Title: Constitutive activation of <scp>DIA</scp> 1 ( <scp>DIAPH</scp> 1) via C‐terminal truncation causes human sensorineural hearing loss
Abstract: Research Article5 October 2016Open Access Source DataTransparent process Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss Takehiko Ueyama Corresponding Author Takehiko Ueyama [email protected] orcid.org/0000-0002-5647-3937 Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan Search for more papers by this author Yuzuru Ninoyu Yuzuru Ninoyu orcid.org/0000-0002-9407-4697 Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan Search for more papers by this author Shin-ya Nishio Shin-ya Nishio Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Japan Search for more papers by this author Takushi Miyoshi Takushi Miyoshi Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Hiroko Torii Hiroko Torii Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Koji Nishimura Koji Nishimura Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Kazuma Sugahara Kazuma Sugahara Department of Otolaryngology, Yamaguchi University Graduate School of Medicine, Ube, Japan Search for more papers by this author Hideaki Sakata Hideaki Sakata Kawagoe Otology Institute, Kawagoe, Japan Search for more papers by this author Dean Thumkeo Dean Thumkeo Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan Search for more papers by this author Hirofumi Sakaguchi Hirofumi Sakaguchi Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan Search for more papers by this author Naoki Watanabe Naoki Watanabe Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan Laboratory of Single-Molecule Cell Biology, Kyoto University Graduate School of Biostudies, Kyoto, Japan Search for more papers by this author Shin-ichi Usami Shin-ichi Usami Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Japan Search for more papers by this author Naoaki Saito Naoaki Saito Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan Search for more papers by this author Shin-ichiro Kitajiri Corresponding Author Shin-ichiro Kitajiri [email protected] Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Takehiko Ueyama Corresponding Author Takehiko Ueyama [email protected] orcid.org/0000-0002-5647-3937 Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan Search for more papers by this author Yuzuru Ninoyu Yuzuru Ninoyu orcid.org/0000-0002-9407-4697 Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan Search for more papers by this author Shin-ya Nishio Shin-ya Nishio Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Japan Search for more papers by this author Takushi Miyoshi Takushi Miyoshi Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Hiroko Torii Hiroko Torii Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Koji Nishimura Koji Nishimura Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Kazuma Sugahara Kazuma Sugahara Department of Otolaryngology, Yamaguchi University Graduate School of Medicine, Ube, Japan Search for more papers by this author Hideaki Sakata Hideaki Sakata Kawagoe Otology Institute, Kawagoe, Japan Search for more papers by this author Dean Thumkeo Dean Thumkeo Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan Search for more papers by this author Hirofumi Sakaguchi Hirofumi Sakaguchi Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan Search for more papers by this author Naoki Watanabe Naoki Watanabe Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan Laboratory of Single-Molecule Cell Biology, Kyoto University Graduate School of Biostudies, Kyoto, Japan Search for more papers by this author Shin-ichi Usami Shin-ichi Usami Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Japan Search for more papers by this author Naoaki Saito Naoaki Saito Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan Search for more papers by this author Shin-ichiro Kitajiri Corresponding Author Shin-ichiro Kitajiri [email protected] Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Author Information Takehiko Ueyama *,1, Yuzuru Ninoyu1, Shin-ya Nishio2, Takushi Miyoshi3, Hiroko Torii3, Koji Nishimura3, Kazuma Sugahara4, Hideaki Sakata5, Dean Thumkeo6, Hirofumi Sakaguchi7, Naoki Watanabe8,9, Shin-ichi Usami2, Naoaki Saito1 and Shin-ichiro Kitajiri *,3 1Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan 2Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Japan 3Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 4Department of Otolaryngology, Yamaguchi University