Phagocytosis of Dictyostelium discoideum studied by the particle-tracking method

• Published in: Experimental cell research 2003-08, Vol.288 (2), p.268-276
• Main Authors: Ishikawa, J, Okano, J, Ohki, K, Amagai, A, Maeda, Y, Miyata, H
• Format: Article
• Language: English
• Subjects: Actin cytoskeleton; Animals; Biological Transport; Cytochalasins - metabolism; Dictyostelium - cytology; Dictyostelium - physiology; Dictyostelium discoideum; Fluorescence microscopy; Intracellular signaling; Microscopy, Video - methods; Optical microscopy; Optical tweezers; Particle Size; Phagocytosis - physiology
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/S0014-4827(03)00212-X

Single phagocytic events of cellular slime mold Dictyostelium discoideum were studied by the method of particle tracking. A 2-μm polystyrene bead, which had been covalently coated with folate, was attached to the advancing edge of a Dictyostelium ameba with the aid of an optical trap. The bead was transported backward on the cell surface. Forty-five percent of the transported beads were internalized. The bead motion was analyzed by determining every 33 ms the x–y coordinate of the centroid of the phase-contrast image of the bead. The x( t) and y( t) traces were smoothed over 1 s and the difference between the smoothed ( x( t) and y( t) ) and the original traces, δ x ≡ x( t) − x( t) and δ y ≡ y( t) − y( t) , were calculated, which represented relatively rapid components of the bead motion. The plot of δ 2 = (δ x 2 + δ y 2) against time could be divided into three phases on the basis of the variance of δ 2. Comparison of the plot with the video sequence indicated that the first phase corresponded to the transport, the second phase to the internalization, and the third phase to the postinternalization process (intracellular movement). Cytochalasin A at 5 μM completely inhibited phagocytosis without affecting the binding of bead to the cell surface, indicating the importance of actin cytoskeleton in all the phases. At 1 μM cytochalasin A the varience of the postinternalization process decreased, and the duration of the transport phase increased. At 0.25 μM cytochalasin A the duration of the internalization phase exhibited a significant increase, but other parameters did not change appreciably. The complex and differential effects of cytochalasin A on the parameters characterizing the three phases in the phagocytic process indicate that various aspects of actin dynamics are involved in the individual process of phagocytosis.