Limits...
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.

Show MeSH

Related in: MedlinePlus

Dot matrix comparison, schematic of the domain organization of p116 and Acan 125, alignment of the verprolin-like sequences, and acceleration of Arp2/3-dependent actin nucleation by the VA domain of p116. (A) Dot matrix comparison between Dictyostelium p116 and Acanthamoeba Acan 125 (window size, 30; stringency, 11). The dark rectangular region in the upper right corner is due to the alignment of their repetitive proline-rich sequences. (B) Schematic of the tripartite domain organization of p116 and Acan 125, showing the percent identity and the percent similarity (identities plus conservative substitutions in parentheses) for each domain. The positions of the 16 LRRs, the verprolin-like sequence that in verprolin has been implicated in binding G-actin, the acidic region, the proline-rich domain, the two PXXP motifs known to be critical for the interaction between Acan 125 and the SH3 domain of Acanthamoeba myosin IC, and the two PXXP motifs deleted from p116 in this study, are indicated. (C) Alignment of a portion of p116 and the homologous sequence in Acan 125 with a region of yeast verprolin that contributes to the binding of G-actin, and with six-residue sequences present in thymosin β4 and actobindin that also contribute to binding monomeric actin. (D) Actin nucleation assays were performed using 4 μM G-actin (5% pyrene actin) with or without the Arp2/3 complex and GST VA (see Materials and Methods). Shown are the rates of polymerization for actin alone (trace a, no symbols), actin plus 50 nM Arp2/3 complex (trace b, squares), actin plus 3 μM GST VA (trace c, open circles), and actin plus 50 nM Arp2/3 complex and 3 μM GST VA (trace d, closed circles). Similar results were obtained using two different preparations of proteins. Addition of 3 μM unfused GST did not accelerate Arp2/3-dependent actin nucleation (data not shown).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2150732&req=5

Figure 5: Dot matrix comparison, schematic of the domain organization of p116 and Acan 125, alignment of the verprolin-like sequences, and acceleration of Arp2/3-dependent actin nucleation by the VA domain of p116. (A) Dot matrix comparison between Dictyostelium p116 and Acanthamoeba Acan 125 (window size, 30; stringency, 11). The dark rectangular region in the upper right corner is due to the alignment of their repetitive proline-rich sequences. (B) Schematic of the tripartite domain organization of p116 and Acan 125, showing the percent identity and the percent similarity (identities plus conservative substitutions in parentheses) for each domain. The positions of the 16 LRRs, the verprolin-like sequence that in verprolin has been implicated in binding G-actin, the acidic region, the proline-rich domain, the two PXXP motifs known to be critical for the interaction between Acan 125 and the SH3 domain of Acanthamoeba myosin IC, and the two PXXP motifs deleted from p116 in this study, are indicated. (C) Alignment of a portion of p116 and the homologous sequence in Acan 125 with a region of yeast verprolin that contributes to the binding of G-actin, and with six-residue sequences present in thymosin β4 and actobindin that also contribute to binding monomeric actin. (D) Actin nucleation assays were performed using 4 μM G-actin (5% pyrene actin) with or without the Arp2/3 complex and GST VA (see Materials and Methods). Shown are the rates of polymerization for actin alone (trace a, no symbols), actin plus 50 nM Arp2/3 complex (trace b, squares), actin plus 3 μM GST VA (trace c, open circles), and actin plus 50 nM Arp2/3 complex and 3 μM GST VA (trace d, closed circles). Similar results were obtained using two different preparations of proteins. Addition of 3 μM unfused GST did not accelerate Arp2/3-dependent actin nucleation (data not shown).

Mentions: Actin was purified from Acanthamoeba castellanii according to Gordon et al. 1976, followed by gel filtration on HiPrep Sephacryl S200 (17-1195-01; Amersham Pharmacia Biotech). Monomeric actin was labeled with N-(1-pyrene)-iodoacetamide (P29; Molecular Probes) and stored lyophil-ized at −80°C. Before use it was resuspended and dialyzed in G buffer (0.1 mM CaCl2, 0.5 mM ATP, pH 7.0, 0.75 mM β-mercaptoethanol, and 3 mM imidazole, pH 7.5). The Arp2/3 complex was purified from Acanthamoeba as described by Kelleher et al. 1998 and stored at −70°C in complex storage buffer supplemented with 200 mM sucrose. The portion of p116 (residues 644–863) that contains a short sequence conserved among G-actin binding proteins (including verprolin), plus an acidic region was amplified by PCR using Pfu polymerase and the following oligonucleotides: 5′-TCAGGATCCATTTGGAAGGAAATCGATTCATGTATC-3′ and 5′-ACTGAATTCTTAATCTGGAATTGGGGTGGCACCACT-3′. The product was digested with BamH1 and EcoR1, cloned into pGEX 2TK (27-4587-01; Amersham Pharmacia Biotech), and the fusion protein, referred to below as “GST VA,” expressed and purified as described above. Actin nucleation assays were performed essentially as described by Higgs et al. 1999. In brief, G-actin was mixed with pyrene-labeled actin at a ratio of 20:1, converted to Mg++-actin, and polymerized at a final concentration of 4 μM by the addition of 10× polymerization buffer (500 mM KCL, 10 mM MgCl2, 10 mM EGTA, and 100 mM imidazole, pH 7.0). Additional proteins (e.g., Arp2/3, GST VA; see the legend to Fig. 5) were dialyzed into G buffer supplemented with 1 mM MgCl2 and added to the actin solution just before the initiation of polymerization. Fluorescence intensities were measured every second for 10 min using a PTI QuantaMaster fluorometer (Photon Technology International) and wavelengths of 365 and 407 nm for excitation and emission, respectively.


