Journal: Human molecular genetics

Article Title: ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

doi: 10.1093/hmg/ddv497

Figure Lengend Snippet: Figure 1. Analysis of ACTG2 variants in MMIHS patients. (A) Pedigrees of the 10 families included in this study. (B) Overview of the 10 exons of ACTG2 with all the variants

Article Snippet: Immunostainings were performed using specific antibodies against ACTG2 (1:100; Novus Biologicals), neurofilament (1:600; Monosan), smooth muscle actin α2 (ACTA2; ready to use; Dako), tyrosin protein kinase kit (c-Kit/CD117) (1:200; Cell Marque), tryptase (1:1600; Dako) and synaptophysin (SP11) (ready to use; Ventana).

Techniques:

Journal: Human molecular genetics

Article Title: ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

doi: 10.1093/hmg/ddv497

Figure Lengend Snippet: Figure 2. Expression of ACTG2 in human intestinal material. (A) Immunohistochemistry analysis shows that ACTG2 is expressed in the human intestine during different

Article Snippet: Immunostainings were performed using specific antibodies against ACTG2 (1:100; Novus Biologicals), neurofilament (1:600; Monosan), smooth muscle actin α2 (ACTA2; ready to use; Dako), tyrosin protein kinase kit (c-Kit/CD117) (1:200; Cell Marque), tryptase (1:1600; Dako) and synaptophysin (SP11) (ready to use; Ventana).

Techniques: Expressing, Immunohistochemistry

Journal: Human molecular genetics

Article Title: ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

doi: 10.1093/hmg/ddv497

Figure Lengend Snippet: Figure 3. Molecular modeling and MD simulations of ACTG2 WT and mutant proteins. (A) Ribbon representation of the secondary structure of ACTG2. Residues of interest have been depicted as van der Waals’ spheres. The representation on the bottom has been obtained by 180° rotation around the vertical axis. (B) RMSD of ACTG2 alpha-

Article Snippet: Immunostainings were performed using specific antibodies against ACTG2 (1:100; Novus Biologicals), neurofilament (1:600; Monosan), smooth muscle actin α2 (ACTA2; ready to use; Dako), tyrosin protein kinase kit (c-Kit/CD117) (1:200; Cell Marque), tryptase (1:1600; Dako) and synaptophysin (SP11) (ready to use; Ventana).

Techniques: Mutagenesis

Journal: Human molecular genetics

Article Title: ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

doi: 10.1093/hmg/ddv497

Figure Lengend Snippet: Figure 4. Cellular distribution and function of ACTG2 WT and mutant proteins. (A) Confocal images of U2OS cells stained for Phalloidin (a F-actin marker), and expressing

Article Snippet: Immunostainings were performed using specific antibodies against ACTG2 (1:100; Novus Biologicals), neurofilament (1:600; Monosan), smooth muscle actin α2 (ACTA2; ready to use; Dako), tyrosin protein kinase kit (c-Kit/CD117) (1:200; Cell Marque), tryptase (1:1600; Dako) and synaptophysin (SP11) (ready to use; Ventana).

Techniques: Mutagenesis, Staining, Marker, Expressing

Journal: Human molecular genetics

Article Title: ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

doi: 10.1093/hmg/ddv497

Figure Lengend Snippet: Figure 5. Effect of the R148S to the structure and function of ACTG2. (A) Molecular modeling and MD simulations of ACTG2 WT and the R148S mutant show moderate structural differences. (B) Confocal images of U2OS cells stained for Phalloidin (a F-actin marker), and expressing a Myc-tagged version of ACTG2 WT and of the R148S

Article Snippet: Immunostainings were performed using specific antibodies against ACTG2 (1:100; Novus Biologicals), neurofilament (1:600; Monosan), smooth muscle actin α2 (ACTA2; ready to use; Dako), tyrosin protein kinase kit (c-Kit/CD117) (1:200; Cell Marque), tryptase (1:1600; Dako) and synaptophysin (SP11) (ready to use; Ventana).

Techniques: Mutagenesis, Staining, Marker, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Differing distribution of non-muscle isoactins in A375 cells. ( A ) The cells were stained with antibodies detecting β and γ actin isoforms. Pink arrows point at invadopodia, red ones at stress fibers and blue ones at lamellipodia. ( B ) A375 cells growing on coverslips were fixed and stained to detect β or γ actin and globular actin by using appropriate antibodies and DNase I coupled to Alexa Fluor™ 594. Red arrows highlight stress fibers and blue ones lamellipodia. ( C ) Total protein extract from A375 cells (twenty μg of protein) was subjected to 2-D PAGE. The gel was stained with silver. ( D ) Immunocytochemically stained cells to detect β and γ actin. Three lines were drawn on a merged photo of the cell and the fluorescence histograms representing signal intensities for every fluorochrome were prepared on this basis.

Article Snippet: Recombinant human β actin (04-CE15) was purchased from Novoprotein (Summit, NJ, USA), whereas recombinant human γ actin (TP301730) was from OriGene (Rockville, MD, USA).

