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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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mACF7-mini cross-links MFs and MTs. pFLAG-mACF7-mini, encoding a chimeric protein of ABD and the COOH-terminal domain of mACF7, was transfected into COS-7 cells. Transfected cells were triple-labeled with rhodamine-conjugated phalloidin (A), mouse monoclonal anti-FLAG M2 antibody (B), and rat monoclonal anti–Tyr-tubulin antibody (C). Cells expressing mACF7-mini contain more actin filaments that are morphologically distinct from stress fibers. These actin filaments colocalized with mACF7-mini and MTs, demonstrating the MF-MT cross-linking property of mACF7-mini. Bar, 20 μm.
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Figure 8: mACF7-mini cross-links MFs and MTs. pFLAG-mACF7-mini, encoding a chimeric protein of ABD and the COOH-terminal domain of mACF7, was transfected into COS-7 cells. Transfected cells were triple-labeled with rhodamine-conjugated phalloidin (A), mouse monoclonal anti-FLAG M2 antibody (B), and rat monoclonal anti–Tyr-tubulin antibody (C). Cells expressing mACF7-mini contain more actin filaments that are morphologically distinct from stress fibers. These actin filaments colocalized with mACF7-mini and MTs, demonstrating the MF-MT cross-linking property of mACF7-mini. Bar, 20 μm.

Mentions: To investigate whether mACF7 might be able to cross-link MFs and MTs in vivo, a construct (pFLAG-mACF7-mini) encoding for a chimeric protein, that contained the ABD and the COOH-terminal domain of mACF7 connected by a FLAG epitope tag, was used for transient transfection studies. Triple-labeling with phalloidin, anti-FLAG, and antitubulin was performed and the results are shown in Fig. 8. Similar to cells expressing mACF7-C protein, long MT-forming whorls and bundles were observed in cells transfected with pFLAG-mACF7-mini. In contrast to the straight stress fibers and membrane ruffles stained by phalloidin in nontransfected cells, many curly actin filaments were detected in cells expressing mACF7-mini. These unusual actin filaments were perfectly colocalized with MTs and decorated with mACF7-mini (Fig. 8). These results show that mACF7-mini could enhance the formation of actin filaments and efficiently cross-link them to MTs.


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)

mACF7-mini cross-links MFs and MTs. pFLAG-mACF7-mini, encoding a chimeric protein of ABD and the COOH-terminal domain of mACF7, was transfected into COS-7 cells. Transfected cells were triple-labeled with rhodamine-conjugated phalloidin (A), mouse monoclonal anti-FLAG M2 antibody (B), and rat monoclonal anti–Tyr-tubulin antibody (C). Cells expressing mACF7-mini contain more actin filaments that are morphologically distinct from stress fibers. These actin filaments colocalized with mACF7-mini and MTs, demonstrating the MF-MT cross-linking property of mACF7-mini. Bar, 20 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168091&req=5

Figure 8: mACF7-mini cross-links MFs and MTs. pFLAG-mACF7-mini, encoding a chimeric protein of ABD and the COOH-terminal domain of mACF7, was transfected into COS-7 cells. Transfected cells were triple-labeled with rhodamine-conjugated phalloidin (A), mouse monoclonal anti-FLAG M2 antibody (B), and rat monoclonal anti–Tyr-tubulin antibody (C). Cells expressing mACF7-mini contain more actin filaments that are morphologically distinct from stress fibers. These actin filaments colocalized with mACF7-mini and MTs, demonstrating the MF-MT cross-linking property of mACF7-mini. Bar, 20 μm.
Mentions: To investigate whether mACF7 might be able to cross-link MFs and MTs in vivo, a construct (pFLAG-mACF7-mini) encoding for a chimeric protein, that contained the ABD and the COOH-terminal domain of mACF7 connected by a FLAG epitope tag, was used for transient transfection studies. Triple-labeling with phalloidin, anti-FLAG, and antitubulin was performed and the results are shown in Fig. 8. Similar to cells expressing mACF7-C protein, long MT-forming whorls and bundles were observed in cells transfected with pFLAG-mACF7-mini. In contrast to the straight stress fibers and membrane ruffles stained by phalloidin in nontransfected cells, many curly actin filaments were detected in cells expressing mACF7-mini. These unusual actin filaments were perfectly colocalized with MTs and decorated with mACF7-mini (Fig. 8). These results show that mACF7-mini could enhance the formation of actin filaments and efficiently cross-link them to MTs.

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