Figure 10.
Dbl3Y570D rescues phagocytosis in mutant MerTK iPSC-derived RPE cells. (a) Schematic outline of RPE cell generation from the MerTK mutant fibroblasts. Note, fibroblasts were derived from a blind individual suffering from retinitis pigmentosa (RP). (b) Fundus image taken from a MerTK-deficient RP individual’s right eye showing the retinal vessels (black arrowheads) and macula (white circle). (c) A red-free image of the macula with the green arrow representing the position of the line scan in b. (d) A spectral domain optical coherence tomography image of the left fundus, through the fovea, showing a thinning of the retina at the fovea (normally over 200 µm) and a loss of retinal lamination, especially centrally (total loss of ellipsoid zone, black arrowhead); the white arrowheads show an epiretinal membrane. (e–g) Confocal immunofluorescence z-section analysis of MerTK mutant RPE transfected with wild-type Dbl3 or Dbl3Y570D and exposed to POS-FITC. Note, RPE cells expressing Dbl3Y570D undergo efficient recovery of phagocytosis. White arrowheads highlight the apical F-actin cortex. (h–k) Analysis of POS adhesion sites to cup population ratios in mutant cells and cells expressing Dbl3Y570D. Note, colocalization analysis by Pearson’s coefficient calculation reveals that MerTK-deficient RPE cells contain almost exclusively Dbl-rich adhesion sites that fail to transform into protrusions depicted in white, whereas mutant cells expressing Dbl3Y570D undergo cup maturation with characteristic Dbl-POS colocalization at contact sites depicted as white rings at contact points. Scale bars represent 10 μm, unless highlighted otherwise. All quantifications are based on n = 3 independent experiments and show the data points, means ±1SD, the total number of cells analyzed for each type of sample across all experiments, and P values derived from t tests are indicated. (l) Schematic illustration of proposed model of MRCKβ-controlled morphodynamic signaling in MerTK receptor-mediated RPE cells. Briefly, MerTK-Cdc42–driven MRCKβ signaling progresses through distinct phases with a lower activity during initial pseudopod induction and high activity, concomitant with increased F-actin, during deformation of protrusions to mediate particle wrapping. Continued MRCKβ-driven myosin-II activity stimulates FAK activation of paxillin (dashed outline represents a putative link based on the established mechanosensory bridge in migratory cells), recruitment of mechanotransducers vinculin and talin, and receptor clustering at cups to coordinate particle wrapping.

Dbl3Y570D rescues phagocytosis in mutant MerTK iPSC-derived RPE cells. (a) Schematic outline of RPE cell generation from the MerTK mutant fibroblasts. Note, fibroblasts were derived from a blind individual suffering from retinitis pigmentosa (RP). (b) Fundus image taken from a MerTK-deficient RP individual’s right eye showing the retinal vessels (black arrowheads) and macula (white circle). (c) A red-free image of the macula with the green arrow representing the position of the line scan in b. (d) A spectral domain optical coherence tomography image of the left fundus, through the fovea, showing a thinning of the retina at the fovea (normally over 200 µm) and a loss of retinal lamination, especially centrally (total loss of ellipsoid zone, black arrowhead); the white arrowheads show an epiretinal membrane. (e–g) Confocal immunofluorescence z-section analysis of MerTK mutant RPE transfected with wild-type Dbl3 or Dbl3Y570D and exposed to POS-FITC. Note, RPE cells expressing Dbl3Y570D undergo efficient recovery of phagocytosis. White arrowheads highlight the apical F-actin cortex. (h–k) Analysis of POS adhesion sites to cup population ratios in mutant cells and cells expressing Dbl3Y570D. Note, colocalization analysis by Pearson’s coefficient calculation reveals that MerTK-deficient RPE cells contain almost exclusively Dbl-rich adhesion sites that fail to transform into protrusions depicted in white, whereas mutant cells expressing Dbl3Y570D undergo cup maturation with characteristic Dbl-POS colocalization at contact sites depicted as white rings at contact points. Scale bars represent 10 μm, unless highlighted otherwise. All quantifications are based on n = 3 independent experiments and show the data points, means ±1SD, the total number of cells analyzed for each type of sample across all experiments, and P values derived from t tests are indicated. (l) Schematic illustration of proposed model of MRCKβ-controlled morphodynamic signaling in MerTK receptor-mediated RPE cells. Briefly, MerTK-Cdc42–driven MRCKβ signaling progresses through distinct phases with a lower activity during initial pseudopod induction and high activity, concomitant with increased F-actin, during deformation of protrusions to mediate particle wrapping. Continued MRCKβ-driven myosin-II activity stimulates FAK activation of paxillin (dashed outline represents a putative link based on the established mechanosensory bridge in migratory cells), recruitment of mechanotransducers vinculin and talin, and receptor clustering at cups to coordinate particle wrapping.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal