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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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Overexpression of mACF7 COOH-terminal domain stabilizes cellular MTs. pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) and transfected COS-7 cells were treated with nocodazole (10 μM) for 1.5 h before being fixed and double-labeled with monoclonal anti-FLAG M2 antibody (A, C, and E) polyclonal anti–Tyr-tubulin antibody (B), polyclonal anti–Glu-tubulin antibody (D), and polyclonal antitubulin antibody (F). The overexpressed mACF7-C protein stabilized MTs from depolymerization by nocodazole (compare positive FLAG-staining cell with its neighboring cells in A and B). Bar, 20 μm.
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Figure 6: Overexpression of mACF7 COOH-terminal domain stabilizes cellular MTs. pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) and transfected COS-7 cells were treated with nocodazole (10 μM) for 1.5 h before being fixed and double-labeled with monoclonal anti-FLAG M2 antibody (A, C, and E) polyclonal anti–Tyr-tubulin antibody (B), polyclonal anti–Glu-tubulin antibody (D), and polyclonal antitubulin antibody (F). The overexpressed mACF7-C protein stabilized MTs from depolymerization by nocodazole (compare positive FLAG-staining cell with its neighboring cells in A and B). Bar, 20 μm.

Mentions: Within each cell there are dynamic and stable MTs. Stable MTs are a small subset of dynamic MTs and are thought to be selectively generated from dynamic MTs. These stable long-lived MTs accumulate a posttranslationally modified form of tubulins known as detyrosinated or Glu-tubulins. These MTs are distinct from their dynamic counterparts that contain predominantly tyrosinated tubulin (Tyr-tubulin). In transfected cells, the overexpressed mACF7-C proteins colocalized with many but not all Tyr-MTs (Fig. 5A and Fig. B). Interestingly, mACF7-C proteins (Fig. 5C and Fig. D) decorated all the Glu MTs. The dynamic Tyr MTs at the periphery of the cell did not appear to colocalize with mACF-7, although it is possible that the bound mACF-7 was present in low amounts too scarce to be detected. As expected, the ABD of mACF7 did not associate with MTs (Fig. 5E and Fig. F). Since long MT-forming whorls were frequently observed in the transfected cells, we considered the possibility that mACF7-C proteins might not only bind to, but also stabilize MTs. To explore this possibility, cells transfected with mACF7-C cDNA were treated with the MT depolymerization agent, nocodazole (10 μM) for 1.5 h before being fixed for immunofluorescence microscopy. These conditions have been shown to be sufficient to cause the complete depolymerization of the endogenous MTs (Khawaja et al. 1988). In contrast to cells without mACF7-C protein, the MT networks of the transfected cells remained intact and were decorated with mACF7-C proteins (Fig. 6, A–D), implying that the COOH-terminal domain of mACF7 can associate with and stabilize MTs. In similar assays, the ABD of mACF7 still only associated with the actin network (Fig. 6E and Fig. F).


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)

Overexpression of mACF7 COOH-terminal domain stabilizes cellular MTs. pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) and transfected COS-7 cells were treated with nocodazole (10 μM) for 1.5 h before being fixed and double-labeled with monoclonal anti-FLAG M2 antibody (A, C, and E) polyclonal anti–Tyr-tubulin antibody (B), polyclonal anti–Glu-tubulin antibody (D), and polyclonal antitubulin antibody (F). The overexpressed mACF7-C protein stabilized MTs from depolymerization by nocodazole (compare positive FLAG-staining cell with its neighboring cells in A and B). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Overexpression of mACF7 COOH-terminal domain stabilizes cellular MTs. pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) and transfected COS-7 cells were treated with nocodazole (10 μM) for 1.5 h before being fixed and double-labeled with monoclonal anti-FLAG M2 antibody (A, C, and E) polyclonal anti–Tyr-tubulin antibody (B), polyclonal anti–Glu-tubulin antibody (D), and polyclonal antitubulin antibody (F). The overexpressed mACF7-C protein stabilized MTs from depolymerization by nocodazole (compare positive FLAG-staining cell with its neighboring cells in A and B). Bar, 20 μm.
Mentions: Within each cell there are dynamic and stable MTs. Stable MTs are a small subset of dynamic MTs and are thought to be selectively generated from dynamic MTs. These stable long-lived MTs accumulate a posttranslationally modified form of tubulins known as detyrosinated or Glu-tubulins. These MTs are distinct from their dynamic counterparts that contain predominantly tyrosinated tubulin (Tyr-tubulin). In transfected cells, the overexpressed mACF7-C proteins colocalized with many but not all Tyr-MTs (Fig. 5A and Fig. B). Interestingly, mACF7-C proteins (Fig. 5C and Fig. D) decorated all the Glu MTs. The dynamic Tyr MTs at the periphery of the cell did not appear to colocalize with mACF-7, although it is possible that the bound mACF-7 was present in low amounts too scarce to be detected. As expected, the ABD of mACF7 did not associate with MTs (Fig. 5E and Fig. F). Since long MT-forming whorls were frequently observed in the transfected cells, we considered the possibility that mACF7-C proteins might not only bind to, but also stabilize MTs. To explore this possibility, cells transfected with mACF7-C cDNA were treated with the MT depolymerization agent, nocodazole (10 μM) for 1.5 h before being fixed for immunofluorescence microscopy. These conditions have been shown to be sufficient to cause the complete depolymerization of the endogenous MTs (Khawaja et al. 1988). In contrast to cells without mACF7-C protein, the MT networks of the transfected cells remained intact and were decorated with mACF7-C proteins (Fig. 6, A–D), implying that the COOH-terminal domain of mACF7 can associate with and stabilize MTs. In similar assays, the ABD of mACF7 still only associated with the actin network (Fig. 6E and Fig. F).

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