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. 2022 Sep 26;5(1):1014.
doi: 10.1038/s42003-022-03988-4.

Kinetics of phagosome maturation is coupled to their intracellular motility

Yanqi Yu #  1 , Zihan Zhang #  1 , Glenn F W Walpole  2   3 , Yan Yu  4
Affiliations

Kinetics of phagosome maturation is coupled to their intracellular motility

Yanqi Yu et al. Commun Biol. .

Abstract

Immune cells degrade internalized pathogens in phagosomes through sequential biochemical changes. The degradation must be fast enough for effective infection control. The presumption is that each phagosome degrades cargos autonomously with a distinct but stochastic kinetic rate. However, here we show that the degradation kinetics of individual phagosomes is not stochastic but coupled to their intracellular motility. By engineering RotSensors that are optically anisotropic, magnetic responsive, and fluorogenic in response to degradation activities in phagosomes, we monitored cargo degradation kinetics in single phagosomes simultaneously with their translational and rotational dynamics. We show that phagosomes that move faster centripetally are more likely to encounter and fuse with lysosomes, thereby acidifying faster and degrading cargos more efficiently. The degradation rates increase nearly linearly with the translational and rotational velocities of phagosomes. Our results indicate that the centripetal motion of phagosomes functions as a clock for controlling the progression of cargo degradation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Simultaneous measurement of phagosome dynamics and acidification.
a Schematics showing internalization of pH-sensitive RotSensors into phagosomes in macrophage cells. b Schematic illustration of the RotSensor design. Each RotSensor contains a 1 µm silica particle tethered with a 100 nm yellow-green fluorescent particle. The silica particles were coated with streptavidin (SAv)-pHrodo Red (pH reporter) and SAv-CF640R (reference). The azimuthal (φ) and polar (θ) angles of each pH-RotSensor were analyzed based on its projection fluorescence image. d and r denote the projection inter-particle distance and the physical inter-particle distance, respectively. c A representative trajectory of a pH-RotSensor-containing phagosome. Vectors indicate the orientation of the pH-RotSensor and are color-coded based on time. Scale bar, 2 µm. Insets show fluorescence images of the RotSensor (top) and the pH response (bottom). Scale bar, 1 µm. Line plots showing acidification (d), translational (e) and rotational velocities (f) of the pH-RotSensor-containing phagosome in c. The acidification profile is fitted with sigmoidal function to determine the initial pH, final pH, the period of rapid acidification (tinitial to tfinal), and acidification rate. The period of rapid acidification is highlighted in gray. Darker lines in e and f are velocity values after wavelet denoising. Scattered plots showing acidification rates against translational (g) and rotational velocities (h) of single phagosomes. Each data point represents one single phagosome. Data points from multiple phagosomes within the same cells are shown in the same solid color. Data points from cells containing only one phagosome are shown as black circles. N = 42 phagosomes from 24 cells for translational tracking, and 17 phagosomes from 12 cells for rotational tracking. Pearson’s coefficients of 0.78 and 0.81 were obtained in g and h, respectively. Scatter plots showing acidification rates against percentage of active rotation (i) and maximum rotation amplitude (j) of single phagosomes during rapid acidification period. N = 17 phagosomes from 12 cells. Pearson’s coefficients of 0.76 and 0.61 are obtained in i and j.
Fig. 2
Fig. 2. Simultaneous measurement of phagosome-lysosome fusion and phagosome transport dynamics.
a Schematic illustration of the Förster resonance energy transfer (FRET)-based phagosome-lysosome fusion assay. The FRET-RotSensor was coated with SAv-Alexa568 (FRET donor). BSA-Alexa647-biotin (FRET acceptor) was loaded into lysosomes. Phagosome fusion with lysosomes leads to intermixing between donor fluorophore (Alexa568) and fluid phase acceptor fluorophore (Alexa647) generating FRET emission (680 nm) under the donor excitation of 561 nm. b and c Fluorescence images and intensity plots showing the change of FRET emission (ex/em: 561/680 nm) and donor emission (magenta, ex/em: 561/586 nm) from a FRET-RotSensor (100 nm yellow-green nanoparticle in green) in a RAW264.7 macrophage. Scale bar, 2 μm. d FRET ratio vs. time plot is fitted with sigmoidal function (shown as the black solid line) to determine the initial time point of phagosome-lysosome fusion (tinitial), the time point where FRET-signal reaches a plateau (tfinal), and the FRET rate, as indicated by the red dotted line. Scatter plots showing normalized FRET rate against translational (e) and rotational velocities (f) of single phagosomes during the period of its fusion with lysosomes. Each data point represents data from a single phagosome. Data points from multiple phagosomes within the same cells are shown in the same solid color. Data points from cells containing only one phagosome are shown as black circles. For translational tracking, N = 40 phagosomes from 22 cells. For rotational tracking, N = 11 phagosomes from 10 cells. The black lines indicate linear regression with a Pearson’s coefficient of 0.75 in e and of 0.86 in f.
Fig. 3
Fig. 3. Correlation of phagosome-lysosome fusion kinetics with the centripetal motility of phagosomes.
a Fluorescence image showing a cell in which lysosomes were labeled with BSA-Alexa647-biotin. Yellow line indicates the cell periphery, and the asterisk indicates the centroid of the nucleus. The initial positions of two representative phagosomes were indicated by the two number-labeled white dots and their trajectories are shown in magenta. Scale bar, 10 μm. b Plots showing the phagosome-to-nucleus distance as a function of time for both the phagosomes indicated in a. Segments of active centripetal runs are highlighted in green. Gray shaded area indicates the effective transport distance of the phagosome from the time where it begins to fuse with lysosomes to the time when it reaches the nucleus boundary. The starting time of phagosome lysosome fusion was obtained based on sigmoidal fitting the FRET ratio vs. time plot. Phagosome #1 underwent active centripetal runs in 48% of the time during effective transport, whereas phagosome #2 had no active centripetal run. c FRET ratio vs. time plots for both phagosomes marked in a. Solid lines indicate sigmoidal fitting to the data. Scatter plots showing normalized FRET rate of single phagosomes plotted against the percentage of active centripetal motion (d) and centripetal velocity (e). Each data point represents data from a single phagosome. Data from multiple phagosomes inside the same cell are shown in the same solid color. Data from cells in which only one phagosome was studied are shown as black circles. N = 40 phagosomes from 22 cells. The black lines indicate linear regression with a Pearson’s coefficient of 0.68 and 0.49, respectively.
Fig. 4
Fig. 4. Phagosome degradation measurements during magnetically manipulated transport.
a Schematic illustration of the experimental setup. The magnetic gradient is generated by a homemade magnetic tweezers setup built on an inverted fluorescence microscope system. Magnetic pulling force was applied on MagSensors after their internalization into phagosomes. The 1 µm MagSensors were coated with SAv-pHrodo Red (pH indicator), SAv-CF640 (reference), and physically adsorbed IgG. b Calibration plot showing the magnetic force exerted on each MagSensor as a function of its distance to the tip of the magnetic tweezers solenoid (shown in inset). Error bars are standard deviations from 5 samples. c Bright-field image of a cell overlaid with the trajectory of a MagSensor-containing phagosome under magnetic pulling. The start and end time points of the exertion of magnetic force are indicated. The blue-colored segment of the trajectory indicates the movement of the MagSensor before cell entry; the red-colored segment of the trajectory indicates the intracellular movement of a MagSensor-containing phagosome under magnetic manipulation. Scale bar, 5 µm. d Mean-square displacements (MSD) calculated from trajectories of individual MagSensors under different conditions as indicated. Each line is an average of results from N = 10 phagosomes from 8 cells (no force), 9 cells (magnetic pulling), and 7 cells (nocodazole). Shaded areas indicate standard deviation of the mean.
Fig. 5
Fig. 5. Phagosome acidification and phagosome-lysosome fusion under magnetic pulling.
a Line plots showing the average phagosome pH as a function of time with or without magnetic pulling as indicated. Each line plot is an average of 20 phagosomes. Shaded areas represent standard deviations. b Box graph showing the average acidification rate of phagosomes with or without magnetic pulling. The average acidification rate is 0.48 ± 0.34 pH unit/min without magnetic pulling (N = 33 phagosomes in 29 cells from 11 independent experiments) and 0.79 ± 0.54 pH unit/min with magnetic pulling (N = 38 phagosomes in 38 cells from 13 independent experiments). c Box graph showing the average final pH in different experiment conditions as indicated. The average final pH is 4.7 ± 0.3 without magnetic manipulation (N = 33) and 4.7 ± 0.4 with magnetic pulling (N = 38). d Line plots showing the average normalized FRET ratio as a function of time with or without magnetic pulling as indicated. The line curves are averaged from 20 individual phagosomes in each experimental condition. Shaded areas represent standard deviations. e Box graph showing the average normalized FRET rate in different experiment conditions as indicated. The average FRET rate is 0.15 ± 0.07 without magnetic manipulation (N = 20 phagosomes in 16 cells from 4 independent experiments) and 0.28 ± 0.21 with magnetic pulling (N = 20 phagosomes in 19 cells from 8 independent experiments). In b, c, and e, each box plot indicates the mean (horizontal line) and the interquartile range from 25% to 75% of the corresponding data set. Statistical significance is highlighted by p values (Mann-Whitney U Test) as follows: **p < 0.01, ***p < 0.001, NS p > 0.05.
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