Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Abstract

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.

Keywords: GTPases; PALM; endocytosis; phosphoinositides; single-molecule.

Fig. 1.

Intracellular calibration of superresolution microscopy allows counting of biomolecules in subdiffraction-limit structures. (A) Intracellular structure size and number of biomolecules span several orders of magnitude. (B) Resolution and counting accuracy are determined using constructs with different mEos2 stoichiometry. (Left) Superresolution images of calibration constructs (red) are superimposed on transmitted light images of yeast cells (gray). Single (1×, Top), double (2×, Middle) and triple (3×, Bottom) repeats of mEos2 were constitutively expressed as fusions with the membrane-localized PH domain of Plcδ. (Center) The magnified fields depict uncorrected (+) and corrected (X) single molecule positions. Each molecule and localization is color-coded by frame number. The number of molecules per cluster is fitted to a binomial distribution B(N,F) (red line). (Right) Pair-correlation functions of corrected images reflect the average distance between molecules, which is constant for the single mEos2 repeat and peaked for the 2× and 3× repeat owing to colocalization. The peak width of corrected 2× and 3× data are narrower than for uncorrected data (only shown for 1×) and reflects the increase in resolution by combining photons from all fluorescent bursts to the molecule's position. (C) Summary of calibration results. The ensemble values of corrected molecules per cell exhibit a linear relation with an expected two- and threefold higher number of mEos2 molecules of the 2× and 3× repeat compared with the single repeat. The table summarizes fraction of observable mEos2 molecules F from the single molecule counting data.

Fig. 2.

Vesicles in the endocytic pathway show characteristic relationship between vesicle size and PI3P content. (A) A tandem repeat of the FYVE domain of EEA1 fused to mEos2 serves as a specific probe to detect accessible PI3P binding sites on endocytic/endosomal vesicles. mEos2 (red) localizes to distinct, circular vesicles within a yeast cell (gray). (B) By applying our calibrated superresolution microscopy approach, the number of mEos2 molecules (N) bound to PI3P on each vesicle is determined (x’s). The molecular distribution exhibits a circular shape (two peaks in a 1D projection). The diameter of vesicles (D) is determined by measuring the spatial spread of molecules. (C) Vesicles display variability in PI3P content and size. Representative vesicles are shown displaying the expected circular shape. The histogram of vesicle diameters exhibits a fitted maximum at 82 nm. (D) PI3P content and vesicle surface area fall on a characteristic curve. Vesicles with fewer than 100 molecules display relatively tight size regulation, whereas structures above this threshold are significantly larger. Vesicles from each part of this characteristic distribution can be found within one individual cell (red). This path is also displayed as an exponential function (black line) fitted to box-smoothed data with 95% confidence envelope.

Fig. 3.

Counting of PI3P-binding sites is validated with conventional fluorescence. (A) Schematic for validation. One PI3P probe (A) is tagged with mEos2, allowing for high-resolution measurements of structure and quantification. The other (B) is fused to GFP, which can be quantified by calculating the total fluorescence intensity. (B) Quantified conventional fluorescence validates superresolution counting. FYVE–mEos2 and FYVE–GFP constructs were expressed in the same cells. A colocalization image shows transmitted light image (gray), mEos2 (red), and GFP (green). The number of mEos2 molecules determined in the red superresolution channel and the amount of GFP quantified by total fluorescence intensity on each vesicle is tightly correlated (Pearson’s coefficient, ρ= 0.91).

Fig. 4.

Variation in the relationship between PI3P-binding site number and vesicle size is due to both intrinsic and extrinsic noise in the endocytic pathway. (A) Measurements made on several vesicles within one individual cell display variation around the characteristic curve. Vesicles from all regions of the PI3P number-size distribution can be found within the same cell. Vesicles within the same cell may be located above (red) or below (pink) the characteristic curve (dotted line, 95% confidence interval as gray shading). (B) Schematics showing two components of noise in PI3P content and size. (Upper) Intrinsic noise is due to a variation between vesicles within a cell and (Lower) extrinsic noise is due to systematic variations between cells.

Fig. 5.

Benchmarking with endocytic/endosomal landmark proteins reveals distinct phases of PI3P production and vesicle fusion. (A) Colocalization of vesicles with landmark proteins of the endocytic pathway. Representative superresolution images (red, left column of each pair) and GFP images (green, right column of each pair) for each class of protein are shown. (B) Colocalization with endocytic pathway proteins reveals distinct subclasses of vesicles. FYVE–mEos2 colocalizes with GFP-tagged clathrin (green) and GTPases Vps21p (orange) and Ypt7p (purple). For each dataset, the median, upper, and lower quartiles are displayed. (C) Model of PI3P production on vesicles in endocytic pathway followed by fusion. Phases of endocytic/endosomal protein association, enzymatic production of PI3P, and fusion to early and late endosomes (1–3) are described in B.