Journal: Journal of molecular cell biology

Article Title: Actin nucleator formins regulate the tension-buffering function of caveolin-1.

doi: 10.1093/jmcb/mjab070

Figure Lengend Snippet: Figure 3 The effects of formin components FHOD1 and Dia1 on the density, size, and motility of cytoplasmic CAV-1 vesicles. (A) Western blotting analysis of endogenous FHOD1 and Dia1 levels in total cell lysates of wild-type (Ctrl), FHOD1 knockdown (FD1 siRNA), Dia1 knock- down (Dia1 siRNA), and FHOD1/Dia1 double knockdown (FD1 þ Dia1 siRNA) U2OS cells, respectively. GAPDH is probed for equal sample loading. (B) Quantification of the number of CAV-1-positive vesicles per mm2 in the Ctrl (n ¼ 24), FD1 siRNA (n ¼ 12), Dia1 siRNA (n ¼ 18), and FD1 þ Dia1 siRNA (n ¼ 23) U2OS cells. The data are presented as mean ± SEM. **P 0.01 (t-test). (C) The length distribution of CAV-1- positive vesicles. The number of vesicles in each size group is divided by the total CAV-1 number of the same cell. n ¼ 17602 vesicles from 24 cells (Ctrl), 6432 vesicles from 12 cells (FD1 siRNA), 9543 vesicles from 18 cells (Dia1 siRNA), and 10231 vesicles from 23 cells (FD1 þ Dia1 siRNA). Data are represented as mean ± SEM. (D) Quantification of the movement rate of CAV-1-marked vesicles in the Ctrl (n ¼ 25), FD1 siRNA (n ¼ 13), Dia1 siRNA (n ¼ 14), and FD1 þ Dia1 siRNA (n ¼ 24) cells. (E) Quantification of the ratio of ‘go and dwelling’ to ‘dwelling’ in the Ctrl (n ¼ 9), formin-inhibited (n ¼ 9), FD1 siRNA (n ¼ 9), and Dia1 siRNA (n ¼ 9) cells. The data are presented as mean ± SEM. **P 0.01 (t-test). (F) Immunofluorescence staining of endogenous F-actin and CAV-1-positive vesicles in U2OS cells express- ing GFP, active GFP-FHOD1, and active GFP-Dia1, respectively. Scale bar, 10 and 5 mm in images and magnified images, respectively. (G) Quantification of the number of CAV-1-positive vesicles per mm2 in each group. n ¼ 30 cells. The data are presented as mean ± SEM. ***P 0.001 (t-test). (H) The length distribution of CAV-1-positive vesicles. The number of vesicles in each size group is divided by the to- tal CAV-1 number of the same cell. n ¼ 16597 vesicles from 20 cells (Ctrl), 15737 vesicles from 20 cells (GFP), 20623 vesicles from 20 cells (active GFP-FHOD1), and 19405 vesicles from 20 cells (active GFP-mDia1). Data are represented as mean ± SEM.

Article Snippet: The following antibodies were used in this study: CAV-1 (D46G3) rabbit monoclonal antibody (1:1000 dilution; #3267, Cell Signaling), FHOD1 rabbit polyclonal antibody (1:1000 dilution; SAB4200147, Sigma-Aldrich), beta actin monoclonal antibody (1:5000 dilution; 66009-1-Ig, Proteintech), Dia1 rabbit polyclonal antibody (1:500 dilution; NBP2-14667, Novus Biologicals Inc.), and GAPDH mouse polyclonal antibody (1:1000 dilution; #G8795, Sigma-Aldrich).

Techniques: Western Blot, Knockdown, Staining

Journal: Journal of molecular cell biology

Article Title: Actin nucleator formins regulate the tension-buffering function of caveolin-1.

