Fig 1.
mCLING labels the plasma and flagellar membranes.
Trypanosomes were incubated with mCLING-488 for 15 min on ice, fixed, and the kinetoplast (K) and nuclear (N) DNA was stained with DAPI. Panels presented are maximum intensity projections of 3D SR-SIM images. The “magenta hot” look-up table in Fiji (that colors areas of intense signal white) was applied to the DAPI channel. (A) Plasma and flagellar membrane staining of a whole trypanosome. (B) Plasma and flagellar membranes of a 1K1N trypanosome. (C) Plasma and flagellar membranes of a trypanosome that has two flagella, labeled with arrowheads. (D) Further growth of a nascent flagellum and apparent division of an elongated kinetoplast (Ke). (E) Elongation of the nucleus (compare to panel A) during mitosis, following kinetoplast division. Arrow points to the anterior tip of a growing flagellum. (F) Post-mitotic cell with two kinetoplasts and two nuclei. Arrow points to the tip of a growing flagellum. Arrowheads indicate the posterior bases of flagella. K = kinetoplast. Kdiv = dividing kinetoplast. N = nucleus. Scale bar = 2 μm.
Fig 2.
Double-labeling of trypanosomes with antibodies and mCLING.
Trypanosomes were incubated with mCLING-488 on ice, then fixed and subjected to “antigen retrieval” before incubation with primary and fluorescent secondary antibodies. The kinetoplast and nucleus were stained with DAPI. Panels presented are maximum intensity projections of 3D SR-SIM images. (A): The trypanosome basal body protein TbRP2 was labeled with YL1/2 primary antibody and AlexaFluor-594 secondary antibody. (B): Trypanosome mitochondrial alternative oxidase (TAO) was labeled with anti-TAO primary antibody and AlexaFluor-594 secondary antibody. Scale bar = 2 μm.
Fig 3.
A fraction of mCLING colocalizes with acidified intracellular compartments.
Uptake of mCLING and LysoTracker was observed in real-time with imaging flow cytometry. Colocalization of mCLING and LysoTracker was assessed using IDEAS software Colocalization Wizard. (A) Flow chart of the experimental procedure. (B) Box and whisker plot of the colocalization score over time. Data points collected within 2.5 seconds of the indicated time points from 3 biological replicates were combined and graphed. N>30 for each replicate at each time point. N≥133 for each time point. Statistical significance between means (1.578, 1.673, 1.935, 2.106, and 2.124, respectively) was determined using one-way ANOVA (p < 0.0001). Annotations in red above each box and whisker plot represent the results of Tukey’s test between time 0 and the respective time point (**** = p <0.0001, ns = not significant). Whiskers represent 5th and 95th percentiles. (C) Example images of trypanosomes with colocalization scores close to the medians (1.498, 1.657, 1.93, and 1.967, respectively) acquired during the collection periods for the indicated time points. Images representing 5th and 95th percentiles of the 0 min and 5 min time points, respectively, are presented.
Fig 4.
Labeling of endosomes with ConA and mCLING.
Trypanosomes were incubated with mCLING-488 and ConA-594 for 15 minutes on ice, then exogenous mCLING was removed. Cells were incubated at 37°C with ConA-594 for 5 or 15 minutes and fixed. DNA in the kinetoplast and nucleus was stained with DAPI. (A) Simplified flow chart of the experimental procedure. (B) Maximum intensity projections of 3D SR-SIM images. 0 min represents the “No uptake” control (kept on ice); 5 min and15 min images include internalized mCLING-positive endosomes. Boxes (in 5 min and 15 min samples) indicate areas of the trypanosome containing endocytosed mCLING and ConA. ConA is observed at the base of the flagellum in the 0 min and 5 min samples. Small image panels below the larger images show the area enclosed by the box, at a higher magnification. K = kinetoplast. N = nucleus. Arrowheads indicate the posterior bases of flagella. Scale bar in large panels = 2 μm. Scale bare in small panels = 1 μm.