Graduate School of Medicine, Ube, Japan 5Kawagoe Otology Institute, Kawagoe, Japan 6Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan 7Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan 8Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan 9Laboratory of Single-Molecule Cell Biology, Kyoto University Graduate School of Biostudies, Kyoto, Japan *Corresponding author. Tel: +81 78 803 5962; Fax: +81 78 803 5971; E-mail: [email protected] *Corresponding author. Tel: +81 75 751 3346; Fax: +81 75 751 7225; E-mail: [email protected] EMBO Mol Med (2016)8:1310-1324https://doi.org/10.15252/emmm.201606609 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C-terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N-terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient-derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR1204–1207) at the DAD C-terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID-DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG-tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID-DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1. Synopsis A novel clinical DIAPH1 mutation (c.3610C>T) is found in two unrelated families, resulting in a constitutively active DIA1. Patients and mice expressing the DIA1(R1204X) protein show progressive hearing loss beginning in the high-frequency range and mimicking a novel DFNA1 subtype. A novel DIAPH1 mutation results in early termination of DIA1 at the diaphanous autoregulatory domain (DAD), R1204X. DIA1(R1204X) disrupts the diaphanous inhibitory domain (DID)–DAD interaction and is constitutively active. Mice expressing DIA1(R1204X) show progressive deafness and OHC loss at the basal turn. DIA1(R1204X) causes a novel DFNA1 subtype showing progressive hearing loss beginning in the high-frequency range and in childhood. Introduction In the past two decades, extensive research on the genetics of nonsyndromic hereditary deafness (NSHD) has been conducted, leading to the discovery of about 100 genes essential for hearing (http://hereditaryhearingloss.org/): about 30 of these encode proteins that interact directly or indirectly with actin (Dror & Avraham, 2010; Drummond et al, 2012). Stereocilia, which are deflected by sound stimulation and are thus the key structures for hearing, are actin-based protrusions and exquisitely organized microvilli/filopodia composed of hundreds of parallel actin filaments with the plus ends at the distal tip; they are organized into precise rows of graded height (characteristic staircase patterns) on the apical surface of cochlear hair cells (HCs) (Frolenkov et al, 2004). Cochlear HCs are arranged in a single row of inner HCs (IHCs) and three rows of outer HCs (OHCs). Although IHCs and OHCs are believed to share common mechanotransduction machinery, they have distinct roles during sound detection: IHCs are true sensors, whereas OHCs function as amplifiers through an active process that involves stereociliary and somatic motility (Schwander et al, 2010). The high sensitivity of HCs depends on the coordinated/synchronized movement of stereocilia upon mechanical stimulation; thus, the precise organization of the length and shape of stereocilia, which is regulated by a large battery of genes, is indispensable. Fifteen formin proteins, which nucleate and elongate unbranched/straight actin, are found in humans and can be classified into eight subfamilies (Campellone & Welch, 2010). The diaphanous-related formin (DRF) subfamily contains proteins commonly referred to as DIAs (also known as DIAPHs and mammalian homologs of Drosophila Diaphanous), which are the best-characterized class of formins. DIAPH1 gene encodes one of three DIA isoforms, DIA1 (DIAPH1). A mutation in DIAPH1 (c.3634+1G>T), in which the canonical splicing donor sequence is disrupted (AAGgtaagt becomes AAGttaagt), moves the splicing donor site to a cryptic site four base pairs (bp) downstream of the original site, resulting in a 4 bp (ttaa) insertion in the gene transcript. The insertion results in a frameshift near, but outside, the C-terminal end of the diaphanous autoregulatory domain (DAD) of DIA1, hereafter referred to as DIA1(ttaa). The frameshift introduces 21 aberrant amino acids (aa) and removes 32 aa of wild-type (WT) DIA1 (p.Ala1212ValfsX22) (Fig 1A) and causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1 (Lynch et al, 1997). DIAs have a modular domain organization consisting of the GTPase-binding