BMB Reports - Manuscript Submission Manuscript Draft Manuscript Number: BMB-15-185 Title: MACF1, a versatile spectraplakin: isoforms, unique structures, and functions Article Type: Mini Review Keywords: MACF1; Spectraplakin; Isoform; Structure; Function Corresponding Author: Airong Qian Authors: Lifang Hu1, Peihong Su1, Runzhi Li1, Chong Yin1, Yan Zhang1, Peng Shang1, Tuanmin Yang2, Airong Qian1,* Institution: 1School of Life Sciences, Northwestern Polytechnical University, 2Honghui Hospital, Xi’an Jiaotong University College of Medicine, UNCORRECTED PROOF 1 Manuscript Type: Mini Review 2 3 Title: MACF1, a versatile spectraplakin: isoforms, unique structures, and functions 4 5 Author's name: Lifang Hu1, Peihong Su1, Runzhi Li1, Chong Yin1, Yan Zhang1, Peng 6 Shang1, Tuanmin Yang2 & Airong Qian1, * 7 8 Affiliation: 1Key Laboratory for Space Bioscience and Biotechnology, Institute of 9 Special Environmental Biophysics, School of Life Sciences, Northwestern 10 Polytechnical University, Xi’an, Shaanxi 710072, P. R. China, 2Honghui Hospital, 11 Xi’an Jiaotong University College of Medicine, Xi’an, Shaanxi 710054, P. R. China 12 13 Running Title: MACF1, a versatile spectraplakin 14 15 Keywords: MACF1, Spectraplakin, Isoform, Structure, Function 16 17 Corresponding Author's Information: Airong Qian, Tel: +86-29-88491840; Fax: 18 +86-29-88491840; E-mail: [email protected]; Address: 127 Youyi Xilu, Xi’an, 19 Shaanxi, 710072, P.R. China 20 21 22 UNCORRECTED1 PROOF 1 ABSTRACT 2 Spectraplakins are crucially important communicators linking cytoskeletal 3 components to each other and to cellular junctions. Microtubule actin crosslinking 4 factor 1 (MACF1), also termed actin crosslinking family 7 (ACF7), is a member of 5 spectraplakin family and is widely expressed in numerous tissues and cells. As one 6 extensively studied spectraplakin, MACF1 has several isoforms with unique 7 structures and is well-known for its capacity to crosslink F-actin and microtubules. 8 MACF1 is one versatile spectraplakin and shows functions ranging from cell 9 processes, embryo development, and tissue-specific functions to human diseases. 10 Therefore, the importance of MACF1 has become more apparent in recent years. Here, 11 we summarize current knowledge on the presence and function of MACF1 and 12 provide perspectives on the future research of MACF1, based on our studies and 13 others. 14 Keywords: MACF1, Spectraplakin, Isoform, Structure, Function 15 16 17 18 19 20 21 22 UNCORRECTED2 PROOF 1 INTRODUCTION 2 The cytoskeleton in most multicellular organisms is a highly organized, 3 interconnected network of filaments that is composed of microfilaments (F-actin), 4 microtubules (MTs), and intermediate filaments (IFs). The cytoskeletal network, as a 5 cytoplasmic scaffold, defines the cell shape and mechanical properties, and functions 6 in many other cellular events including cell polarization, migration, division, adhesion, 7 intracellular trafficking, and organelle locomotion. Thus, the cytoskeleton is both 8 static and adaptive, providing a dynamic cellular architecture that is able to remodel 9 itself to respond to cellular changes. The tight organization and dynamics of these 10 filaments strongly depends on their associated proteins. Spectraplakins have been 11 demonstrated to be the cytoskeletal cross-linkers with the ability to interact with all 12 the three types of cytoskeletal filaments (F-actin, MTs, and IFs). 13 Spectraplakins are exceptionally long, gigantic (>500 kD), multi-domain 14 cytoskeletal proteins that are master orchestrators for coordinating cytoskeletal 15 elements by binding to F-actin, MTs, and IFs (1, 2). Spectraplakins are evolutionarily 16 conserved and belong within both the spectrin superfamily and plakin superfamily (3). 17 “Spectraplakins” are named as combinations of “spectrin” and “plakin” for their 18 sharing features of both spectrins and plakins (3, 4). So far, mammalian spectraplakins 19 only comprise two members: microtubule actin crosslinking factor 1 (MACF1) and 20 bullous pemphigoid antigen 1 (BPAG1). Moreover, the spectraplakin family also 21 consists of Shot/Kakapo (in Drosophila), Magellan (in zebrafish), and Vab-10 (in 22 Caenorhabditis elegans). UNCORRECTED3 PROOF 1 MACF1, also named actin crosslinking family 7 (ACF7), MACF, macrophin, and 2 trabeculin-α, is a widely expressed critical spectraplakin (2). By crosslinking both 3 MTs and F-actin and controlling the cytoskeletal dynamics, MACF1 plays key roles 4 in regulating cell migration (5-8) and cell proliferation and in maintaining tissue 5 integrity (9, 10). In addition, MACF1 mediates signal transduction and is pivotal for 6 embryo development (11). More recently, a human disease caused by MACF1 7 mutation was reported (12). 8 In this paper, the basic molecular characteristics of MACF1 are first introduced. 9 Then, the current knowledge on MACF1 is reviewed highlighting its versatile 10 functions. Finally, we provide perspectives on the future research of MACF1 based on 11 our studies and others, providing researchers with the current status of MACF1 12 research. 13 MACF1: GENE, ISOFORM, AND TISSUE DISTRIBUTION 14 Gene and isoforms of MACF1 15 MACF1, a large ~600 kD protein was first discovered by Byers et al. in screening for 16 additional members of the actin crosslinker superfamily (13). The authors isolated a 17 partial cDNA of MACF1 using degenerate primer-mediated PCR and named it ACF7. 18 Subsequently, murine ACF7 was further characterized (14), and its full-length cDNA 19 was cloned and found to encode a 608-kD protein (15). For the association of murine 20 ACF7 with both actin and MTs, ACF7 was renamed MACF, representing microtubule 21 actin cross-linking factor. Meanwhile, the human cDNA was cloned independently by 22 two groups and was called macrophin and trabeculin-α, respectively (16, 17). UNCORRECTED4 PROOF 1 MACF1/ACF7 is encoded by MACF1 gene, which is located on human 2 chromosome 1p32 and mouse chromosome 4 (13, 14, 18). The human MACF1 gene 3 contains at least 102 exons and spans over 270 kb. Genomic organization analysis of 4 human MACF1 gene shows that MACF1 and ACF7 are the same gene (18). MACF1 5 gene is a hybrid between genes encoding plakins and spectrins/dystrophins (15, 18). 6 MACF1 shares similar gene sequences with plectin, a well-characterized plakin, 7 presenting the same exon-intron boundaries for the N-terminal actin-binding domain 8 (ABD), one large exon encoding plectin repeats, and a similar serine/glycine-rich 9 C-terminus containing GSR repeats. In addition, the spectrin repeats in MACF1 are 10 similar to those in the dystrophin gene. This hybrid gene structure of MACF1 reveals 11 its relationship with the Drosophila gene shot and the Caenorhabditis elegans gene 12 vab-10. Because of the hybrid structure of these genes and the combined features of 13 both the spectrin and plakin family members, they belong to the spectraplakin family 14 (3). 15 Like dystonin/BPAG1 (19, 20), MACF1 displays isoform diversity, and several 16 MACF1 isoforms produced by alternative splicing and different promoter usage have 17 been reported (Fig. 1). Bernier et al. first identified three murine ACF7/MACF1 18 isoforms with a common 3’ sequence but a unique 5’ region (14). Two of the isoforms 19 (ACF7-1 and ACF7-2) contain identical actin-binding domains (ABDs) but different 20 5’UTRs, while isoform 3 (ACF7-3) contains a unique 5’UTR, a longer N-terminal 21 sequence, and just the second half of the ABD (14). Subsequently, MACF1-4, a fourth 22 isoform of MACF1 was cloned (18). Different from the above three MACF1 isoforms, UNCORRECTED5 PROOF 1 MACF1-4 contains no ABD at the N-terminus, but instead consists of plectin repeats 2 (Fig. 1). Later, another murine alternatively spliced isoform of MACF1 that encodes a 3 gigantic protein of approximately 800-kD was revealed (21). The authors named this 4 isoform MACF1b and renamed the original isoform MACF1a. MACF1b harbors 5 extra plakin repeats between the plakin domain and the spectrin repeats of MACF1a 6 (Fig. 1). With the different N-terminal domain, the first three isoforms ACF7-1, 7 ACF7-2, and ACF7-3 were renamed as MACF1a1, MACF1a2, and MACF1a3 (21). 8 The Liem group further found the existence of a novel isoform of MACF1, MACF1c, 9 when studying the role of MACF1 in the nervous system by adopting MACF1a 10 conditional knockout (cKO) mice (22). MACF1c is identical to MACF1a except for 11 the absence of the N-terminal ABD domain (Fig. 1). 12 In addition to the above MACF1 isoforms, a putative MACF/MACF1 isoform, 13 MACF2, was reported by the Liem group (23). By performing a sequence homology 14 analysis, the authors found a partial human cDNA clone (KIAA0728) that encoded a 15 polypeptide with 68% amino acid identity to human MACF and called it MACF2 due 16 to the similarity. However, according to the unigene database, the authors found 17 differences in the chromosomal locations and nucleotide sequences of the transcripts 18 between MACF and MACF2, suggesting that human MACF and MACF2 represented 19 two distinct protein products from two different genes (23). Consequently, MACF2 20 was demonstrated as an isoform of BPAG1 and renamed as BPAG1-a (20). 21 Therefore, six isoforms of MACF1, including MACF1a1, MACF1a2, MACF1a3, 22 MACF1-4, MACF1b, and MACF1c (Fig. 1), have been reported at present. UNCORRECTED6 PROOF 1 MACF1’s tissue distribution 2 As a cytoskeletal linker protein, MACF1 is ubiquitously expressed with some 3 isoforms distributed in different tissues (Table 1). 4 Bernier et al. first showed that MACF1 was widely expressed in postnatal mouse 5 tissues, including brain, spinal cord, spleen, liver, heart, skeletal muscle, stomach, 6 lung, kidney, and skin, presenting the strongest expression in lung followed by brain, 7 spinal cord, cardiac/skeletal muscle, and skin (14). Their further determination 8 demonstrated the predominant expression of MACF1 in neural, muscle, and lung 9 tissue at beginning during embryonic development and continuing into adulthoods 10 (24). A striking difference is observed in tissue distribution for different MACF1 11 transcripts.