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)

Dot matrix comparison, schematic of the domain organization of p116 and Acan 125, alignment of the verprolin-like sequences, and acceleration of Arp2/3-dependent actin nucleation by the VA domain of p116. (A) Dot matrix comparison between Dictyostelium p116 and Acanthamoeba Acan 125 (window size, 30; stringency, 11). The dark rectangular region in the upper right corner is due to the alignment of their repetitive proline-rich sequences. (B) Schematic of the tripartite domain organization of p116 and Acan 125, showing the percent identity and the percent similarity (identities plus conservative substitutions in parentheses) for each domain. The positions of the 16 LRRs, the verprolin-like sequence that in verprolin has been implicated in binding G-actin, the acidic region, the proline-rich domain, the two PXXP motifs known to be critical for the interaction between Acan 125 and the SH3 domain of Acanthamoeba myosin IC, and the two PXXP motifs deleted from p116 in this study, are indicated. (C) Alignment of a portion of p116 and the homologous sequence in Acan 125 with a region of yeast verprolin that contributes to the binding of G-actin, and with six-residue sequences present in thymosin β4 and actobindin that also contribute to binding monomeric actin. (D) Actin nucleation assays were performed using 4 μM G-actin (5% pyrene actin) with or without the Arp2/3 complex and GST VA (see Materials and Methods). Shown are the rates of polymerization for actin alone (trace a, no symbols), actin plus 50 nM Arp2/3 complex (trace b, squares), actin plus 3 μM GST VA (trace c, open circles), and actin plus 50 nM Arp2/3 complex and 3 μM GST VA (trace d, closed circles). Similar results were obtained using two different preparations of proteins. Addition of 3 μM unfused GST did not accelerate Arp2/3-dependent actin nucleation (data not shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150732&req=5

Figure 5: Dot matrix comparison, schematic of the domain organization of p116 and Acan 125, alignment of the verprolin-like sequences, and acceleration of Arp2/3-dependent actin nucleation by the VA domain of p116. (A) Dot matrix comparison between Dictyostelium p116 and Acanthamoeba Acan 125 (window size, 30; stringency, 11). The dark rectangular region in the upper right corner is due to the alignment of their repetitive proline-rich sequences. (B) Schematic of the tripartite domain organization of p116 and Acan 125, showing the percent identity and the percent similarity (identities plus conservative substitutions in parentheses) for each domain. The positions of the 16 LRRs, the verprolin-like sequence that in verprolin has been implicated in binding G-actin, the acidic region, the proline-rich domain, the two PXXP motifs known to be critical for the interaction between Acan 125 and the SH3 domain of Acanthamoeba myosin IC, and the two PXXP motifs deleted from p116 in this study, are indicated. (C) Alignment of a portion of p116 and the homologous sequence in Acan 125 with a region of yeast verprolin that contributes to the binding of G-actin, and with six-residue sequences present in thymosin β4 and actobindin that also contribute to binding monomeric actin. (D) Actin nucleation assays were performed using 4 μM G-actin (5% pyrene actin) with or without the Arp2/3 complex and GST VA (see Materials and Methods). Shown are the rates of polymerization for actin alone (trace a, no symbols), actin plus 50 nM Arp2/3 complex (trace b, squares), actin plus 3 μM GST VA (trace c, open circles), and actin plus 50 nM Arp2/3 complex and 3 μM GST VA (trace d, closed circles). Similar results were obtained using two different preparations of proteins. Addition of 3 μM unfused GST did not accelerate Arp2/3-dependent actin nucleation (data not shown).
Mentions: Actin was purified from Acanthamoeba castellanii according to Gordon et al. 1976, followed by gel filtration on HiPrep Sephacryl S200 (17-1195-01; Amersham Pharmacia Biotech). Monomeric actin was labeled with N-(1-pyrene)-iodoacetamide (P29; Molecular Probes) and stored lyophil-ized at −80°C. Before use it was resuspended and dialyzed in G buffer (0.1 mM CaCl2, 0.5 mM ATP, pH 7.0, 0.75 mM β-mercaptoethanol, and 3 mM imidazole, pH 7.5). The Arp2/3 complex was purified from Acanthamoeba as described by Kelleher et al. 1998 and stored at −70°C in complex storage buffer supplemented with 200 mM sucrose. The portion of p116 (residues 644–863) that contains a short sequence conserved among G-actin binding proteins (including verprolin), plus an acidic region was amplified by PCR using Pfu polymerase and the following oligonucleotides: 5′-TCAGGATCCATTTGGAAGGAAATCGATTCATGTATC-3′ and 5′-ACTGAATTCTTAATCTGGAATTGGGGTGGCACCACT-3′. The product was digested with BamH1 and EcoR1, cloned into pGEX 2TK (27-4587-01; Amersham Pharmacia Biotech), and the fusion protein, referred to below as “GST VA,” expressed and purified as described above. Actin nucleation assays were performed essentially as described by Higgs et al. 1999. In brief, G-actin was mixed with pyrene-labeled actin at a ratio of 20:1, converted to Mg++-actin, and polymerized at a final concentration of 4 μM by the addition of 10× polymerization buffer (500 mM KCL, 10 mM MgCl2, 10 mM EGTA, and 100 mM imidazole, pH 7.0). Additional proteins (e.g., Arp2/3, GST VA; see the legend to Fig. 5) were dialyzed into G buffer supplemented with 1 mM MgCl2 and added to the actin solution just before the initiation of polymerization. Fluorescence intensities were measured every second for 10 min using a PTI QuantaMaster fluorometer (Photon Technology International) and wavelengths of 365 and 407 nm for excitation and emission, respectively.

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