doi: 10.1093/jmcb/mjab070

Figure Lengend Snippet: Figure 4 The linear elongated actin network by formins is critical for CAV-1 disappearance upon hypo-osmotic shock. (A) Time-lapse imag- ing of U2OS cells expressing CAV-1-mCherry cultured under routine culture condition (iso-osmosis) with formin inhibition or FHOD1 þ Dia1 double knockdown, respectively. (B) Time-lapse imaging of U2OS cells expressing CAV-1-mCherry upon hypo-osmotic shock under Ctrl, for- min inhibition, or FHOD1 þ Dia1 double knockdown condition, respectively. In A and B, white dash lines indicate the outline of the cells. CAV-1-positive vesicles were detected by Imaris at different time points. Vanished CAV-1 vesicles upon 2- and 5-min iso-osmotic (A) or hypo-osmotic (B) shock are labeled by green and orange dots, respectively. Red dots illustrate the remaining CAV-1-positive vesicles after 5-min hypo-osmotic treatment. Scale bar, 10 mm. (C) Quantification of the percentage of CAV-1-positive vesicles left upon 5-min hypo- or iso-osmotic shock under Ctrl, formin inhibition, or FHOD1 þ Dia1 double knockdown conditions, respectively. The data are presented as mean ± SEM. **P 0.01, ***P 0.001 (t-test). (D) Western blotting analysis of CAV-1, cavin-1, and actin levels in U2OS cells with formin in- hibition upon hypo-osmotic shock. The blot is also probed with GAPDH antibody to verify equal sample loading. The obtained intensity value from wild-type cells was set to 1. n ¼ 3. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

Article Snippet: The following antibodies were used in this study: CAV-1 (D46G3) rabbit monoclonal antibody (1:1000 dilution; #3267, Cell Signaling), FHOD1 rabbit polyclonal antibody (1:1000 dilution; SAB4200147, Sigma-Aldrich), beta actin monoclonal antibody (1:5000 dilution; 66009-1-Ig, Proteintech), Dia1 rabbit polyclonal antibody (1:500 dilution; NBP2-14667, Novus Biologicals Inc.), and GAPDH mouse polyclonal antibody (1:1000 dilution; #G8795, Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Inhibition, Knockdown, Imaging, Labeling, Western Blot

Journal: Scientific Reports

Article Title: Caveolin-1α regulates primary cilium length by controlling RhoA GTPase activity

doi: 10.1038/s41598-018-38020-5

Figure Lengend Snippet: Cav1α controls cilium length via ROCK and Dia1. (A , B) Control and Cav1α KO cells were untreated (−) or treated with 10 µM Y27632 for 24 h and then stained for acetylated tubulin, F-actin and γ-tubulin ( A ). The scatter-plot represents the total length of cilia measured in µm; more than 700 cells were analyzed for each condition ( B ). (C) Control and DIA1 KO cells were analyzed by immunoblotting for DIA1. GAPDH was used as a loading control. (D , E) Control and DIA1 KO cells were grown for 5 days and stained for acetylated tubulin, F-actin, γ-tubulin and nuclei ( D ). The scatter-plot represents ciliary lengths measured in µm; more than 700 cells were analyzed in control and DIA1 KO cells ( E ). (F–H) Control and Cav1α KO cells were transiently transfected with the indicated constructs, then fixed and stained after 72 h for F-actin and acetylated tubulin ( F ). The scatter-plots represent the ciliary lengths measured in µm; more than 100 cells were analyzed for each condition ( G , H ). Scale bars, 5 µm. Data in B, E, G and H were pooled from at least three independent experiments and are represented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, non-significant.

Article Snippet: The sources of the antibodies to the different markers were as follows: Cav1 (mouse mAb IgG1, used at 1/2,000 for immunoblot analysis; 610406; and the rabbit polyclonal antibody used at 1/200 for immunofluorescence analysis; 610059) and DIA1 (mouse mAb IgG1, used at 1/1000 for immunoblotting; clone51/mDia1; 610484), were from BD Transduction Laboratories. γ-tubulin (mouse mAb IgG1, used at 1/500 for immunofluorescence analysis; clone GTU-88, T3559) and acetylated tubulin (mouse mAb IgG2b, used at 1/500 for immunofluorescence analysis; clone 6-11B-1; T7451) were obtained from Sigma-Aldrich.

Techniques: Staining, Western Blot, Transfection, Construct

Journal: Scientific Reports

Article Title: Caveolin-1α regulates primary cilium length by controlling RhoA GTPase activity

doi: 10.1038/s41598-018-38020-5

Figure Lengend Snippet: Model of Cav1α function in the regulation of primary cilia length. In control cells (left), Cav1α localizes in non-caveolar domains at the apical membrane of MDCK cells, where it positively regulates RhoA activity. Active RhoA subsequently promotes apical actin polymerization through its effectors ROCK1 and DIA1. Actin meshwork rearrangement regulates ciliary lengthening by controlling the access of transport vesicles to the centrosomal zone. In the absence of Cav1α (right), RhoA activation is impaired and less apical actin polymerizes, allowing the arrival of more material at the centrosome that is used to assemble longer cilia.

