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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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Association of ACF7 COOH-terminal domain with MTs. Transient transfections of pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) were performed in COS-7 cells. Transfected cells were stained 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), and were examined by immunofluorescence microscopy. mACF7-C proteins colocalize with most of the Tyr-MTs (A and B) and all of the Glu-MTs (C and D). The noncolocalized Tyr-MTs are indicated by an arrow (B). Frequently, long MTs forming whorls were observed in pFLAG–mACF7-C transfected cells (B and D). The overexpressed ABD protein did not associate with the MT network (E and F). Bar, 20 μm.
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Figure 5: Association of ACF7 COOH-terminal domain with MTs. Transient transfections of pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) were performed in COS-7 cells. Transfected cells were stained 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), and were examined by immunofluorescence microscopy. mACF7-C proteins colocalize with most of the Tyr-MTs (A and B) and all of the Glu-MTs (C and D). The noncolocalized Tyr-MTs are indicated by an arrow (B). Frequently, long MTs forming whorls were observed in pFLAG–mACF7-C transfected cells (B and D). The overexpressed ABD protein did not associate with the MT network (E and F). Bar, 20 μm.

Mentions: Full-length Gas2 was shown previously to be a component of the MF network, although the interaction domain had not been fully characterized (Brancolini et al. 1992; Collavin et al. 1998). Therefore, we examined possible interactions between the COOH-terminal domain of mACF7 (mACF7-C) and MFs by transient transfection assays. In addition, we also analyzed the actin-binding properties of the putative NH2-terminal ABD of mACF7. To facilitate the detection of the truncated proteins, the NH2 termini of mACF7-C and ABD were fused to FLAG epitope tags, which also provided the translational start codons. Transient transfections were performed on COS-7 cells. As shown in Fig. 4A and Fig. B, overexpressed ABD protein colocalized with filamentous actin in stress fibers and membrane ruffles of transfected cells, confirming the interaction of this highly conserved domain with MFs. In contrast, the mACF7-C protein, displayed a filamentous staining pattern that exhibited no significant correlation with the actin network (Fig. 4C and Fig. D). Therefore, we compared this staining pattern with that of the other two cytoskeletal networks, MTs and IFs, and found that mACF7-C proteins codistributed with MTs (Fig. 5), but not with vimentin (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)

Association of ACF7 COOH-terminal domain with MTs. Transient transfections of pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) were performed in COS-7 cells. Transfected cells were stained 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), and were examined by immunofluorescence microscopy. mACF7-C proteins colocalize with most of the Tyr-MTs (A and B) and all of the Glu-MTs (C and D). The noncolocalized Tyr-MTs are indicated by an arrow (B). Frequently, long MTs forming whorls were observed in pFLAG–mACF7-C transfected cells (B and D). The overexpressed ABD protein did not associate with the MT network (E and F). Bar, 20 μm.
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Related In: Results  -  Collection

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

Figure 5: Association of ACF7 COOH-terminal domain with MTs. Transient transfections of pFLAG-mACF7-C (A–D) and pFLAG-ABD (E and F) were performed in COS-7 cells. Transfected cells were stained 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), and were examined by immunofluorescence microscopy. mACF7-C proteins colocalize with most of the Tyr-MTs (A and B) and all of the Glu-MTs (C and D). The noncolocalized Tyr-MTs are indicated by an arrow (B). Frequently, long MTs forming whorls were observed in pFLAG–mACF7-C transfected cells (B and D). The overexpressed ABD protein did not associate with the MT network (E and F). Bar, 20 μm.
Mentions: Full-length Gas2 was shown previously to be a component of the MF network, although the interaction domain had not been fully characterized (Brancolini et al. 1992; Collavin et al. 1998). Therefore, we examined possible interactions between the COOH-terminal domain of mACF7 (mACF7-C) and MFs by transient transfection assays. In addition, we also analyzed the actin-binding properties of the putative NH2-terminal ABD of mACF7. To facilitate the detection of the truncated proteins, the NH2 termini of mACF7-C and ABD were fused to FLAG epitope tags, which also provided the translational start codons. Transient transfections were performed on COS-7 cells. As shown in Fig. 4A and Fig. B, overexpressed ABD protein colocalized with filamentous actin in stress fibers and membrane ruffles of transfected cells, confirming the interaction of this highly conserved domain with MFs. In contrast, the mACF7-C protein, displayed a filamentous staining pattern that exhibited no significant correlation with the actin network (Fig. 4C and Fig. D). Therefore, we compared this staining pattern with that of the other two cytoskeletal networks, MTs and IFs, and found that mACF7-C proteins codistributed with MTs (Fig. 5), but not with vimentin (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