Measuring the hierarchy of molecular events during clathrin-mediated endocytosis

Abstract

A well-orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin-coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics. To address this question, we have used multi-channel image acquisition and automated analysis of CCP dynamics in combination with a new method to quantify the time courses of recruitment of endocytic factors to CCPs of different lifetimes. Using this approach we have extracted the kinetics of recruitment and disassembly of fluorescently labeled clathrin and/or AP-2 throughout the entire lifetime of temporally defined CCP cohorts. On the basis of these analyses, we can (i) directly correlate recruitment profiles of these two proteins; (ii) define five distinct CCP maturation phases, i.e. initiation, growth, maturation, separation and departure; (iii) distinguish events with absolute versus fractional timing and (iv) provide information on the spatial distribution of fluorophores during CCP maturation. Emerging from these analyses is a more clearly defined role for AP-2 in determining the temporal hierarchy for clathrin recruitment and CCP maturation. This method provides a new means to identify other such hierarchies during CCP maturation.

Figure 1. Discrete stages of CCP maturation defined by lifetime binning and aligned average

(A) Example of raw intensity time courses (grey) aligned to the point of appearance, and their average (green). (B) Intensity time courses as in A aligned to the point of disappearance, and their average (red). (C) Average intensity time courses derived from CCPs within a single lifetime cohort. The final curve (purple) is the weighted average of the appearance-aligned (green) and disappearance-aligned (red) curves; the relative weights of the two curves follows a sigmoid distribution (inset). (D) Temporally-defined landmarks during CCP maturation defined in an average intensity time course of LCa-EGFP (illustrated for a 60s cohort). Reference points and phase segmentation as described in the main text.

Figure 2. Differential behaviors of AP-2 and clathrin during CCP maturation

(A) Single-channel LCa-EGFP time courses. (B) Single-channel σ2-EGFP time courses. (C) Example of time point-vs-lifetime plots for events of known timing. (D) Time points of segmented references in LCa-EGFP intensity time courses. (E) Time points of segmented reference points in σ2-EGFP intensity time courses. (F) Zoom-in of the initiation phase in LCa-EGFP; intensity time courses are normalized to the intensity at t_first. (G) Zoom-in of the initiation phase in σ2-EGFP; intensity time courses are normalized to the intensity at t_first. (H) Measured slope of LCa- and σ2-EGFP time courses during initiation phase. (I) Zoom-in of the departure phase in LCa-EGFP; intensity time courses are normalized to the intensity at t_last. (J) Zoom-in of the departure phase in σ2-EGFP; intensity time courses are normalized to the intensity at t_last. (K) Measured slope of LCa- and σ2-EGFP time courses during departure phase.

Figure 3. AP-2 is a determinant of clathrin behavior during CCP maturation

(A) LCa-mCherry time courses, TIRF illumination (slave channel). (B) σ2-EGFP time courses, TIRF illumination (master channel). (C) Time points of segmented reference points and phases in LCa-mCherry TIRF intensity time courses. (D) Time points of segmented reference points and phases of σ2-EGFP TIRF intensity time courses. (E) Example of superimposed LCa-TIRF and σ2- TIRF time courses; time courses are segmented independently to establish temporal hierarchy of reference points and phases in both channels.

Figure 4. Stages of CCP internalization revealed by dual channel TIRF/Epi illumination

(A) LCa-EGFP time courses, TIRF illumination. (B) LCa-EGFP time courses, Epi-illumination. (C) Representative superimposed TIRF/Epi clathrin time courses. The lower panels show the corresponding relative distances calculated from the TIRF/Epi intensity ratio; the time point of elongation onset P_e is determined as described in the text. (D) Time points of intensity maxima and elongation onset in LCa-EGFP intensity time courses. (E) LCa-EGFP distance time courses calculated from TIRF/Epi intensity ratio aligned to either t_first (brown traces) or t_last (purple traces).

Figure 5. Schematic of the temporal hierarchy of molecular dynamics of AP-2 and clathrin

Our analysis of the relative behaviors of AP-2 and clathrin defines both ‘fractional’ events, i.e. they occur at a fixed fraction of total CCP lifetime, and ‘absolute’ events, i.e. they occur at a fixed time relative to initiation or departure, regardless of CCP lifetime.