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Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.

Leung CL, Sun D, Zheng M, Knowles DR, Liem RK - J. Cell Biol. (1999)

Bottom Line: However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats.More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules.The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

ABSTRACT
We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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Interaction of mACF7 with polymerized actin and MTs in vitro. (A) Actin spin-down binding assays were performed with [35S]methionine ABD and mACF7-C proteins. In vitro translated mACF7-C protein appeared as a doublet in SDS-PAGE. These two different sized proteins might be the result of degradation or alternative initiation. In the presence of polymerized actin filaments (+MF), a significant portion of ABD was found in the pellet (p). Without actin (−MF), most of the ABD remained in the supernatant (s). A small portion of mACF7-C protein was also found in the pellet, indicating weak interaction between mACF7-C protein and actin filaments. (B) Microtubule spin-down binding assays were performed with [35S]methionine-labeled ABD and mACF7-C proteins. In the presence of polymerized MTs (+MT), mACF7-C but not ABD protein cosediment with the MT pellet (p). Without MTs (−MT), both of the proteins remained in the supernatant (s).
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Figure 7: Interaction of mACF7 with polymerized actin and MTs in vitro. (A) Actin spin-down binding assays were performed with [35S]methionine ABD and mACF7-C proteins. In vitro translated mACF7-C protein appeared as a doublet in SDS-PAGE. These two different sized proteins might be the result of degradation or alternative initiation. In the presence of polymerized actin filaments (+MF), a significant portion of ABD was found in the pellet (p). Without actin (−MF), most of the ABD remained in the supernatant (s). A small portion of mACF7-C protein was also found in the pellet, indicating weak interaction between mACF7-C protein and actin filaments. (B) Microtubule spin-down binding assays were performed with [35S]methionine-labeled ABD and mACF7-C proteins. In the presence of polymerized MTs (+MT), mACF7-C but not ABD protein cosediment with the MT pellet (p). Without MTs (−MT), both of the proteins remained in the supernatant (s).

Mentions: In vitro spin-down binding assays were carried out to ascertain interactions between ABD and actin filaments, and between mACF7-C and MTs. 35S-labeled proteins were synthesized in vitro and incubated with polymerized actin and tubulin before centrifugation. The bound proteins in the spin-down pellets were resolved on SDS-PAGE and visualized by autoradiography. As demonstrated in Fig. 7, the taxol stabilized MTs pull down mACF7-C protein but not ABD protein, whereas polymerized actin filament pulls down a significant amount of ABD protein and a small amount of mACF7-C protein. The interaction between mACF7-C and polymerized tubulins is most likely direct, because no MT-associated proteins (MAPs) are present in the taxol stabilized MTs, although proteins in the reticulocyte lysate system could enhance this binding. Since polymerized actin filaments were not able to pull down a partial BPAG1 COOH-terminal protein in similar assays (data not shown), the in vitro interaction between mACF7-C and actin filaments may also be specific. However, no obvious colocalization of mACF7-C and actin structures was observed in transfection studies; therefore, it is not clear that this interaction happens in vivo.


Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.

Leung CL, Sun D, Zheng M, Knowles DR, Liem RK - J. Cell Biol. (1999)

Interaction of mACF7 with polymerized actin and MTs in vitro. (A) Actin spin-down binding assays were performed with [35S]methionine ABD and mACF7-C proteins. In vitro translated mACF7-C protein appeared as a doublet in SDS-PAGE. These two different sized proteins might be the result of degradation or alternative initiation. In the presence of polymerized actin filaments (+MF), a significant portion of ABD was found in the pellet (p). Without actin (−MF), most of the ABD remained in the supernatant (s). A small portion of mACF7-C protein was also found in the pellet, indicating weak interaction between mACF7-C protein and actin filaments. (B) Microtubule spin-down binding assays were performed with [35S]methionine-labeled ABD and mACF7-C proteins. In the presence of polymerized MTs (+MT), mACF7-C but not ABD protein cosediment with the MT pellet (p). Without MTs (−MT), both of the proteins remained in the supernatant (s).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168091&req=5

Figure 7: Interaction of mACF7 with polymerized actin and MTs in vitro. (A) Actin spin-down binding assays were performed with [35S]methionine ABD and mACF7-C proteins. In vitro translated mACF7-C protein appeared as a doublet in SDS-PAGE. These two different sized proteins might be the result of degradation or alternative initiation. In the presence of polymerized actin filaments (+MF), a significant portion of ABD was found in the pellet (p). Without actin (−MF), most of the ABD remained in the supernatant (s). A small portion of mACF7-C protein was also found in the pellet, indicating weak interaction between mACF7-C protein and actin filaments. (B) Microtubule spin-down binding assays were performed with [35S]methionine-labeled ABD and mACF7-C proteins. In the presence of polymerized MTs (+MT), mACF7-C but not ABD protein cosediment with the MT pellet (p). Without MTs (−MT), both of the proteins remained in the supernatant (s).
Mentions: In vitro spin-down binding assays were carried out to ascertain interactions between ABD and actin filaments, and between mACF7-C and MTs. 35S-labeled proteins were synthesized in vitro and incubated with polymerized actin and tubulin before centrifugation. The bound proteins in the spin-down pellets were resolved on SDS-PAGE and visualized by autoradiography. As demonstrated in Fig. 7, the taxol stabilized MTs pull down mACF7-C protein but not ABD protein, whereas polymerized actin filament pulls down a significant amount of ABD protein and a small amount of mACF7-C protein. The interaction between mACF7-C and polymerized tubulins is most likely direct, because no MT-associated proteins (MAPs) are present in the taxol stabilized MTs, although proteins in the reticulocyte lysate system could enhance this binding. Since polymerized actin filaments were not able to pull down a partial BPAG1 COOH-terminal protein in similar assays (data not shown), the in vitro interaction between mACF7-C and actin filaments may also be specific. However, no obvious colocalization of mACF7-C and actin structures was observed in transfection studies; therefore, it is not clear that this interaction happens in vivo.

Bottom Line: However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats.More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules.The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

ABSTRACT
We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

Show MeSH
Related in: MedlinePlus