domain (GBD), a partially overlapping N-terminal diaphanous inhibitory domain (DID), formin homology (FH) domains FH1 and FH2, and the C-terminal DAD (Fig 1A). Through an autoinhibitory intramolecular interaction between the DID and the DAD, which is regulated by Rho family GTPases (Amano et al, 2010), DIAs are held inactive in the resting state (Watanabe et al, 1999; Campellone & Welch, 2010; Kuhn & Geyer, 2014). DRFs including mouse Dia1 show directional movement over the distance of several micrometers in cells as they processively elongate actin filaments via their FH2 domain (Watanabe & Mitchison, 2002; Higashida et al, 2004). Given the location of the mutation, it has been speculated that disruption of the autoinhibitory intramolecular DID-DAD interaction, and consequent activation of DIA1, might underlie DFNA1 pathology. However, it is also possible that the DIA1(ttaa) mutation might compromise other functions of DIA1 that are not related to autoregulation. Thus, the underlying pathogenesis of DFNA1 remains to be determined at the molecular level. Figure 1. Structural domains of DIA1 and patients with a novel DFNA1 mutation The positions of M1190D, c.3610C>T (p.R1204X), and c.3634+1G>T (p.A1212VfsX22) with the latter being the only mutation previously associated with DFNA1. Bidirectional arrow shows the autoinhibitory intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous autoregulatory domain (DAD). GBD, GTPase-binding domain; FH1 and FH2, formin homology domain 1 and 2. Electropherograms of DNA sequencing, family pedigrees, and audiograms of two unrelated patients. Electropherograms show a heterozygous c.3610C>T substitution resulting in a stop codon (R1204X). Arrows in family pedigrees indicate the patients identified in the present study. In audiograms, red/pink and blue indicate right and left, respectively; solid and dashed lines show air conduction hearing; and square brackets show bone conduction hearing. The bold line (at the age of 48 years) and thin line (at the age of 50 years) in the audiogram of patient 1 indicate progressive hearing loss (green arrow). Download figure Download PowerPoint Patients in the original DFNA1 pedigree demonstrate mild- and low-frequency deafness starting in childhood (about 10 years of age), which develops into profound deafness involving all frequencies by adulthood (Lynch et al, 1997). Although DFNA1 was thought to originate due to sensorineural defects, evaluation of a young (8-year-old) DFNA1 patient with mild- and low-frequency deafness revealed a normal auditory brainstem response (ABR). It was therefore suggested that endolymphatic hydrops was involved in the early pathophysiology of the condition (Lalwani et al, 1998). Moreover, to date DFNA1 has only been reported in one Costa Rican family (called the "M" family because of the founder name, Monge). Thus, which of the two proposed underlying mechanisms is responsible for the development and clinical manifestations of DFNA1 remains unclear. Here, we identified a novel DIAPH1 point mutation (c.3610C>T), which results in early termination just before four sequential basic amino acids (RRKR1204–1207) in the DAD, p.R1204X. This mutation (which we define as "R1204X") was found in two unrelated Japanese families and showed autosomal dominant progressive deafness in the high-frequency range that began in childhood. DIA1(R1204X) behaved as a constitutively active DIA1 mutant, as the mutation disrupted the autoinhibitory intramolecular DID-DAD interaction. This was associated with frequent directional actin polymerization movement and an increased number of elongated microvilli in cells. Furthermore, transgenic (TG) mice expressing FLAG-tagged DIA1(R1204X) showed progressive deafness that began in the high-frequency range in parallel with cochlear HC loss, predominantly in OHCs at the basal turn. Stereocilia were sparse and abnormally short, elongated, and fused. Comparison of in vivo models, including the novel and clinically relevant one we present here, will provide further insight into the molecular mechanisms that underlie the development of DFNA1 and the diverse clinical symptoms with which it is associated. Results Patients By performing genomic sequencing, we identified a novel DIAPH1 point mutation (c.3610C>T) generating the DIA1(R1204X) mutant, which has an early translation termination site just before four sequential basic amino acids (RRKR1204–1207) in the C-terminal end of the DAD (Fig 1A and B). The location of the mutation is near the 4-bp (ttaa) insertion in DIA1 (DIAPH1) mRNA causing original DFNA1, but the ttaa insertion itself is located outside the DAD. This mutation was found in two unrelated Japanese families. Family pedigrees of both patients showed an autosomal dominant inheritance of deafness (Fig 1B). Patient 1 had a history of hearing loss that started in her childhood. She experienced slowly progressive deafness and was referred to an otologist at the age of 48. Magnetic resonance imaging of her brain, including the inner ear and VIII nerve, showed no abnormalities. Her audiograms (both air and bone conduction) showed bilateral deafness, predominantly in the high-frequency range, although all frequencies were affected (Fig 1B). Two years after her admission, her hearing deteriorated significantly, particularly in the low-frequency range (Fig 1B). Patient 2 passed newborn hearing screening. She felt progressive deafness starting around 3 years old and was referred to an otologist at the age of 6. Her audiograms (both air and bone conduction) showed bilateral and high-tone deafness (Fig 1B). Elongation of microvilli by DIA1(R1204X) mutant To examine the effect of DIA1(R1204X) on stereocilia, which are an exquisitely organized subtype of microvilli/filopodia, we studied the microvilli of MDCK cells cultured on membrane inserts. Because of the difficulties in observing and comparing the length of microvilli in reconstituted lateral images obtained by a confocal laser microscope, we established MDCK cells stably expressing mCherry-ESPIN1, which have elongated microvilli (about 2–3 μm) labeled by the mCherry red fluorescent protein. GFP-DIA1(M1190D), a constitutively active DIA1 mutant (Lammers et al, 2005), was localized to microvilli, induced microvilli elongation and altered cell shape (Fig 2). Unlike GFP-DIA1(M1190D), the GFP-DIA1(R1204X) mutant was not discretely localized to microvilli, although it induced elongated and congested microvilli compared with surrounding nontransfected cells and WT GFP-DIA1-transfected cells (Fig 2). These results suggest that DIA1(R1204X) may be a constitutively active mutant of DIA1, albeit with weaker activity than DIA1(M1190D). Figure 2. DIA1(R1204X) induces elongation of microvilli in MDCKmCherry-ESPIN1 cellsGFP-tagged DIA1, DIA1(M1190D), and DIA1(R1204X) plasmids were transfected into MDCKmCherry-ESPIN1 cells cultured on membrane inserts. Twenty-four hours after transfection, a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope. Arrows indicate the elongated microvilli visualized by mCherry-ESPIN1 with GFP-hDIA1(R1204X) expression. Scale bars: 5 μm. Representative of three experiments. Download figure Download PowerPoint Induction of elongated microvilli by DIA1(R1204X) To confirm that DIA1(R1204X) is a mildly active DIA1 mutant, we utilized HeLa cells, which have previously been used in studies of DIA/Dia (Watanabe et al, 1999; Sakamoto et al, 2012). Although GFP-DIA1(M1190D) localized to the plasma membrane (PM) and induced stress fiber (SF) formation, GFP-DIA1, GFP-DIA1(R1204X), or GFP-DIA1(ttaa) induced no remarkable SF formation (Fig 3A). However, GFP-DIA1(R1204X) had a dot-like localization adjacent to the PM (Fig 3A). Furthermore, 3D imaging revealed that GFP-DIA1(R1204X) localized to microvilli, and induced elongated and thick microvilli on the surface (Fig 3B, Movie EV1). Nontransfected cells (Fig 3B, Movie EV1) or those expressing GFP-DIA1 or GFP-DIA1(ttaa) (Movies EV2 and EV3) had no effect on microvilli phenotypes. Statistical analysis revealed that the longest microvilli in GFP-DIA1(R1204X)-expressing cells were significantly longer than those in GFP-DIA1-expressing and nontransfected cells [R1204X (n = 10): 6.25 ± 0.53 μm, WT (n = 10): 3.09 ± 0.23 μm, nontransfected: 3.12 ± 0.22 (n = 10); five independent experiments; P < 0.0001 (between WT and R1204X, nontransfected and R1204X) by one-way ANOVA followed by Bonferroni's post hoc test]. These results confirm that DIA1(R1204X) is a mildly active DIA1 that alters microvilli phenotypes, but that has no remarkable effects on SF formation. Figure 3. DIA1(R1204X) induces elongated and thick microvilli in HeLa cellsVarious GFP-tagged DIA1 plasmids were transfected into HeLa cells and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (blue) (A, B: representative of five experiments). Stress fiber formation was observed with a confocal laser microscope. Note the induction of stress fibers by GFP-DIA1(M1190D) (asterisks). Arrow and arrowheads indicate a plasma membrane (PM) localization of GFP-DIA1(M1190D) and a dot-like localization of GFP-DIA1(R1204X) adjacent to the PM, respectively. Inset in the top panel of DIA1(R1204X) shows the magnified image of the region indicated by the rectangle. Scale bars: 10 μm. Microvilli with or without GFP-DIA1(R1204X) expression were observed under a confocal laser microscope. Note elongated and thick microvilli (arrowheads) in GFP-DIA1(R1204X)-transfected cells, but not in nontransfected cells (circles). 