Journal: The Journal of Biological Chemistry
Article Title: Ninein isoform contributions to intracellular processes and macrophage immune function
doi: 10.1016/j.jbc.2025.108419
Figure Lengend Snippet: Ninein ISO2 plays a major role in facilitating F-actin recruitment and particle internalization in macrophages. A , quantification of the average duration (in seconds) from the binding of a particle to the cell surface (t = 0 s) to the initial F-actin recruitment at the contact site (n ≥ 12) in RAW cells treated with ninein TOT , ninein CAN , ninein ISO2 , or control siRNA. B , representative confocal images showing the binding of IgG-sRBCs to the cell surface of RAW cells treated with the indicated siRNAs. Arrows indicate F-actin enrichment at the IgG-sRBC-contact site on the macrophage surface, with a box highlighting the location of ninein at the centrosome. C , quantification of the percentage of bound IgG-sRBCs with F-actin polymerization at the contact site (n ≥ 181) under the indicated conditions. D , representative epifluorescence images of RAW cells treated with the indicated siRNAs. Cells were challenged with 6.92 μm IgG-coated beads for an 8 min pulse, washed, and chased for 12 min to assess internalization efficiency. Cells were fixed, immunostained for external beads, permeabilized, and immunostained for ninein. Green asterisks indicate bound and/or incompletely internalized particles labeled with Cy5-anti-IgG. E , percentage of internalized beads (number of completely internalized beads/total bound plus internalized beads) in cells treated with the indicated siRNAs (n ≥ 1148 beads). p -values were calculated using one-way ANOVA followed by Tukey's multiple comparisons test or Kruskal-Wallis test from two experiments ( B and C ) and three experiments ( A , D , and E ). Error bars represent SEM ( A ) and SEP ( C and E ). Scale bars = 10 μm. F , proposed roles for ninein CAN and ninein ISO2 isoforms. Both ninein isoforms localize at the centrosome to enhance dynein-dynactin complex recruitment to the centrosome. The presence of ninein CAN is important for the Golgi morphology and positioning close to the centrosome, likely through interactions with the MT cytoskeleton. After particle binding, ninein ISO2 plays a primary role in the initial F-actin recruitment to the particle contact site to promote timely cup formation and efficient internalization during FcγR-mediated phagocytosis in macrophages.
Article Snippet: The 12-well plate was then mounted into Etaluma (LS720) automated live cell inverted epifluorescence microscope (Etaluma's LS Microscopes) enclosed within a 37 °C 5% CO 2 incubator and cells were imaged for 22 h with 20 min interval.
Techniques: Binding Assay, Control, Labeling