Article Snippet: The sources of the antibodies to the different markers were as follows: Cav1 (mouse mAb IgG1, used at 1/2,000 for immunoblot analysis; 610406; and the rabbit polyclonal antibody used at 1/200 for immunofluorescence analysis; 610059) and DIA1 (mouse mAb IgG1, used at 1/1000 for immunoblotting; clone51/mDia1; 610484), were from BD Transduction Laboratories. γ-tubulin (mouse mAb IgG1, used at 1/500 for immunofluorescence analysis; clone GTU-88, T3559) and acetylated tubulin (mouse mAb IgG2b, used at 1/500 for immunofluorescence analysis; clone 6-11B-1; T7451) were obtained from Sigma-Aldrich.

Techniques: Activity Assay, Activation Assay

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: Physical characteristics of DIA1 proteins and similarity to orthologues from key species.

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques:

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: DIA1 is absent from the genome sequences of nematodes (grey font) as well as fungi, plants, amoebozoa and chromalveolates (not shown). Due to a paucity of sequence data, it is unclear whether a DIA1 homologue is absent from the Porifera (grey font). DIA1L was exclusively found in echinoderm and cephalochordate genomes (underlined), which also encode DIA1 . DIA1L is absent from tunicates, but a current dearth of sequence data precludes evaluation of hemichordate genomes for DIA1L homologues (indicated by a dotted bold grey line on right hand side, and a lack of underline). A bold dotted black line (right-hand side) indicates that the presence of DIA1R has been confirmed in cartilaginous fish but, probably due to a lack of sequence data, DIA1 has yet to be identified in this class of chordates. Both a DIA1 and DIA1R gene are present in vertebrate genomes (bold font), with a notable absence of DIA1R in acanthopterygian fish (asterisk). Furthermore, two DIA1 paralogues were identified in the genomes of fish from the superorder Ostariophysi, but not in fish from other superorders (see ). Data for the schematic metazoan phylogeny were from numerous sources – . Proposed rounds of whole-genome duplication (WGD) are indicated by filled black spheres, where two WGDs occurred early in the vertebrate lineage (1R/2R) and a third WGD (3R) in the ray-finned fish lineage before the diversification of teleosts , , . Proposed duplications of DIA1 -family genes are indicated by red circles, and ‘loss’ of DIA1 -family genes by grey squares. Dashed arrows are used to annotate events occurring in our current model of DIA1 -family evolution. Further details of two different models of DIA1 -family duplication and ‘loss’ events in the fish lineage (*) can be found in , where some fish species encode DIA1 paralogues, while others lack DIA1R . Accession numbers of DIA1 , DIA1R , and DIA1L sequences can be found in -S5, and .

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: The sequence alignment was generated using CLUSTALW . Identical amino acids are highlighted in red font and indicated below the alignment with an asterisk (*). Strongly similar amino acids are highlighted in green font and indicated below the alignment with a colon (:). Weakly similar amino acids are highlighted in blue font and indicated below the alignment with a full stop (.). Dissimilar amino acids are in black font. Amino acids conserved in all DIA1 proteins, as determined by alignment of DIA1 gene products from all species , are underlined ( * ). Amino acid numbering is provided above the alignment. Gaps required for optimal alignment are indicated by dashes. Standard single-letter amino acid abbreviations are used. Organism abbreviations use the first letter of the genus name, followed by the first four letters of the species (e.g. Homo sapiens DIA1 is abbreviated to HsapiDIA1). The two D. rerio DIA1 paralogues are abbreviated as DreriDIA1a and DreriDIA1b. Full species names and accession numbers can be found in .

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques: Sequencing, Generated

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: In both models (A and B), the genome of the hypothetical chordate ancestor encodes two DIA1 -family genes: DIA1 and DIA1L . The DIA1L gene has been ‘lost’ in the urochordate/vertebrate lineage, preceding the 1/2R whole genome duplications (WGDs). A duplicated copy of DIA1 , which we have called DIA1R , was retained subsequent to the 1/2R WGD event, with both DIA1 and DIA1R identified in lamprey, fish, and tetrapod genomes. In the fish lineage, however, two different models, (A) and (B), could account for our current knowledge of DIA1 family members. In model (A), the DIA1 duplication generating DIA1a and DIA1b coincides with the 3R WGD. Two lineage-specific ‘losses’ of DIA1a have then occurred: the first in the G. morhua lineage, and the second in the Protacanthopterygian/Acathopterygian lineage. There are too few data available to determine whether the channel catfish encodes DIA1a , DIA1b , both, or neither. In model (B), the DIA1 duplication leading to DIA1a and DIA1b in ostariophysans does not coincide with 3R but, instead, is specific to the ostariophysan lineage. Both model (A) and (B) both predict DIA1R gene loss in the acanthopterygian lineage. Proposed rounds of WGD , , are indicated by filled black spheres: numbering of the WGDs is provide in black boxes: those occurring early in the vertebrate lineage marked as 1R/2R and that in the ray-finned fish lineage marked as 3R. Proposed duplications of DIA1 -family genes are indicated by red circles, and ‘loss’ of DIA1 -family genes by grey squares. Data for the schematic fish phylogeny were from numerous sources , – .