3D movies are available in Movie EV1 (R1204X), Movie EV2 (WT), and Movie EV3 (ttaa). Scale bar: 10 μm. Download figure Download PowerPoint Disrupted intramolecular DID-DAD interaction in DIA1(R1204X) To further confirm that DIA1(R1204X) is constitutively active due to the disrupted autoinhibitory interaction between the DID and the DAD, we performed pull-down assays using GST-tagged DID and biotin-tagged DADs: DAD(WT), DAD(R1204X), and DAD(M1190D) (Fig 4A). The interaction between GST-DID and biotin-DAD(R1204X) was significantly weaker than that between GST-DID and biotin-DAD(WT), and similar to that between GST-DID and biotin-DAD(M1190D) (Fig 4B). These results suggest that the DIA1(R1204X) mutation disrupts the autoinhibitory intramolecular DID-DAD interaction, thereby rendering it constitutively active. However, DIA1(R1204X) was less active than DIA(M1190D) in cells, suggesting that factors other than the DID-DAD interaction may also regulate DIA1 activity. Figure 4. R1204X mutation in the DAD disrupts the interaction between the DID and the DAD Amino acid sequences of the biotin-labeled WT-, R1204X-, and M1190D-DAD. Underlined and red letters indicate the consensus motif (MDxLLxxL, essential for DID binding) and the unstructured basic region (enhances DID binding), respectively (Lammers et al, 2005). Purified GST and GST-tagged DID proteins (50 nM) were mixed with biotin-labeled DADs (WT, R1204X, and M1190D; 50 nM) in binding buffer. After rotation, streptavidin-coupled magnetic beads were added to the solution, and the mixture was agitated. The material absorbed to the beads was eluted in Laemmli sample buffer and was subjected to SDS–PAGE, followed by immunoblotting using an HRP-conjugated GST Ab. Comparable levels of input (GST proteins) are confirmed in the left panel. Representative of five experiments. Source data are available online for this figure. Source Data for Figure 4B [emmm201606609-sup-0010-SDataFig4.pptx] Download figure Download PowerPoint Importance of electrostatic interaction for the DID-DAD interaction An acidic groove formed by acidic aa of the DID (E358, D361, E362, and D366 in mouse Dia1; E358, E361, E362, and D366 in human DIA1) has been identified by crystal structure analysis (Lammers et al, 2008; Nezami et al, 2010). Given that the DAD has four sequential basic aa (RRKR1204–1207 in DIA1; RRKR is conserved in Dia1) at its C-terminus, electrostatic interactions may play an important role in the intramolecular DID-DAD interaction (Fig EV1A) (Lammers et al, 2008; Nezami et al, 2010; Kuhn & Geyer, 2014). Based on the prediction that exchange of RRKR1204–1207 into EEEE would render DIA1 more active than DIA1(R1204X), we constructed a mutant DIA1(RRKR1204–1207/EEEEX) (Fig EV1A). GFP-DIA1(RRKR1204–1207/EEEEX) was localized to both the PM and microvilli, and its expression induced both microvilli elongation and SF formation (Fig EV1B, Movie EV4). These data suggest that this mutant is more active than DIA1(R1204X) and instead has similar activity to DIA1(M1190D) (Fig 3A). Click here to expand this figure. Figure EV1. DIA1(RRKR1204–1207/EEEEX) induces elongated microvilli and enhances stress fiber formation in HeLa cells Illustrations of WT DIA1 (contains four basic amino acids at the DAD C-terminus), and the R1204X (basic amino acids absent) and RRKR1204–1207/EEEEX (basic-to-acidic amino acid swaps) mutants. GFP-tagged DIA1(RRKR1204–1207/EEEEX) was transfected into HeLa cells and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (blue) and observed under a confocal laser microscope. Note the localization of GFP-DIA1(RRKR1204–1207/EEEEX) to the plasma membrane (arrow) and microvilli (double arrows) and induction of enhanced stress fiber (asterisks) and elongated/thick microvillus formation (arrowheads) by GFP-DIA1(RRKR1204–1207/EEEEX) expression, compared