FIGURE 9.

Knockdown of clavesins in neurons alters lysosome morphology. A, hippocampal neurons at 4 DIV were transduced with lentivirus encoding a non-targeting miRNA or with lentivirus encoding two different miRNAs targeting clavesin 1 (1-1, 1-2), two different miRNAs targeting clavesin 2 (2-1, 2-2), or different combinations of the clavesin 1 and 2 miRNAs, as indicated. At 12 DIV, lysates were prepared from the cultures, and equal protein aliquots were processed for Western blots with antibodies against CHC, AP-1, or clavesin 1/2. B, hippocampal neurons at 4 DIV were transduced with lentivirus encoding clavesin 1-1 and 2-1 miRNAs. At 21 DIV, cells were processed for indirect immunofluorescence with polyclonal antibody against clavesin (red) and monoclonal antibody against AP-1. The AP-1 signal was processed in the CY5 channel (633) and was pseudocolored green for the purpose of merging with the red clavesin signal. The lentivirus also drives expression of GFP (leftmost panels). Bar, 10 μm. C, hippocampal neurons at 7 DIV were transduced with lentivirus encoding non-targeting miRNA, clavesin 1-1 miRNA, clavesin 2-1 miRNA, or both clavesin miRNAs. At 14 DIV, cells were processed for indirect immunofluorescence with antibodies against LAMP1 (red) and MAP2 (blue). The lentivirus also drives expression of GFP. Bar, 10 μm. D, the total LAMP1-positive area was quantified. Each bar represents the mean ± S.E. with n = 40. ***, a significance of p < 0.001 using Welch's t test.