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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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Full-length mACF7 associates with actin and MTs. COS-7 cells were transiently transfected with pFLAG-mACF7-fl and triple-labeled with rhodamine-conjugated phalloidin (A and D), mouse monoclonal anti-FLAG M2 antibody (B and E), and rat monoclonal anti–Tyr-tubulin antibody (C and F). In a small population of transfected cells, overexpressed mACF7 and endogenous MTs coaligned with some of the actin stress fibers (A–C, arrows). In most cases, colocalization of mACF7, actin, and tubulin was only observed in patches. Insets in D–F show high magnifications of squared areas that contain patches. In addition, full-length mACF7 also coaligned with MTs and partially colocalized with actin at the membrane ruffles (D and E, arrows). Bar, 20 μm.
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Figure 9: Full-length mACF7 associates with actin and MTs. COS-7 cells were transiently transfected with pFLAG-mACF7-fl and triple-labeled with rhodamine-conjugated phalloidin (A and D), mouse monoclonal anti-FLAG M2 antibody (B and E), and rat monoclonal anti–Tyr-tubulin antibody (C and F). In a small population of transfected cells, overexpressed mACF7 and endogenous MTs coaligned with some of the actin stress fibers (A–C, arrows). In most cases, colocalization of mACF7, actin, and tubulin was only observed in patches. Insets in D–F show high magnifications of squared areas that contain patches. In addition, full-length mACF7 also coaligned with MTs and partially colocalized with actin at the membrane ruffles (D and E, arrows). Bar, 20 μm.

Mentions: To further characterize the function of mACF7, a COOH-terminal FLAG-tagged full-length mACF7 construct was prepared for transient transfections. In some transfected cells, we observed partial coalignment of mACF7, MTs, and actin filaments (Fig. 9, A–C, arrows). However, in most cases, colocalization of mACF7, MTs, and actin was only observed in patches (Fig. 9, insets). These differences appear to be due to the differential expression levels of mACF7 in the transfected cells. Cells expressing lower levels of full-length mACF7 had more stress fibers that colocalized with mACF7. A subset of these stress fibers also colocalized with MTs (Fig. 9, A–C). In contrast, in most transfected cells there were few actin stress fibers (Fig. 9 D). In addition, mACF7 also colocalized with actin at membrane ruffles (Fig. 9D and Fig. E, arrows) and decorated all the MTs. These data show that mACF7 can bind to both actin filaments and MTs, and may have the ability to cross-link these cytoskeletal elements. Since vimentin has also been shown to associate with MTs (Gurland and Gundersen 1995), we studied the distribution of vimentin in mACF7-overexpressing cells, but found no obvious colocalization of IFs with mACF7 (data not shown).


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)

Full-length mACF7 associates with actin and MTs. COS-7 cells were transiently transfected with pFLAG-mACF7-fl and triple-labeled with rhodamine-conjugated phalloidin (A and D), mouse monoclonal anti-FLAG M2 antibody (B and E), and rat monoclonal anti–Tyr-tubulin antibody (C and F). In a small population of transfected cells, overexpressed mACF7 and endogenous MTs coaligned with some of the actin stress fibers (A–C, arrows). In most cases, colocalization of mACF7, actin, and tubulin was only observed in patches. Insets in D–F show high magnifications of squared areas that contain patches. In addition, full-length mACF7 also coaligned with MTs and partially colocalized with actin at the membrane ruffles (D and E, arrows). Bar, 20 μm.
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Related In: Results  -  Collection

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

Figure 9: Full-length mACF7 associates with actin and MTs. COS-7 cells were transiently transfected with pFLAG-mACF7-fl and triple-labeled with rhodamine-conjugated phalloidin (A and D), mouse monoclonal anti-FLAG M2 antibody (B and E), and rat monoclonal anti–Tyr-tubulin antibody (C and F). In a small population of transfected cells, overexpressed mACF7 and endogenous MTs coaligned with some of the actin stress fibers (A–C, arrows). In most cases, colocalization of mACF7, actin, and tubulin was only observed in patches. Insets in D–F show high magnifications of squared areas that contain patches. In addition, full-length mACF7 also coaligned with MTs and partially colocalized with actin at the membrane ruffles (D and E, arrows). Bar, 20 μm.
Mentions: To further characterize the function of mACF7, a COOH-terminal FLAG-tagged full-length mACF7 construct was prepared for transient transfections. In some transfected cells, we observed partial coalignment of mACF7, MTs, and actin filaments (Fig. 9, A–C, arrows). However, in most cases, colocalization of mACF7, MTs, and actin was only observed in patches (Fig. 9, insets). These differences appear to be due to the differential expression levels of mACF7 in the transfected cells. Cells expressing lower levels of full-length mACF7 had more stress fibers that colocalized with mACF7. A subset of these stress fibers also colocalized with MTs (Fig. 9, A–C). In contrast, in most transfected cells there were few actin stress fibers (Fig. 9 D). In addition, mACF7 also colocalized with actin at membrane ruffles (Fig. 9D and Fig. E, arrows) and decorated all the MTs. These data show that mACF7 can bind to both actin filaments and MTs, and may have the ability to cross-link these cytoskeletal elements. Since vimentin has also been shown to associate with MTs (Gurland and Gundersen 1995), we studied the distribution of vimentin in mACF7-overexpressing cells, but found no obvious colocalization of IFs with mACF7 (data not shown).

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