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The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.

Jung G, Remmert K, Wu X, Volosky JM, Hammer JA - J. Cell Biol. (2001)

Bottom Line: Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content.These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin.We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH(2)-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.

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Localization of the Arp2/3 complex and p116 in vegetative cells. (A) Z series (0.7 μm sections) of a vegetative cell double stained for actin and Arp3 (top, ventral; bottom, dorsal). The corresponding overlaid (actin, red; Arp3, green; colocalization, yellow) and DIC images are also shown. (B) A single, ventral, 1 μm-confocal section through vegetative cells double stained for actin and Arp3 (top) or actin and p116 (bottom). The corresponding overlaid (red, actin; green, Arp3 or p116; colocalization, yellow) and DIC images are also shown. The arrows in the two Actin panels point to the actin-rich ventral lamella, whereas the arrowheads in the Arp3 and p116 panels point to the prominent spots of staining seen for both antibodies just inside the lamella. Bars: (A) 4.6; (B) 7.2 μm.
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Figure 6: Localization of the Arp2/3 complex and p116 in vegetative cells. (A) Z series (0.7 μm sections) of a vegetative cell double stained for actin and Arp3 (top, ventral; bottom, dorsal). The corresponding overlaid (actin, red; Arp3, green; colocalization, yellow) and DIC images are also shown. (B) A single, ventral, 1 μm-confocal section through vegetative cells double stained for actin and Arp3 (top) or actin and p116 (bottom). The corresponding overlaid (red, actin; green, Arp3 or p116; colocalization, yellow) and DIC images are also shown. The arrows in the two Actin panels point to the actin-rich ventral lamella, whereas the arrowheads in the Arp3 and p116 panels point to the prominent spots of staining seen for both antibodies just inside the lamella. Bars: (A) 4.6; (B) 7.2 μm.

Mentions: With regard to vegetative cells, Fig. 6 A shows a Z series of a cell that had been double stained for Arp3 and actin. Striking colocalization between the two (yellow) is seen almost exclusively in dorsal confocal sections, where both are concentrated throughout a cup-shaped macropinocytic crown. Very similar images were obtained by double staining vegetative cells for Arp3 and coronin, a bona fide marker for these dorsal macropinocytic extensions (De Hostos 1999), and by using the antibody to p19 instead of Arp3 (data not shown). Examination of the ventral confocal sections in Fig. 6 A, and the single ventral confocal section shown for the two cells in Fig. 6 B (top), indicates that the Arp2/3 complex, while present within the ventral actin–rich lamellae of vegetative cells (marked by arrows in upper “Actin” panel), is not highly concentrated there. This may reflect the fact that the requirement for actin assembly in these lamellae, which are not driving rapid cell migration, is significantly less than in dorsal crowns, which are driving constitutive endocytosis. Interestingly, Arp3 is often concentrated in bright spots that accumulate just inside the ventral lamellae (see arrowheads in Fig. 6 B, top, “Arp3” panel). The nature and function of these spots is currently unknown.


The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.

Jung G, Remmert K, Wu X, Volosky JM, Hammer JA - J. Cell Biol. (2001)

Localization of the Arp2/3 complex and p116 in vegetative cells. (A) Z series (0.7 μm sections) of a vegetative cell double stained for actin and Arp3 (top, ventral; bottom, dorsal). The corresponding overlaid (actin, red; Arp3, green; colocalization, yellow) and DIC images are also shown. (B) A single, ventral, 1 μm-confocal section through vegetative cells double stained for actin and Arp3 (top) or actin and p116 (bottom). The corresponding overlaid (red, actin; green, Arp3 or p116; colocalization, yellow) and DIC images are also shown. The arrows in the two Actin panels point to the actin-rich ventral lamella, whereas the arrowheads in the Arp3 and p116 panels point to the prominent spots of staining seen for both antibodies just inside the lamella. Bars: (A) 4.6; (B) 7.2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2150732&req=5

Figure 6: Localization of the Arp2/3 complex and p116 in vegetative cells. (A) Z series (0.7 μm sections) of a vegetative cell double stained for actin and Arp3 (top, ventral; bottom, dorsal). The corresponding overlaid (actin, red; Arp3, green; colocalization, yellow) and DIC images are also shown. (B) A single, ventral, 1 μm-confocal section through vegetative cells double stained for actin and Arp3 (top) or actin and p116 (bottom). The corresponding overlaid (red, actin; green, Arp3 or p116; colocalization, yellow) and DIC images are also shown. The arrows in the two Actin panels point to the actin-rich ventral lamella, whereas the arrowheads in the Arp3 and p116 panels point to the prominent spots of staining seen for both antibodies just inside the lamella. Bars: (A) 4.6; (B) 7.2 μm.
Mentions: With regard to vegetative cells, Fig. 6 A shows a Z series of a cell that had been double stained for Arp3 and actin. Striking colocalization between the two (yellow) is seen almost exclusively in dorsal confocal sections, where both are concentrated throughout a cup-shaped macropinocytic crown. Very similar images were obtained by double staining vegetative cells for Arp3 and coronin, a bona fide marker for these dorsal macropinocytic extensions (De Hostos 1999), and by using the antibody to p19 instead of Arp3 (data not shown). Examination of the ventral confocal sections in Fig. 6 A, and the single ventral confocal section shown for the two cells in Fig. 6 B (top), indicates that the Arp2/3 complex, while present within the ventral actin–rich lamellae of vegetative cells (marked by arrows in upper “Actin” panel), is not highly concentrated there. This may reflect the fact that the requirement for actin assembly in these lamellae, which are not driving rapid cell migration, is significantly less than in dorsal crowns, which are driving constitutive endocytosis. Interestingly, Arp3 is often concentrated in bright spots that accumulate just inside the ventral lamellae (see arrowheads in Fig. 6 B, top, “Arp3” panel). The nature and function of these spots is currently unknown.

Bottom Line: Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content.These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin.We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH(2)-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.

Show MeSH
Related in: MedlinePlus