Techniques: Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Successful inactivation of ACTB and ACTG1 genes and altered actin cytoskeleton architecture in CR- ACTB [clones with inactivated ACTB with the help of CRISPR/Cas9(D10A) technique] and CR- ACTG1 [clones with inactivated ACTG1 with the help of CRISPR/Cas9(D10A) technique] clones. ( A ) Western blot analysis of control cells and clones without either β or γ actin. To identify isoactins and total actin, appropriate antibodies were used. Corresponding Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. Green rectangles represent statistically higher level of protein in comparison to control cells. ( B ) Densitometric analysis of β, γ, and total actin level in tested clones done on membranes shown in ( A ) ( n = 3). ( C ) The qRT-PCR analysis of ACTB and ACTG1 expression level in CR- ACTB and CR- ACTG1 clones ( n = 9). ( D ) Clones devoid of β or γ actin were immunostained with antibodies recognizing non-muscle actins. All pictures were taken at the same settings used during microscopic observations. Pictures of two other clones for every condition are shown in . ( E ) Representative plots presenting fluorescence distribution of stained isoactins in tested clones shown in ( D ). Quantitative analysis of area under curve (AUC) from plots ( n = 6). ( F ) Clones were immunostained with anti-β and anti-γ actin antibodies to show cellular distribution of both isoactins. Red arrows point at stress fibers and green ones at actin mesh. ( G ) Western blot analysis of filamentous and globular actin levels in clones without β or γ actin growing on coverslips for 72 h. Nitrocellulose membranes were incubated with mouse anti-total actin antibodies. Quantitative estimation of filamentous ( F ) to globular ( G ) ratio ( n = 8–9). ( H ) Clones were stained with fluorescently labelled phalloidin and DNase I to detect F- and G-actin. White arrows highlight F-actin accumulation. Results are expressed as the mean ±SD ( B , E , G ) or ±SEM ( C ), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***).

Article Snippet: Recombinant human β actin (04-CE15) was purchased from Novoprotein (Summit, NJ, USA), whereas recombinant human γ actin (TP301730) was from OriGene (Rockville, MD, USA).

Techniques: Clone Assay, CRISPR, Western Blot, Control, Comparison, Quantitative RT-PCR, Expressing, Fluorescence, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Cells devoid of β or γ actin had impaired invasion abilities, but only CR- ACTG1 cells’ 2-D migration was affected. ( A – D ) Spontaneous 2-D migration of tested cells. The cells of studied clones growing on IncuCyte ® ImageLock plates were recorded over 72 h with IncuCyte ® Live Cell Analysis Imaging System ( n = 30). ( A ) Trajectories of single cells’ migration. ( B ) Calculated covered distances of the cells. ( C ) Estimation of velocity and ( D ) directionality of cells’ migration. ( E ) Wound healing assay was performed on control (CR-CTRL), CR- ACTB, and CR- ACTG1 cells to analyze collective migration over 72 h ( n = 3). ( F ) Analysis of 3-D migration/invasion of control cells and cells without β or γ actin ( n = 16). The cells were seeded onto Matrigel™ gel placed in the upper compartment of a Transwell™ and to the lower compartment a medium enriched in serum was added. ( G ) Gelatin digestion assay. The cells seeded on fluorescently labeled gelatin were 12 h later fixed and stained with Alexa Fluor™ 488 phalloidin. Bright spots represent digested gelatin by cells. Pink arrows point at invadopodia. ( H ) Quantitative analysis of invadopodia number and ( I ) dimensions of digested area ( n = 36). ( J ) Cells after fixation were stained with antibodies recognizing cofilin and cortactin. Across the area rich in invadopodia a line was drawn and intensities of fluorescence for detected cortactin and cofilin were plotted on corresponding graphs. Pink arrows point at invadopodia. ( K ) Western blot analysis of cell lysates. Membranes were probed with antibodies detecting cofilin and cofilin phosphorylated at Ser3 (p-cofilin 3 ). Corresponding densitometric analysis and Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. Green rectangles represent statistically higher level of protein in comparison to control cells. ( L ) Calculated p-cofilin 3 :cofilin ratio ( n = 3). Results are expressed as the mean ± SD ( B – F , H , K ) or median ( I ), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.0001 (****).

Article Snippet: Recombinant human β actin (04-CE15) was purchased from Novoprotein (Summit, NJ, USA), whereas recombinant human γ actin (TP301730) was from OriGene (Rockville, MD, USA).

