full-length ca2 coding sequence (imaGenes GmbH)
90
Structured Review
imaGenes GmbH
full-length ca2 coding sequence
Full Length Ca2 Coding Sequence, supplied by imaGenes GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/length+coding+sequence/pmc08025036-182-5-14?v=imaGenes+GmbH
Average 90 stars, based on 1 article reviews
Full Length Ca2 Coding Sequence, supplied by imaGenes GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/length+coding+sequence/pmc08025036-182-5-14?v=imaGenes+GmbH
Average 90 stars, based on 1 article reviews
full-length ca2 coding sequence - by Bioz Stars,
2026-08
90/100 stars
Images
1) Product Images from "Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density"
Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density
Journal: EMBO Reports
doi: 10.15252/embr.202050145
Figure Legend Snippet: A NIH3T3 fibroblasts co‐expressing DsRed‐CA7 and EGFP‐CA2 ( n = 4 independent replicates). B–E Co‐localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing (B) EGFP, (C) EGFP‐CA2, or (D, E) EGFP‐CA7 and stained with phalloidin‐594 to visualize F‐actin ( n = 4, 10, and 8 independent replicates, respectively). A magnification of the area marked with the yellow rectangle in (C) and (D) shows the localization of EGFP‐CA2 and EGFP‐CA7 compared to phalloidin‐594. (E) EGFP‐CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles (arrow) and plasmalemmal protrusions (arrowhead). F–H The normalized fluorescence emission intensity profiles for F‐actin (red line) and (F) EGFP, (G) EGFP‐CA2, or (H) EGFP‐CA7 (black line). Data information: For the plots, pixel intensities were measured through the cross‐section of the cell indicated by the yellow line in panels (B–D). Scale bar in (A–E) 20 µm.
Techniques Used: Expressing, Staining, Over Expression, Fluorescence
Figure Legend Snippet: A–C NIH3T3 fibroblasts expressing EGFP‐CA7‐mutant1 (A), EGFP‐CA7‐mutant2 (B), and EGFP‐CA7‐mutant3 (C). F‐actin is visualized with Phalloidin‐594. In the right‐most panels of (A–C) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal (black) and actin (red), and the yellow line in left‐most panels indicates the cross‐section from which the pixel intensities were measured. Scale bars 20 µm. D Analysis of the mutated CA7 and F‐actin co‐localization in cultured fibroblasts. Scatterplots of fluorescent intensities per pixel (EGFP vs. Phalloidin‐594) along a cross‐section through a representative cell. Pearson’s correlation coefficient ( r ) for the analyzed cells is given in each panel. E Pearson’s correlation coefficient values calculated for the depicted constructs and compared to CA7. F‐actin had a strong positive correlation coefficient with EGFP‐CA7 ( r = 0.91 ± 0.02, n = 26 cells). Neither EGFP alone ( r = 0.01 ± 0.03, n = 56) nor EGFP‐CA2 ( r = 0.09 ± 0.03, n = 53) co‐localized with F‐actin ( P < 0.0001 for both constructs, when compared to CA7). From the five mutated CA7 constructs, EGFP‐CA7‐mutant1 ( r = 0.51 ± 0.04, P < 0.0001, n = 25) and EGFP‐CA7‐mutant3 ( r = 0.72 ± 0.03, P = 0.05, n = 21) co‐localized less with F‐actin actin when compared to CA7. The co‐localization of the other four mutated CA7 constructs, EGFP‐CA7‐mutant2 ( r = 0.92 ± 0.02, P = 0.99, n = 18), EGFP‐CA7‐R223E ( r = 0.86 ± 0.02, P = 0.99, n = 24), and EGFP‐CA7‐H96/98C ( r = 0.79 ± 0.04, P = 0.14, n = 18) did not differ significantly from that of CA7. Data are shown as mean ± SEM. Data did not pass Shapiro–Wilk test for normality, statistical comparison against CA7 was done with Kruskal–Wallis test corrected for multiple comparisons. Source data are available online for this figure.
Techniques Used: Expressing, Fluorescence, Cell Culture, Construct, Comparison
Figure Legend Snippet: Actin co‐sedimentation assay shows that CA7 binds to F‐actin. The binding is enhanced at more acidic pH (6.5 vs. 7.4). n = 3 independent replicates at each actin concentration, two‐way ANOVA, P < 0.001. Fluorescence time‐lapse images of F‐actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labeled non‐muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5, and 23 min). Intensity‐based fire‐coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. Quantification of the mean increase in filament length ( n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross‐sections for individual filaments /bundles ( n = 30–31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within‐unit factor and the presence of CA7 as a between‐unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non‐significant. Kymographs showing different time points in the line of interest (line width 1 µm) from the experiments analyzed in (C). Kymographs were generated with Fiji Multi Kymograph function. Total time is 159 frames = 26.5 min. Scale bar 5 μm (full height is 20 μm). F‐actin bundles can be detected as clear lines in kymographs. Fluorescence time‐lapse images of F‐actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labeled non‐muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22–23 min 20 s). Intensity‐based fire‐coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. These data are from a representative video ( n = 8 independent repeats). Data information: Data are presented as mean ± SEM in (A) and mean ± SD in (C). Source data are available online for this figure.
