Journal: bioRxiv

Article Title: Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

doi: 10.1101/2021.09.09.459609

Figure Lengend Snippet: A- Western blot analysis of Myo1b expression in extracts from non-targeted (NT) control and myo1b targeted Caco-2 cells (KO) using CRISPR/Cas9. B- Confocal sections of Caco-2 3D cultures stained for Myo1b, F-actin (phalloidin) and nuclei (Dapi). C- Phylogenetic tree based on protein sequence of zebrafish, chick, human and mouse Myo1b and Myo1a and drosophila Myosin95E. D- In situ hybridization for myo1b transcripts on 3 and 5dpf zebrafish larvae whole mounts (left panel) and cross-sections at the level of the intestinal bulb (right panels). On sections, the forming intestinal bulb is circled with white dashed lines. E- Live, longitudinal (antero-posterior axis) confocal section of the intestinal bulb of a 5dpf zebrafish larva expressing the transcription activator KalT4 driving the expression of the eGFP-Myo1b transgene under the control of an upstream activating sequence (UAS). The precise construction of the transgenes is annotated in the panel. Scale bars 30µm.

Article Snippet: Sections were incubated for 30 min at room temperature in blocking buffer (3% BSA in PBS) and then overnight at 4°C with anti Myo1b antibody (1:200, Novus Biologicals NBP1-87739) in blocking.

Techniques: Western Blot, Expressing, Control, CRISPR, Staining, Sequencing, In Situ Hybridization

Journal: bioRxiv

Article Title: Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

doi: 10.1101/2021.09.09.459609

Figure Lengend Snippet: Confocal sections of NT control and myo1b KO Caco-2 3D cultures stained for the apical and microvilli markers phospho-Ezrin (pERM, A ) and Villin ( B ). F-actin (phalloidin) and nuclei (Dapi) are stained, scale bars 30µm, boxed areas showed in insets are enlarged 2.5x. C- Quantification of the percentage of well-formed cysts with a single central lumen in NT control and myo1b KO Caco-2 3D cultures. Data represented are median and interquartile range from n=4 replicates, Wilcoxon test, *p<0.05.

Article Snippet: Sections were incubated for 30 min at room temperature in blocking buffer (3% BSA in PBS) and then overnight at 4°C with anti Myo1b antibody (1:200, Novus Biologicals NBP1-87739) in blocking.

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

doi: 10.1101/2021.09.09.459609

Figure Lengend Snippet: A- Schematics of the design and B- DNA gel of the RT-PCR performed to control Myo1b-MO knock-down efficiency. Higher bands amplified in PCR1 MO compared to control and bands amplified in PCR2 MO correspond to Myo1b cDNA retaining the intron targeted by Myo1b-sMO, as verified by sequencing. In B, the multiple bands amplified, both in control and MO conditions, correspond to expected splicing variants of exons 23, 24 and 25 (highlighted in grey) as checked by sequencing. RT-is the control RT without superscript compared to RT+. C- Bright field pictures of 3 and 5dpf larvae presenting the phenotypes of control and Myo1b Morpholinos, WT and myo1b -/-larvae. D- Schematics of CRISPR/Cas9-mediated gene disruption at the myo1b genomic locus. The sgRNA (sgB, blue arrow) was targeting exon 2 downstream the start codon (ATG, green arrow). Compared to the WT sequence, the mutated allele displayed an insertion of 1bp generating a frame shift from amino-acid 21 and a premature STOP codon after 29 amino-acids. E- Western Blot with antibodies against Myo1b and Tubulin on lysates of dissected guts from WT and -/- adults. F- Negative control with a sense probe and in situ hybridisation with a myo1b anti-sense probe on wild-type embryos at 2 and 4 cell-stages showing maternal contribution for myo1b mRNA. G- RT-QPCR of myo1eb expression at 3dpf with EF1a used as reference gene, normalised on expression of the WT samples. Shown are mean and sem (WT=100.0±9.2, -/- =160.1±39.5, n=6).

Article Snippet: Sections were incubated for 30 min at room temperature in blocking buffer (3% BSA in PBS) and then overnight at 4°C with anti Myo1b antibody (1:200, Novus Biologicals NBP1-87739) in blocking.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Amplification, Sequencing, CRISPR, Disruption, Western Blot, Negative Control, In Situ, Hybridization, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

doi: 10.1101/2021.09.09.459609

Figure Lengend Snippet: A- Confocal single optical sections stained with nuclear labelling (Dapi) of 5dpf larvae injected with control and Myo1b Moprholinos (MO), and of 5dpf wild-type (WT) and myo1b -/-(-/-) larvae. ib intestinal bulb (circled with dashed lines), m muscles, n notochord, nt neural tube, sb swim bladder, y yolk. Scale bar=100µm. B- Quantifications from Dapi stained sections of the total number of cells per section at 3 and 5dpf in the four conditions. Data represented are median and interquartile range, n=10 to 15, Wilcoxon test, *p<0.05, ***p<0.001. C- Confocal optical sections of the intestinal bulb of 5dpf larvae in the four conditions stained for the microvilli marker Villin, F-actin (phalloidin) and nuclei (Dapi) showing the preserved apico-basal polarity of enterocytes when Myo1b is affected. Scale bar 20µm. D- Single confocal planes of live 5dpf larvae expressing Cldn15la-GFP injected with control MO (left) and Myo1b MO (right). Note the flat epithelium in the Myo1b MO condition. Scale bar=100µm. E- Single confocal planes of live 5dpf WT and myo1b -/- larvae expressing Cldn15la-GFP and quantification of the average length of the 3 longest folds per intestinal bulb analysed. Scale bar=100µm. Data presented are median and interquartile range, n WT =8 and n -/- =11, Wilcoxon test, **p<0.01.