Techniques: Migration, Clone Assay, Live Cell Imaging, Wound Healing Assay, Control, Labeling, Staining, Fluorescence, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Stress fibers’ formation is disturbed in clones devoid of β or γ actin. ( A ) CR-CTRL, CR- ACTB, and CR- ACTG1 cells upon stimulation with LPA were fixed and stained with Alexa Fluor™ 488 phalloidin. Red arrows point at stress fibers. ( B ) The number of thick stress fibers was calculated for every condition and presented as a bar chart ( n = 61–81). ( C ) Activity of RhoA, Rac1, and Cdc42 was estimated after lysophosphatidic acid (LPA) stimulation ( n = 3). ( D ) F:G actin ratio was evaluated in the cells on the base of Western blot analysis of F- and G-actin levels in studied clones ( n = 9). Membranes were incubated with anti-total actin antibodies. ( E ) The cells were immunostained with antibodies detecting the human FH1/FH2 domain-containing protein 1 (FHOD1) and Myosin IIa. Yellow arrows highlight Myosin IIa accumulation. ( F ) Plots of averaged ( n = 9) and individual fluorescence intensity distribution for FHOD1 and Myosin IIa staining across a cell. ( G ) Western blot analysis of cell lysates. Membranes were probed for FHOD1, Myosin IIa, and myosin light chain phosphorylated at Thr18 and Ser19 (pMLC 18/19 ). Corresponding densitometric analysis and Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. ( H ) The cells were stained with anti-pMLC 19 antibodies and fluorescently labeled phalloidin. Results are expressed as the mean ± SD ( C , D ) or ± SEM ( B ), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.0001 (****).

Article Snippet: Recombinant human β actin (04-CE15) was purchased from Novoprotein (Summit, NJ, USA), whereas recombinant human γ actin (TP301730) was from OriGene (Rockville, MD, USA).

Techniques: Clone Assay, Staining, Activity Assay, Western Blot, Incubation, Fluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Lamellipodial dynamics are altered in cells devoid of β or γ actin. ( A ) Control clones and clones devoid β or γ actin after 5 min of incubation with phorbol-12-myristate-13-acetate (PMA) were fixed and F-actin was stained with the help of Alexa Fluor™ 488 phalloidin. ( B ) Quantification of leading edge width upon incubation with PMA ( n = 32–34). ( C ) Estimation of leading edge thickness of cells exposed to PMA ( n = 23–32). ( D ) Representative movie frame with marked lines from which kymographs were created. ( E ) Plots showing positon of the leading edge represented by F-actin detection under control and PMA exposition conditions ( n = 6). ( F ) Calculated AUC from plots presented in ( E ). ( G ) Estimation of activity of RhoA, Rac1, and Cdc42 was upon PMA stimulation ( n = 3). ( H ) F:G actin ratio estimation ( n = 9). The membranes were probed for total actin. Blue arrows point at lamellipodia. Results are expressed as the mean ± SD ( B , C , F – H ) or ± SEM ( E ), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).

Article Snippet: Recombinant human β actin (04-CE15) was purchased from Novoprotein (Summit, NJ, USA), whereas recombinant human γ actin (TP301730) was from OriGene (Rockville, MD, USA).

Techniques: Control, Clone Assay, Incubation, Staining, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Focal adhesion formation is altered in clones devoid of β or γ actin. ( A ) Immunostaining detecting α-parvin, VASP, and total actin. The genetically nonmanipulated A375 cells were grown under full medium conditions. White arrows point at focal adhesions, while yellows ones at nascent focal adhesions (FAs)/accumulation of VASP. ( B , E , H ) The clones growing either in full medium or incubated with LPA or PMA were fixed and stained with antibodies recognizing α-parvin or VASP. ( C , F , I ) The number of α-parvin-rich focal adhesions as well as their surface area were calculated for every tested condition ( n = 30). ( D , G ) The number of VASP-rich focal adhesions was calculated ( n = 30). Results are expressed as the mean ± SD, p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).

Article Snippet: Recombinant human β actin (04-CE15) was purchased from Novoprotein (Summit, NJ, USA), whereas recombinant human γ actin (TP301730) was from OriGene (Rockville, MD, USA).

Techniques: Clone Assay, Immunostaining, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Proposed mechanism of influence of disturbed actin organization in CR- ACTB and CR- ACTG1 cells on melanoma cells’ motility and adhesion. Cells devoid of β actin that grew in full medium exhibited more nascent (VASP-rich) focal adhesions (nFAs) and mature (VASP- and α-parvin-rich) focal adhesions (FAs) than did CR-CTRL cells. Although the CR- ACTG1 and CR- ACTB cells possess more mature FAs under the same conditions than control cells, FAs of CR- ACTG1 cells are smaller. Moreover, CR- ACTG1 cells have many more nascent FAs than both control and CR- ACTB cells. Alterations in FA dynamics, stress fiber formation, and the distribution of some ABPs at the leading edge results in changes in Rac1 activation, improved 2-D migration, and impaired invasion in CR- ACTB cells. In the case of CR- ACTG1 cells, the impaired FA turnover, increased number of thick stress fibers, and decreased level of Arp3 at the leading edge led to lowered activation status of RhoA and finally to impeded 2-D and 3-D migration. For details, please see the discussion. In summary, the lack of γ actin has more severe effects on melanoma cells than the lack of β actin. We believe that changes in the formation/turnover of FAs might be caused by the different properties of non-muscle actins’ interactions with ABPs.

Article Snippet: Recombinant human β actin (04-CE15) was purchased from Novoprotein (Summit, NJ, USA), whereas recombinant human γ actin (TP301730) was from OriGene (Rockville, MD, USA).

Techniques: Control, Activation Assay, Migration