Techniques Used: Sedimentation, Binding Assay, Concentration Assay, Fluorescence, In Vitro, Labeling, Control, Generated
Figure Legend Snippet: A, B Actin co‐sedimentation assay was carried out at five different concentrations of β/γ‐actin and with 1 µM (A) CA7 or (B) CA2 at two different pH (7.4 or 6.5). After centrifugation, the supernatant (S) and pellet (P) fractions were separated and resolved by SDS‐PAGE. Staining the gels with Coomassie Blue showed that CA7 co‐sedimented in the pellets with actin, whereas CA2 was found only in the supernatant fraction. CA7; three repetitions and CA2; one repetition at each of the four actin concentrations/pH. C Analysis of the CA2 gels confirmed that the isoform does not interact with actin at either pH tested. D In the presence of mCA7, existing filaments assemble thus increasing elongation velocity in a stepwise manner. Fluorescence time‐lapse images of F‐actin bundling in the in vitro bundling assay. A mixture of unlabeled and Rhodamine labeled non‐muscle actin was polymerized in the absence (PBS control, upper row) or in the presence of mCA7 (1.12 µM, lower row). Numbers above images indicate the time after the onset of the experiment (images every 10 s) (First frame 200 s = 3 min 20 s, last frame 500 = 8 min 20 s). The filament/bundle ends that were followed over the experiment are indicated with white arrows. Intensity‐based fire‐coloring (Fiji) was used. Scale bar 5 μm. E Quantification of the filament/bundle length for shown frames. Measured lengths were plotted to Excel x = time in seconds, y = length in μm.
Techniques Used: Sedimentation, Centrifugation, SDS Page, Staining, Fluorescence, In Vitro, Labeling, Control
Figure Legend Snippet: A, B NIH3T3 cells transfected with DsRed (A) or DsRed‐CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 min, or with an equal amount of DMSO for 60 min. Analyses of experiments show that in cells transfected with DsRed‐CA7 F‐actin structures collapse more slowly (0 min: 83% “normal”; 2 min: 80%, P = 0.44; 5 min: 74%, P = 0.39; 10 min: 27%, P = 0.08; 30 min: 16%, P = 0.02; DMSO: 82%, P = 0.99; tested against 0 min with two‐way ANOVA, Dunnett’s multiple comparison test) than in the DsRed‐transfected ones (0 min: 89% “normal”; 2 min: 48%, P = 0.14; 5 min: 31%, P = 0.04; 10 min: 2%, P = 0.001; 30 min: 0.7%, P = 0.001; DMSO: 88%, P = 0.8; tested against 0 min with two‐way ANOVA, Dunnett’s multiple comparison test). For the analysis, cells were categorized to three groups as “normal”, “some shape/F‐actin left”, and “round”. The upper panel shows example images of the cells in all three categories for (A) DsRed‐ or (B) DsRed‐CA7 transfected cells (actin visualized with Phalloidin‐488). Data information: One hundred cells per each time point from each experiment ( n = 3) were counted and categorized. Scale bar 50 µm. Data are presented as mean ± SEM. Source data are available online for this figure.
Techniques Used: Transfection, Incubation, Comparison
Figure Legend Snippet: A, B Three‐dimensional representation of (A) CA2 and (B) CA7 structure. The amino acids 101–105 (SLDGQ in CA2 and KKHDV in CA7), 113, 115, and 237–242 (GEPEEL in CA2 and DDERIH in CA7) form a ridge at the protein surface (highlighted in yellow). R223 is located close to the ridge. C Superimposing the CA7 α‐helix‐6 (CA7 in blue, areas where mutations1–3 are located is in yellow, and the putative actin‐interacting CA7 helix in red) on the Twf‐C/G‐actin structure (gray) shows a sterically compatible structure. D Sequence alignment of human CA7 and CA2 protein sequences generated using the Clustal O (1.2.1) multiple sequence alignment. Residues forming a ridge at the CA7 protein surface in the CA7 3D structure are highlighted in the CA7 sequence (bold/underlined). R223 is marked in turquoise and H96 and H98 (mutated to gain a catalytically loss‐of‐function mutant) are highlighted in green. The row below sequence alignment indicates structural features of CA7 based on the UniProt database (B = beta strand, H = helix and, T = turn). E Schematic representation of four mutants with a full (mutant1) or partial (mutant2 and mutant3) replacement of the amino acids encoding the ridge in CA7 by the corresponding CA2 sequence. In the reversed mutant (CA2‐revCA7), the amino acids replaced in mutant1 were introduced to CA2. Data information: Panels (A–C) were prepared using PyMOL (PyMOL The PyMOL Molecular Graphics System, Version 1.4.1 Schrödinger, LLC.).