Article Snippet: Sections were incubated for 30 min at room temperature in blocking buffer (3% BSA in PBS) and then overnight at 4°C with anti Myo1b antibody (1:200, Novus Biologicals NBP1-87739) in blocking.

Techniques: Staining, Injection, Control, Muscles, Marker, Expressing

Journal: bioRxiv

Article Title: Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

doi: 10.1101/2021.09.09.459609

Figure Lengend Snippet: A- Quantifications from EDU and Dapi stained sections of the proportion of cells in S-phase at 3dpf do not reveal significant differences in the proliferative rate of Control (n=12) vs Myo1b MO (n=10) and of WT (n=7) vs myo1b -/- (n=12) samples. B- Confocal sections of intestinal bulbs stained for apoptosis (Tunel, red) at 5dpf in WT and myo1b -/-samples and quantifications of the proportion of apoptotic cells in the four conditions (Control n=14, Myo1b MO n=11; WT n=10; myo1b -/-n=11). C, D- Confocal sections of intestinal bulbs stained for differentiation markers in WT and myo1b -/-samples and quantifications of the proportion of differentiated cells in the four conditions. At 5dpf, neither differentiation of the secretory lineage ( C - 2F11, Control n=12, Myo1b MO n=12; WT n=12; myo1b -/-n=18) nor differentiation of the absorptive lineage ( D - 4E8, Control n=10, Myo1b MO n=13; WT n=10; myo1b -/-n=8) are significantly altered. For all quantifications, data represented are median and interquartile range, Wilcoxon test, *p<0.05. For confocal images, nuclei are counterstained with Dapi (blue), bars=20µm.

Article Snippet: Sections were incubated for 30 min at room temperature in blocking buffer (3% BSA in PBS) and then overnight at 4°C with anti Myo1b antibody (1:200, Novus Biologicals NBP1-87739) in blocking.

Techniques: Staining, Control, TUNEL Assay

Journal: bioRxiv

Article Title: Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

doi: 10.1101/2021.09.09.459609

Figure Lengend Snippet: Transmission electron micrographs of sections of intestinal bulbs from WT, Myo1b MO and myo1b-/- 5dpf larvae presenting a general view of the folds of the epithelium ( A , scale bars 10µm) and of the apico-basally polarized enterocytes ( B , scale bar 2µm; b basal, a apical, n nuclei). Insets in B , show higher magnifications of the cytoplasm region to highlight the accumulation of vesicles in Myo1b MO and myo1b -/-samples, scale bar 1µm. C- Transmission electron micrographs of sections of intestinal bulbs from WT, Myo1b MO and myo1b-/- 5dpf larvae illustrating the organization of the brush border, and quantifications of the average length and density of the intestinal microvilli in the different conditions. Data presented are median and interquartile range, n length =77, n density =75 per condition. Scale bar 500 nm.

Article Snippet: Sections were incubated for 30 min at room temperature in blocking buffer (3% BSA in PBS) and then overnight at 4°C with anti Myo1b antibody (1:200, Novus Biologicals NBP1-87739) in blocking.

Techniques: Transmission Assay

Journal: bioRxiv

Article Title: Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

doi: 10.1101/2021.09.09.459609

Figure Lengend Snippet: A- Immunohistochemistry analyses of Myo1b in non-targeted (NT) control and UNC45A deficient (KO) Caco-2 cells show decreased Myo1b levels. Pictures are maximal projections of confocal stacks, Hoechst labels nuclei, scale bar 30µm. B- Confocal sections of NT control and UNC45A KO Caco-2 cells treated with the proteasome inhibitor MG132 and stained for Myo1b and the aggresome probe Proteostat. Hoechst labels nuclei, scale bar 30µm. Boxed areas showed in insets are enlarged 2x, arrowheads point at Proteostat-labelled protein aggregates and highlight colocalisation with Myo1b proteins in UNC45A KO cells. C , D- Confocal sections of a human biopsy from a healthy patient (control, C) and from a UNC45A LOF patient (D) immuno-labelled for the microvilli marker Villin and for Myo1b; Hoechst labels nuclei, scale bar 100µm. Boxed areas showed in insets are enlarged 3x, and highlight the apical localisation of Myo1b in control tissue (C) at the base of the villi (1) and in crypts (2), which is essentially lost in the UNC45A LOF tissue (D).

Article Snippet: Sections were incubated for 30 min at room temperature in blocking buffer (3% BSA in PBS) and then overnight at 4°C with anti Myo1b antibody (1:200, Novus Biologicals NBP1-87739) in blocking.

Techniques: Immunohistochemistry, Control, Staining, Marker

Journal: Frontiers in Zoology

Article Title: Differential expression of myosin heavy chain isoforms in cardiac segments of gnathostome vertebrates and its evolutionary implications

doi: 10.1186/s12983-019-0318-9

Figure Lengend Snippet: Results from peptide sequence analysis by nano HPLC-ESI-MS/MS of the inflow and outflow segments of S. canicula

Article Snippet: , G. gallus , Myosin 1B , MYH1B_CHICK , 223648 , 11536 , 23.

Techniques: Sequencing