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques:

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: Gene products from species with known full-length DIA1 and DIA1R orthologues were aligned using CLUSTALW , with DIA1 from the cnidarian species Nematostella vectensis (NvectDIA1), included for comparative purposes. Identical amino acids are highlighted in red font and indicated below the alignment with an asterisk (*). Strongly similar amino acids are highlighted in green font and indicated below the alignment with a colon (:). Weakly similar amino acids are highlighted in blue font and indicated below the alignment with a full stop (.). Dissimilar amino acids are in black font. Amino acids conserved in all DIA1 and DIA1R proteins, as determined by alignment of the DIA1 and DIA1R gene products from all species , are underlined ( * ). Amino acid numbering is provided above the alignment. Gaps required for optimal alignment are indicated by dashes. Standard single-letter amino acid abbreviations are used. Organism abbreviations use the first letter of the genus name, followed by the first four letters of the species (e.g. Homo sapiens DIA1R is abbreviated to HsapiDIA1R). Full species names and accession numbers can be found in and . Predicted signal peptide cleavage sites for human DIA1 and DIA1R are indicated by arrows above or below the alignment, respectively.

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques:

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: DIA1-family motifs.

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques: Residue

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: All full-length DIA1 , DIA1R , and/or DIA1L gene products were aligned using CLUSTALW , and this figure represents excerpts from this master alignment, where proteins from the following phyla only are represented: Cnidaria ( N. vectensis DIA1: NvectDIA1), Arthopoda (D. melanogaster DIA1: DmelaDIA1), Echinodermata ( S. purpuratus DIA1 and DIA1L: SpurpDIA1 and SpurpDIA1L), Cephalochordata ( B. floridae DIA1 and DIA1L paralogues: BflorDIA1, BflorDIA1La, b, and c), and Chordata. The latter includes representatives of the subphylum Urochordata ( C. intestinalis DIA1: CinteDIA1) and subphylum Vertebrata ( H. sapiens DIA1 and DIA1R: HsapiDIA1 and HsapiDIA1R). Amino acid numbering from the master alignment is provided above the alignment. Gaps required for optimizing the master alignment are indicated by dashes. Standard single-letter amino acid abbreviations are used. Organism abbreviations use the first letter of the genus name, followed by the first four letters of the species (e.g. Homo sapiens DIA1R is abbreviated to HsapiDIA1R). Full species names and accession numbers can be found in , and . The predicted location of the DIA1 and DIA1R signal peptides (SP) are indicated above the alignment . Conserved amino acid motifs detected in the master alignment ( , ) are indicated in numbered boxes above the alignment. Consensus amino acids for each motif are indicated below the alignment. Amino acids absolutely conserved across the whole DIA1-family are indicated in red upper-case letters, those strongly conserved across the whole DIA1-family are in green, and those weakly conserved across the whole DIA1-family in blue . In addition, black lower-case letters indicate amino acids conserved in over 80% of DIA1-family sequences , while grey lower-case letters indicate conservation in 50–80% of DIA1-family sequences .

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques:

Journal: PLoS ONE

Article Title: Characterization of the Deleted in Autism 1 Protein Family: Implications for Studying Cognitive Disorders

doi: 10.1371/journal.pone.0014547

Figure Lengend Snippet: The evolutionary history of the DIA1 family was inferred using the neighbour-joining method . The optimal tree is shown, with statistical reliability of branching assessed using 1000 bootstrap replicates , where percentage values are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and units are the number of amino acid substitutions per site. All positions containing gaps were eliminated from the dataset . There were a total of 258 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 . The tree was rooted on the cnidarian N. vectensis DIA1 sequence (NvectDIA1), as highlighted with an asterisk. Organism abbreviations use the first letter of the genus name, followed by the first four letters of the species. Full species names and accession numbers can be found in , and .

Article Snippet: Secondly, reciprocal pair-wise E-values between C. intestinalis DIA1, and DIA1 and DIA1R of the same key vertebrate species ( H. sapiens , M. musculus , G. gallus , D. rerio ), were generated ( ).

Techniques: Sequencing