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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02774

Journal of Cell Science 119, 550-558 (2006)
Published by The Company of Biologists 2006
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Dictyostelium myosin-IE is a fast molecular motor involved in phagocytosis

Ulrike Dürrwang1, Setsuko Fujita-Becker1, Muriel Erent1, F. Jon Kull2, Georgios Tsiavaliaris3, Michael A. Geeves4 and Dietmar J. Manstein1,3,*

1 Abteilung Biophysik, Max-Planck Institut für medizinische Forschung, Jahnstr. 29, 69120 Heidelberg, Germany
2 Dartmouth College, Department of Chemistry, 6128 Burke Laboratory, Hanover, NH 03755, USA
3 Institut für Biophysikalische Chemie, OE 4350, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30623 Hannover, Germany
4 Department of Biosciences, University of Kent, Canterbury CT2 7NJ, UK


Figure 1
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  		Fig. 1. Cellular localization of YFP-tagged myosin-IE. (A) Myosin-IE is preferentially localized at dynamic actin structures. Colocalization of YFP-Myosin-IE with actin demonstrated by epifluorescence microscopy. Actin was stained with TRITC-Phalloidin. YFP-Myosin-IE is shown in green, actin in red. (B) Localization of YFP-Myosin-IE (green) and coronin (red) at crown like structures as visualized by confocal microscopy. (C) Confocal analysis of the 3D distribution of YFP-Myosin-IE (green) and coronin. The X-Z and Y-Z views show myosin-IE predominantly at the side walls of large invaginations of the plasma membrane. Coronin localizes to the bottom of the invaginations and their upper rim. Bars, 10 µm (A,B); 3 µm (C).

 

Figure 2
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  		Fig. 2. Localization of myosin-IE during phagocytosis. (A) Myosin-IE was observed to be associated with the phagocytic cup during the process of ingesting bits of other cells, and the early phagosome (see Movie 1 in supplementary material). (B) Phagocytosis of yeast cells (red) by D. discoideum amoeba. Myosin-IE associates with the phagocytic cup shortly after particle docking. It remains associated with the phagocytic cup during engulfment and dissociates from the phagosome within 2 minutes of completion of uptake. Bar, 3 µm.

 

Figure 3
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  		Fig. 3. The role of myosin-IE in phagocytosis and pinocytosis. (A) Phagocytotic ability was assayed by following the uptake kinetics of TRITC-labelled yeast cells in D. discoideum overproducing YFP-myosin-IE ({blacksquare}) and wild-type cells ({circ}). (B) Uptake kinetics of the fluid-phase marker FITC-dextran in D. discoideum overproducing YFP-myosin-IE ({blacksquare}) and wild-type cells ({circ}).

 

Figure 4
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  		Fig. 4. Transient kinetic analysis of the interaction of Myosin-IE with actin and nucleotides. (A) ATP-induced dissociation of the actomyosin complex. The observed rate constants for E698 is linearly dependent on the ATP concentration in the range 5-25 µM. The apparent second-order rate constant for ATP binding to actomyosin (K1k+2) was determined from the slope of the line. (B) The data over the range from 5 µM to 8 mM were fitted to a hyperbola. The rate constants for the isomerization step are given by the plateau values. (C) ADP inhibition of ATP-induced dissociation of the actomyosin complex of E698(S336E). Small amounts of ADP in the complex of E698(S336E) and pyrene-actin produced biphasic dissociation reactions. (D) Relative amplitudes of the two exponentials in dependency on the ADP concentration. The data are fitted with hyperbolae resulting in a KAD of 12 µM for the slow phase ({triangleup}) and the fast phase ({blacktriangleup}).

 

Figure 5
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  		Fig. 5. Inhibition of Myosin-IE by free Mg2+. The rate of ADP dissociation from Myosin-IE is dependent on the Mg2+ concentration. The kobs for the displacement of 30 µM ADP from 0.25 µM E698 by 500 µM MgATP is plotted over the range 0 to 20 mM free-Mg2+ concentration. The data were fitted to a hyperbola and indicate a Kd of 0.99±0.3 mM.

 

Figure 6
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  		Fig. 6. The dynamics of ATP binding and hydrolysis by the myosin constructs were analyzed in terms of the models shown in Schemes 1-3, where M refers to myosin head fragment, A to actin, and T, D and Pi to ATP, ADP and phosphate, respectively. In these schemes, a notation is used that distinguishes between the constants in the presence and absence of actin by using bold (K+1, K1) versus italic type (k+1, K1); subscript A and D refer to actin (KA) and ADP (KD), respectively.

 

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© The Company of Biologists Ltd 2006