Techniques Used: Sequencing, Generated, Mutagenesis
Figure Legend Snippet: Sequence alignment of the catalytically active (CA1, CA2, CA3, CA5A, CA5B, CA7, and CA13) and catalytically inactive (CA8 and CA10) human cytosolic CA protein sequences generated using the Clustal O (1.2.1) multiple sequence alignment. The amino acids that were characterized as part of a putative actin‐binding site of CA7 (highlighted in red) are not conserved in the other cytosolic CA isoforms (highlighted in gray). An asterisk below the aligned sequences indicates fully conserved residues, a colon indicates residues with strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix) and a period indicates residues with weakly similar properties (scoring ≤ 0.5 in the Gonnet PAM 250 matrix).
Techniques Used: Sequencing, Generated, Binding Assay
Figure Legend Snippet: A–G (A) Transfection with EGFP‐CA7‐mutant2 modified cellular F‐actin structures in a similar manner than CA7. (B) Introduction of KKHDV and DDERIH motifs to CA2 (EGFP‐CA2‐revCA7) did not affect the diffuse cytosolic localization of the isoform 2. (C) EGFP‐CA7‐R223E and (D) the catalytically loss‐of‐function mutant EGFP‐CA7‐H96/98C co‐localized with F‐actin. F‐actin is visualized with Phalloidin‐594 in (A–D). The normalized fluorescence emission intensity profiles of (E) EGFP‐CA2‐revCA7 (F) EGFP‐CA7‐R223E, and (G) EGFP‐CA7‐H96/98C (black lines) and F‐actin (red line). The yellow line in (B–D) indicates the cross‐section of the cell from which the pixel intensities were measured. Analysis of co‐localization is shown in lower panels of (E–G). Representative single‐cell pixel intensities of EGFP and phalloidin‐594 channels were plotted against each other and the Pearson’s correlation coefficient value ( r ) was calculated. n = 3–4 independent transfections/construct. Scale bars in (A–D) is 20 µm. H A representative Western blot showing the expression levels of EGFP‐CA fusion proteins in 10 µg of lysate collected 24 h after transfection. The EGFP‐tagged CA fusion proteins are visible at approximately 60 kDa, and the β‐actin loading control is visible at 42 kDa. I Quantification of the fusion proteins expression levels in NIH3T3 cells. Expression level of EGFP‐CA7 was set at 1 for each Western blot. n = 5 independent transfections for all fusion proteins except for CA7‐mutant2, for which n = 4 (data passed Shapiro–Wilk test for normality). Transfections’ efficacy of different mutant fusion proteins was compared to CA7 WT using one‐way ANOVA with Dunnett’s multiple comparisons test. Data are given as mean + SEM. Source data are available online for this figure.
Techniques Used: Transfection, Modification, Mutagenesis, Fluorescence, Construct, Western Blot, Expressing, Control
Figure Legend Snippet: A–H (A) Control experiment with neurons co‐expressing mCherry‐actin and EGFP. (B) Neuron transfected with mCherry‐actin and EGFP‐CA2. Compared to the spine‐targeted mCherry‐actin, CA2 localizes more diffusely along dendritic shafts and spines. Both (C) EGFP‐CA7 and (D) EGFP‐CA7‐mutant2 show a highly overlapping localization with mCherry‐actin and disruption of dendritic spine morphology. Spines were replaced by thick, filopodia‐like dendritic protrusions, which lack spine heads. The loss‐of‐function constructs (E) EGFP‐CA7‐mutant3 and (F) EGFP‐CA7‐mutant1 are more homogenously present in both dendrites and spines. (G) EGFP‐CA7‐R223E and (H) the catalytically inactive EGFP‐CA7‐H96/98C showed overlapping localization with mCherry‐actin. Scale bar 5 µm (A–C, G, H), 10 µm (D–F). I EGFP‐CA7 expression disrupted normal spine morphology in cultured neurons. Control, only mCherry‐actin: spines with head 0.34 ± 0.04, thin spines/filopodia 0.21 ± 0.02, abnormal spines 0.00 ± 0.00, total 0.54 ± 0.05 spines/µm; n = 10 cells, 509 spines, 973 µm analyzed dendrite. EGFP‐CA2: spines with head 0.35 ± 0.04, thin spines/filopodia 0.17 ± 0.02, abnormal spines 0.00 ± 0.00, total 0.53 ± 0.06 spines/µm; n = 10 cells, 535 spines, 992 µm analyzed dendrite; EGFP‐CA7: spines with head 0.05 ± 0.02, thin spines /filopodia 0.10 ± 0.02, abnormal spines 0.34 ± 0.03, total 0.49 ± 0.05 spines/µm; n = 10 cells, 493 spines, 10,256 µm dendrite. Analyzed cells were pooled from two independent experiments. The bar diagrams show the mean. Source data are available online for this figure.
