Fig. 1

Patient-derived fibroblasts with mutations in LITAF display enlarged late endocytic compartments. a Schematic diagram to illustrate the domain organisation of the LITAF protein. The N terminus is characterised by a proline-rich region, followed by a ‘LITAF domain’ at the C terminus. Both T115N and L125P mutations are within the hydrophobic region of the LITAF domain (grey). b Diagram of the topology of the LITAF domain within the phospholipid bilayer (created with Adobe Photoshop). The predicted hydrophobic anchor (grey) of the LITAF domain, shown as a Rosetta structural model (from Ho et al. [14]), is embedded in the membrane's phospholipid bilayer, but does not traverse it. c Representative confocal immunofluorescence images of control fibroblasts, and CMT1C patient-derived fibroblasts with mutations in LITAF (L125P, T115N) display enlarged late endocytic compartments, as shown by LAMP1 (green) and Cathepsin D (red) staining. Insets show LAMP1 staining in ‘rings’ as opposed to fine puncti in LITAF mutant cells. d Protein lysates were analysed by immunoblotting with an antibody raised against LITAF to confirm the expression of LITAF from control and CMT1C patient-derived fibroblasts. Alpha-tubulin served as loading control. Full-length blots are shown in Additional file 2: Fig. 2e) Conventional electron microscopy of control and LITAF patient-derived fibroblasts reveal abnormal, swollen, vacuolar compartments in LITAF mutant cells (L125P, T115N). Insets reveal vacuoles to be membrane bound; black arrows show interconnections, and white asterisks show dense core lysosomes