Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives

Abstract

Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent cargo for receptor-mediated endocytosis. We have now synthesized biologically active fluorescent mating-pheromone derivatives and demonstrated that receptor-mediated endocytosis in budding yeast occurs via the clathrin- and actin-mediated endocytosis pathway. We found that endocytic proteins first assemble into patches on the plasma membrane, and then alpha-factor associates with the patches. Internalization occurs next, concomitant with actin assembly at patches. Additionally, endocytic vesicles move toward early endosomes on actin cables. Early endosomes also associate with actin cables, and they actively move toward endocytic sites to capture vesicles being released from the plasma membrane. Thus, endocytic vesicle formation and capture of the newly released vesicles by early endosomes occur in a highly concerted manner, mediated by the actin cytoskeleton.

Fig. 1.

Structure and localization of fluorophor-conjugated α-factor. (A) Diagram of Alexa Fluor-488 (A488)- and Alexa Fluor-594 (A594)-α-factor. (B and C) Ste2p receptor-dependent binding and internalization of Alexa-α-factor. Alexa-α-factor was added to wild-type (B) or ste2Δ (C) cells and was followed through the endocytic pathway for the indicated times. (D, F, and G) A488-α-factor (TIRF optics) appeared in endocytic patches before Abp1p and Sla1p but after Ede1p (epifluorescence). Shown are single frames from the GFP and the RFP channels of the movie and a merged image (Upper) and time series of single patches from wild-type cells expressing the indicated fluorophor-tagged proteins (Lower). The time to acquire one image pair was 2 s. (E) Kymographs of time-lapse images collected at 2-s intervals. Arrows in D mark where the kymograph was generated. Numbers and the direction of the arrows in D correspond to those in E. Arrowheads (E Right) indicate independent A488-α-factor-labeled spots. (H) Localization of A594-α-factor and Sla1-GFP in cells treated with 200 μM LatA. After incubating cells expressing Sla1-GFP with 200 μM LatA at 25°C for 30 min, cells were incubated with A594-α-factor at 0°C for 30 min in minimal medium lacking glucose in the continued presence of 200 μM LatA. The images were acquired at 2 min and 20 min after washing out unbound Alexa-α-factor with glucose-containing medium and warming cells to 25°C in the continued presence of 200 μM LatA. (Scale bars, 2.5 μm.)

Fig. 2.

Dynamic behavior of endosomes and endocytic vesicles. (A) Active movement of endosomes and endocytic vesicles toward each other. Endosomes labeled with A594-α-factor and endocytic vesicles labeled with Sla1-GFP were imaged in wild-type cells. Time to acquire one image pair was 2.8 s. (Scale bar, 2.5 μm.) (B) Tracking of endosomes and endocytic vesicles shown in A. Blue and red frames correspond to blue and red boxes in A. Red and green dots indicate endosomes and endocytic vesicles, respectively. Big and small dots denote the first and last positions, respectively, of endosomes or patches. (Scale bars, 0.5 μm.) (C) Two phases of endosome motility. Velocities of endosomes shown in A were plotted at 2.8-s intervals. The blue and red lines represent the velocities of the endosomes shown in the blue and red boxes, respectively, in A. Green circles indicate the points at which endocytic vesicles merge with endosomes. (D) Quantification of endosome velocity and the timing of patch internalization. Wild-type cells expressing Abp1-GFP were incubated with A594-α-factor, and internalization was induced 3 min before imaging. Endosome velocities were acquired at 0.5-s intervals, and the velocities were categorized according to velocity range. Blue bars indicate velocities of all endosomes (n = 1,268). Red bars indicate the velocities of endosomes that were merging with endocytic vesicles (n = 150). (E) Movements of endosomes in arp3-D11A cells. The time to acquire one image pair was 6.5 s. (Scale bars, 2.5 μm.) (F) Tracking of the endosome in the boxed area in E. Green dots are Abp1-GFP patches. (Scale bar, 0.5 μm.)

Fig. 3.

Endosome motility along actin cables. (A) Localization and motility of endosomes on actin cables. Cells expressing Abp140–3GFP were incubated with A594-α-factor, and internalization was induced 3 min before imaging. Time to acquire one image pair was 1.0 s. (B) Localization and motility of endosomes on actin cables in bni1–12 bnr1Δ cells at the restrictive temperature. Cells were cultured for 1 h at 37°C and then labeled with A594-α-factor on ice for 2 h. Internalization was initiated as described in Materials and Methods. Endosome movement was imaged at room temperature. The time difference between each frame is 10 s. [Scale bars, 2.5 μm (A and B).] (C) Tracking of the endosome in the boxed area in A or B. The time difference between each position along the track is 1.0 s. (Scale bars, 0.5 μm.) (D) Quantification of endosome velocity in cells treated with 200 μM LatA for 30 min or at the permissive temperature and nonpermissive temperature in bni1–12 bnr1Δ cells.

Fig. 4.

Actin cables are necessary for efficient transport of α-factor from endocytic vesicles to the vacuole. (A) Cells were cultured at 37°C for 1 h, and 5 μM A594-α-factor was added for the indicated times. Arrowheads identify vacuoles. (B) Relative fluorescence intensity of vacuoles stained by A594-α-factor (n = 30 cells for each strain). The intensity of A594-α-factor in the vacuole was measured by using the program imagej v1.32. Values were relative to the fluorescence intensity in wild-type cells at 30 min. (C) Internalization of [³⁵S]-labeled α-factor in wild-type cells or bni1–12 bnr1Δ cells at 37°C. Results are the mean of two experiments.

Fig. 5.

Endosomes move along actin cables to endocytic vesicles. (A) Localization of 3GFP-tagged Abp140p and RFP-tagged Abp1p in living cells. Time to acquire one image pair was 2.0 s. Arrowheads indicate examples of colocalization. (B) Higher magnification view of the boxed area in A. Time series of single patch and cable from wild-type cells expressing Abp1-RFP and Abp140–3GFP. Time to acquire one image pair was 1 s. (C) Cells expressing Abp1-GFP and Abp140–3GFP were incubated with A594-α-factor and internalization was induced 5 min before imaging. Yellow arrowheads identify endosomes that move to endocytic vesicles (white arrowheads). (Scale bars, 2.5 μm.)