Techniques Used: Control, Expressing, Transfection, Disruption, Construct, Cell Culture
Figure Legend Snippet: A Isoform‐specific subcellular localization shown in cultured hippocampal neurons (DIV14) co‐expressing DsRed‐CA7 ( left ) and EGFP‐CA2 ( middle ). B, C Representative confocal images of precocious in vivo expression of (B) EGFP‐CA2 and (C) EGFP‐CA7 in P40 mouse cortical layer 2/3 pyramidal neurons. Neurons were transfected at E14.5 with EGFP‐CA2 or EGFP‐CA7 using in utero electroporation and images were taken from fixed slices. Right panels in (B) and (C) show higher magnification of the primary apical dendrite marked with a box. D The strong expression of EGFP‐CA7 disrupted the normal spine morphology and induced the formation of thick, filopodia‐like protrusions. Data information: n = 5 independent repeats for cultured neurons and two animals/construct in vivo . Scale bar in (A) 5 µm; (B and C): 5 µm, insets in (B, C) and panel (D): 25 µm.
Techniques Used: Cell Culture, Expressing, In Vivo, Transfection, In Utero, Electroporation, Construct
Figure Legend Snippet: Comparison of mEPSCs in cortical layer 2/3 pyramidal neurons from P30 to P40 WT and CA7 KO mice. Sample traces of mEPSC recordings from WT and CA7 KO neurons, low‐pass filtered at 1 kHz (left). The data are summarized in the bar diagrams (right). mEPSC frequency ( P = 0.63) and amplitude ( P = 0.90) were not significantly different between WT and CA7 KO and neurons ( n = 7 and 5 neurons, respectively, Student’s independent samples t ‐test). Representative confocal images of apical dendrites from Lucifer Yellow‐injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 to P37 mice. Scale bar 2 µm. Summary of the spine density analysis done from the Lucifer Yellow‐injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites ( n = 28 neurons for both) compared to WT ( n = 29 neurons for apical and n = 30 for basal dendrite analysis) ( P = 0.000002 for apical dendrites, analyzed with Mann–Whitney test, and P = 6.8 × 10 −8 for basal dendrites, Student’s t ‐test with Welch correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice. The spine head width distribution differed significantly between the genotypes ( n = 467 spines from WT and n = 421 spines from CA7 KO animals, 15 neurons analyzed from both genotypes, Wilcoxon rank‐sum test with continuity correction, W = 134,540, P < 0.001). Data information: Data are given as mean ± SEM in (A) and as mean + SEM in (C). Source data are available online for this figure.
Techniques Used: Comparison, Injection, MANN-WHITNEY, Control
![Expression profiles of key differentially expressed genes (DEGs) between nontransgenic (NT) and GmMEKK2 ‐overexpression lines (ZMP) involved in the reactive oxygen species (ROS)‐ and salicylic acid (SA)‐related pathways. (A) KEGG enrichment analysis of DEGs between NT and ZMP plants. Left: NT_CK versus ZMP_CK (uninfected controls); Right: NT_7d versus ZMP_7d (7 days post‐SMV‐inoculation [dpi]). Points represent enriched pathways, with size indicating gene count and colour reflecting −log 10 (adjusted p ‐value). Red arrows highlight defence‐related pathways. (B) Expression dynamics of key components among MAPK, plant hormone signalling and plant–pathogen interaction pathways. Schematic depicts signal transduction from apoplast to cytoplasm, including Ca 2+ sensors (CNGCs and CDPKs), ROS producers (Rbohs) and SA‐induced defence protein (PR1). Heatmaps show expression levels across conditions (NT and ZMP at 0, 7 and 14 dpi), with gene IDs labelled.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5255/pmc12665255/pmc12665255__MPP-26-e70184-g003.jpg)
