Journal: Cell reports

Article Title: Myosin 1b regulates intestinal epithelial morphogenesis via interaction with UNC45A.

doi: 10.1016/j.celrep.2024.114941

Figure Lengend Snippet: Figure 1. MYO1B interacts with UNC45A and is misfolded in UNC45A-depleted cells and in biopsies from UNC45A-deficient patient (A) Coimmunoprecipitation of UNC45A with MYO1b and HSP90 in Caco-2 cells transduced with empty vector (EV) or FLAG/MYC-UNC45A. (B) Western blot analysis of UNC45A Caco-2 KO cells and NT control lysates using anti-MYO1b antibody and relative quantification (n = 5). (C) Immunohistochemistry analyses and quantification of MYO1b intensity in NT control (n = 8) and UNC45A KO (n = 9) Caco-2 cells. Pictures are maximal projections of confocal stacks; Hoechst labels nuclei. Scale bar, 30 mm. (D) Confocal sections of NT control and UNC45A KO Caco-2 cells treated with the proteasome inhibitor MG132 and stained for MYO1b and with the aggresome probe Proteostat and quantification of the colocalization (NT n = 32; KO n = 21). Hoechst labels nuclei. Scale bar, 30 mm. Boxed areas shown in insets are enlarged 23. Arrowheads point at Proteostat-labeled protein aggregates and highlight colocalization with MYO1B proteins in UNC45A KO cells.

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: Transduction, Plasmid Preparation, Western Blot, Control, Immunohistochemistry, Staining, Labeling

Journal: Cell reports

Article Title: Myosin 1b regulates intestinal epithelial morphogenesis via interaction with UNC45A.

doi: 10.1016/j.celrep.2024.114941

Figure Lengend Snippet: Figure 2. MYO1B loss impairs enterocyte 3D organization and trafficking despite normal apicobasal polarization (A and B) 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, 30 mm. Boxed areas shown in insets are enlarged 2.53. (C) Quantification of the percentage of well-formed cysts with a single central lumen in NT control and MYO1B KO Caco-2 3D cultures. In total, 115 cysts from eight fields (NT) and 379 cysts from nine fields (KO) were analyzed. Data are presented as median and interquartile range; Mann-Whitney ****p < 0.0001. (D) Imaging of the synchronized transport of stably expressed SBP-EGFP-ALPI (intestinal alkaline phosphatase) in Caco-2 NT, MYO1B KO, and MYO5B KO cells. Streptavidin-KDEL was used as an ER hook. Release from the ER was induced by addition of biotin at 0 min. Scale bar, 50 mm. (E) Schematics of the RUSH assay. PM, plasma membrane; ER, endoplasmic reticulum; G, Golgi; N, nucleus. (F) Kinetics of arrival of ALPI in Caco-2 NT, MYO1B KO, and MYO5B KO (color) cells to the cell surface after release from the ER. See also Videos S1, S2, and S3. Caco-2 NT n = 30, MYO1B KO n = 26, MYO5B KO n = 22, two-way ANOVA followed by a Bonferroni multiple comparison test. ns, not significant; *p < 0.5, ***p < 0.001, ****p < 0.0001.

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, MANN-WHITNEY, Imaging, Stable Transfection, Clinical Proteomics, Membrane, Comparison

Journal: Cell reports

Article Title: Myosin 1b regulates intestinal epithelial morphogenesis via interaction with UNC45A.

doi: 10.1016/j.celrep.2024.114941

Figure Lengend Snippet: Figure 3. MYO1B loss impairs membrane re- cycling and spindle orientation (A) Confocal sections of NT control, MYO1B KO, and MYO5B KO Caco-2 3D cultures stained for the lysosome marker LAMP1 and DAPI, and quantifi- cation of the percentage of vacuoles containing cells (Caco-2 NT n = 22, MYO1B KO n = 35, MYO5B KO n = 35). Scale bars, 20 mm. (B) Confocal sections of NT control and MYO1B KO Caco-2 3D cultures stained for b-tubulin and DAPI. The b angle between the spindle axis and the substratum in the confocal x-z dimension was quantified in metaphase (Caco-2 NT n = 42, MYO1B KO n = 40, MYO5B KO n = 44). Scale bars, 20 mm. Data are presented as median; one-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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: Membrane, Control, Staining, Marker

Journal: Cell reports

Article Title: Myosin 1b regulates intestinal epithelial morphogenesis via interaction with UNC45A.

doi: 10.1016/j.celrep.2024.114941

Figure Lengend Snippet: Figure 4. Myo1b localizes apically in the zebrafish gut epithelium but is not involved in epithelial cell differentiation (A) Phylogenetic tree based on protein sequence of zebrafish, chick, human, and mouse Myo1b and Myo1a and Drosophila Myo95E. (B) In situ hybridization for myo1b transcripts on 3- and 5-dpf zebrafish larvae whole mounts (left panels) and cross-sections at the level of the intestinal bulb (right panels). On sections, the forming intestinal bulb is circled with white dashed lines. (C) Live, longitudinal (anteroposterior axis) confocal section of the intestinal bulb of a 5-dpf zebrafish larva expressing the transcription activator KalT4 driving the expression of the eGFP-Myo1b transgene under the control of an upstream activating sequence in the gut epithelium. Scale bar, 30 mm. (D) Confocal imaging of zebrafish section sections stained with DAPI of 5-dpf larvae injected with control and Myo1b MO, and 5-dpf 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, 100 mm.

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: Cell Differentiation, Sequencing, In Situ Hybridization, Expressing, Control, Imaging, Staining, Injection, Muscles