with that in nonexpressing cells (circles). Scale bars: 10 μm. 3D movie is available in Movie EV4. Representative of five experiments. Download figure Download PowerPoint To further confirm that electrostatic interactions play critical roles in the DID-DAD interaction, we made three additional mutants: DIA1(RRKR1204–1207/EEEX), DIA1(RRKR1204–1207/EEX), and DIA1(RRKR1204–1207/EX) (Fig EV2A). PM localization was used as surrogate marker of activation, because active mutants, including DIA1(M1190D) and DIA1(RRKR1204–1207/EEEEX), are localized to the PM. DIA1(RRKR1204–1207/EEEX) and DIA1(RRKR1204–1207/EEX) were localized to the PM, whereas DIA1(RRKR1204–1207/EX) and DIA1(R1204X) showed a dot-like localization in the adjacent area to the PM. The degree to which each of the four mutants localized to the PM (or areas adjacent to the PM) depended on the number of acidic aa: DIA1(RRKR1204–1207/EEEEX) ≥ DIA1(RRKR1204–1207/EEEX) > DIA1(RRKR1204–1207/EEX) > DIA1(RRKR1204–1207/EX) > DIA1(R1204X) > WT DIA1 (Fig EV2B). These results indicate that electrostatic interactions play critical roles in the DID-DAD interaction, and that DIA1(R1204X) is a mildly active DIA1 mutant. Click here to expand this figure. Figure EV2. R1204X mutation in the DAD generates a mildly active DIA1, and the C-terminal aa (56 aa) after the DAD is a negative regulator of the intramolecular DID-DAD interaction Illustrations of the C-terminal truncation DIA1 mutants with 0–4 acidic amino acids (aa) at the DAD C-terminus. GFP-tagged WT DIA1 and mutants containing 0–4 acidic aa substitutions were transfected into HeLa cells. Twenty-four hours after transfection, plasma membrane (PM) localization was observed using a confocal laser microscope. The degree of PM localization (arrows) is shown from left to right: RRKR1204–1207/EEEEX (strongest) and WT (no PM localization). Note dot-like localization in areas adjacent to the PM (arrowheads) in GFP-RRKR1204–1207/EX- and GFP-R1204X-expressing cells. Scale bars: 10 μm. Representative of five experiments. Illustrations of DIA1 mutants with mutation of the acidic amino acids in the RRKR1204–1207 motif at the DAD C-terminus. GFP-tagged RRKR1204–1207/E, RRKR1204–1207/EE, RRKR1204–1207/EEE, and RRKR1204–1207/EEEE mutants were transfected into HeLa cells. Twenty-four hours after transfection, plasma membrane (PM) localization was observed using a confocal laser microscope. Increasing PM localization of these mutants (arrows): RRKR1204–1207/EE < RRKR1204–1207/EEE < RRKR1204–1207/EEEE, was observed. Note no apparent (or dot-like) PM localization was observed with GFP-RRKR1204–1207/E, and that the dot-like localization of RRKR1,204–1,207/E was significantly weaker than that of RRKR1204–1207/EX (see B). Scale bars: 10 μm. Representative of five experiments. Download figure Download PowerPoint We made four additional mutants: DIA1(RRKR1204–1207/EEEE), DIA1(RRKR1204–1207/EEE), DIA1(RRKR1204–1207/EE), and DIA1(RRKR1204–1207/E) (Fig EV2C), which all retain the C-terminal 56 aa of DIA1 (1208–1263 aa). DIA1(RRKR1204–1207/E) was less strongly associated with areas adjacent to the PM when compared to DIA1(RRKR1204–1207/EX), with the degree of PM binding among this second set of mutants being DIA1(RRKR1204–1207/E) < Dia1(RRKR1204–1207/EE) < Dia1(RRKR1204–1207/EEE) < Dia1(RRKR1204–1207/EEEE) (Fig EV2D). Thus, we conclude that C-terminal aa (1208–1263 aa) are required to suppress DIA1 activation. Insufficient autoinhibition of DIA1(R1204X) in live cells Previous fluorescence single-molecule speckle microscopy (SiMS) study revealed that the FH1-FH2 domain of mouse Dia1 shows processive movement in living cells based on its constitutive actin polymerization activity (Higashida et al, 2004). In full-length WT Dia1, the polymerization activity of FH1-FH2 domain is suppressed by the autoinhibitory DID-DAD interaction, and binding of Rho family GTPases to the DID relieves this suppression (Watanabe et al, 1999). Compatible with this finding, WT Dia1 showed scarce processive movement in a SiMS study (Higashida et al, 2008), and microinjection of active RhoA(G14V) induced processive movement (Higashida et al, 2004). We expressed GFP-tagged DIA1 molecules in living cells in order to evaluate the actin elongation activity of DIA1(R1204X) using fluorescence SiMS (Watanabe, 2012). GFP-DIA1(R1204X) expressed in XTC cells showed frequent processive movements (Fig 5B, Movie EV6) compared with GFP-DIA1(WT) (Fig 5A, Movie