Fig. 1C) for each recorded immunogold labeling the indicated proteins. n_g = 120 for each protein. The number of invaginations (n_i) associated with the recorded immunogolds is indicated. The x axis corresponds to the level of the basal PM. The red dotted line indicates the position of the IT. The gray lines converging at the origin define constant GRP values.

Fig. 4.

Comparative ultrastructural dynamics of endocytic proteins. (A–C) Graphs representing the frequency of immunogolds labeling the indicated proteins binned according to their GRP (parameter definition in Fig. 1C) associated with short (IL < 70 nm), intermediate (70 ≤ IL < 110 nm), and long (IL ≥ 110 nm) invaginations. n_g = 120 for each protein. Dashed graphs correspond to proteins analyzed in ref. . The gray/green shadow depicts the distribution of Sla1, which indicates the position of the endocytic coat. The black and red dashed vertical lines indicate the positions of the basal PM and the IT, respectively. SI Appendix, Fig. S3 shows analogous GRP graphs, including all components of the endocytic modules analyzed so far by QIEM, and SI Appendix, Fig. S4 shows GRP, GDPM, and GDIT graphs for each of the newly analyzed endocytic factors. SI Appendix, Table S2 shows the homogeneity analysis of the GRP, GDIT, and GDPM parameters as a function of the IL, SI Appendix, Table S3 shows descriptive statistics, and SI Appendix, Table S4 shows the GLM analysis of the 18 endocytic factors.

Fig. 5.

Constriction of the endocytic invaginations correlates with the translocation of F-BAR proteins to the profile neck. (A) Diagram illustrating the two main shapes adopted by the yeast endocytic profiles. The invagination geometry is defined by D_min, D_PM, and D₄₀. Invaginations were defined as ω-shaped when D_min was smaller than D₄₀. Black arrows indicate the position of the constriction. CDPM is the distance from the constriction to the PM. (B) Percentages of ω- vs. U-shaped invaginations as a function of their length. The number of invaginations analyzed was 26, 95, and 46 for the IL bins <70, 70–110, and ≥110 nm, respectively. Data were randomly collected from immuno-EM micrographs of invaginations of WT cells labeled with immunogolds against different HA-tagged endocytic proteins (n = 167). (C) Averages and SDs for the D_min, D_PM, and D₄₀ of endocytic invaginations binned according to their length. Student t test P < 0.005 indicates a very significant difference. Invaginations analyzed were the same as in B. (D) Graph representing the CDPM plotted against the IL. n represents the number of the constricted invaginations analyzed. The x axis represents the level of the basal PM. The red dotted line indicates the positions of the IT. The gray lines converging at the origin define constant CRP values (CRP = CDPM/IL). (E and F) Graphs showing the relative frequency of immunogolds labeling Syp1, Bzz1, and Rvs167 binned according to their GRP and the relative frequency of the constriction according to its relative position (CRP) corresponding to invaginations with 70 ≤ IL < 110 nm (E) and IL ≥ 110 nm (F). Dashed graphs indicate proteins previously analyzed in ref. . SI Appendix, Table S5 shows the statistical analysis of the colocalization of the constriction with the BAR proteins. (G) Representative immuno-EM micrographs showing endocytic invaginations from WT, syp1∆, and bzz1∆ strains labeled with immunogolds against actin. (Scale bar: 100 nm.) (H) Percentages of ω- vs. U-shaped invaginations longer than 70 nm for the indicated strains. The number of the analyzed invaginations was 84, 100, and 106 for the WT, bzz1∆, and syp1∆ strains, respectively. (I) Average and SD for the D_min of endocytic invaginations longer than 70 nm for the indicated strains. Invaginations analyzed were the same as in H. MWW test P < 0.005 indicates very significant differences.

Fig. 6.

Elongation and constriction of the endocytic profiles require the myosin-I ATPase. (A) Representative immuno-EM micrographs showing endocytic invaginations from a myo3Δ myo5Δ strain expressing either the WT MYO5 or the myo5^E414V mutant labeled with immunogolds against Sla1-HA. (B) Scatter graph representing the GDPM of immunogolds labeling Sla1-HA in the strains described in A vs. the corresponding IL (parameter definition in Fig. 1). n_g = 120 for each strain. (C) Histogram representing the relative frequency of the Sla1-HA immunogolds from B binned according to the IL. The MWW test showed very significant differences (P < 0.001) for the Sla1-HA IL distribution of the indicated strains. (D) Representative micrographs showing endocytic invaginations from a myo3Δ myo5Δ strain expressing the myo5^E414V mutant labeled with immunogolds against Syp1-HA. (E) Scatter graph representing the GDPM of 120 immunogolds labeling Syp1-HA for the strain described in D (red circles) vs. the corresponding IL (parameter definition in Fig. 1). For comparison, the graph was superimposed on the graph showing the position of Syp1-HA in WT cells (blue circles) (Fig. 3). (F) Statistical comparison of the Syp1-HA GRP in the myo3Δ myo5^E414V mutant vs. the GRP of Syp1-HA in a WT strain for invaginations shorter (IL < 70 nm) or longer (IL > 70 nm) than 70 nm. The average GRP, SD, and number of immunogolds analyzed (n_g) for each population are indicated. MWW tests (P < 0.005) show very significant differences and are highlighted in red.

Fig. 7.

Scheme of ultrastructural dynamics of endocytic proteins during endocytic budding. The scheme shows the dynamic localization of yeast endocytic proteins along growing endocytic profiles based on ref. and this study. Critical steps during the endocytic process, which are blocked by interfering with actin dynamics (LatA) or the ATPase activity of myosins-I, are shown.

Fig. P1.

(A) Sequential recruitment of proteins (yeast/mammalian homologs) to the endocytic sites as assessed by live-cell fluorescence microscopy (4). The color indicates groups of proteins with similar distributions along the endocytic invaginations in our EM study, which is summarized in B. Steps block by drugs preventing actin polymerization or mutations in the myosin-I ATPase are indicated. (C) Fluorescence micrographs of consecutive 300 × 300-nm frames from double color time-lapse movies showing the transient recruitment of fluorescent GFP-Myo5 (green) and Abp1-RFP (red) at cortical endocytic in living cells. Frames were recorded every 2 s. Modified from ref. . (D) Electron micrographs of plasma membrane invaginations of increasing length specifically labeled with immunogolds (black dots) against endocytic proteins on ultrathin sections of chemically fixed yeast. (Scale bar: 100 nm.)