Skip to main content
. 2020 Nov 13;9:e56058. doi: 10.7554/eLife.56058

Figure 3. ZDHHC14 is required for PSD93 targeting to the AIS in hippocampal neurons.

Hippocampal neurons transduced with lentivirus to express GFP alone (A; ‘Control’), or together with Zdhhc14 shRNA #1 (B; ‘14 sh#1’) were fixed and immunostained with antibodies against PSD93 (left column and green in merged images on right), AnkG (AIS marker, middle column and magenta in merged images on right), and GFP (marker of infected neurons, blue in merged images, right column). Lower panel images are magnified views of dashed red or white boxed area of top row images and arrowheads indicate additional non-magnified AISs in image. (C) Mean gray value of PSD93 signal within AnkG-defined AIS, normalized to control condition (unpaired Student’s t-test ****p=0.012, N = 3 independent cultures, 95% CI [0.072, 0.32]). (D) Quantified AIS lengths (defined by AnkG staining; unpaired Student’s t-test **p=0.0026, N = 3 independent cultures, 95% CI [8.42, 20.39]). (E) Integrated density of PSD93 signal within AnkG-defined AIS, normalized to control condition (Student’s t-test ***p=0.0008, N = 3 independent cultures, 95% CI [0.32, 0.60]). Scale bar in full and magnified views: 10 μm. Additional example images are provided in Figure 3—figure supplement 3.

Figure 3—source data 1. Source data for Figure 3C–E and for Figure 3—figure supplement 2C–E.

Figure 3.

Figure 3—figure supplement 1. A gentle fixation method highlights AIS-localized PSD93 in hippocampal neurons.

Figure 3—figure supplement 1.

Hippocampal neurons were fixed using a standard fixation method (A) or with a previously reported gentle fixation method (Ogawa et al., 2008) (B) and immunostained with antibodies against PSD93 (left column and green in merged images on right) and AnkG (AIS marker, middle column and magenta in merged images on right). Lower panel images are magnified views of dashed red or white boxed area of top row images and arrowheads indicate additional non-magnified AISs in image. Under standard fixation, PSD93 is visible at the AIS and at presumptive synapses, but gentle fixation highlights AIS-localized PSD93.
Figure 3—figure supplement 2. Additional Evidence that ZDHHC14 is required for PSD93 targeting to the AIS in hippocampal neurons.

Figure 3—figure supplement 2.

Hippocampal neurons were transduced with (A) control or (B) Zdhhc14 sh#2-expressing lentiviruses on DIV9 and fixed on DIV18 to determine PSD93 targeting to the AIS. Neurons were immunostained with antibodies against PSD93 (left column and green in merged images on right), AnkG (AIS marker, middle column and magenta in merged images on right), and GFP (marker of infected neurons, blue in merged images, right column). Lower panel images are magnified views of dashed red or white boxed area of top row images. Arrowheads indicate additional non-magnified AISs in image. (C) Mean gray value of PSD93 signal within AnkG-defined AIS, normalized to control condition (Mann Whitney p=0.99; N = 3 independent cultures, U = 4.00). (D) Quantified AIS lengths (defined by AnkG staining; unpaired Student’s t-test **p=0.0068, N = 3 independent cultures, 95% CI [4.35, 14.55]). (E) Integrated density of PSD93 signal within AnkG-defined AIS, normalized to control condition (unpaired Student’s t-test *p=0.030; N = 3 independent cultures, 95% CI [0.50, 0.59]). Scale bar in full and magnified views: 10 μm. Additional example images are provided in Figure 3—figure supplement 3.
Figure 3—figure supplement 3. Additional example images confirm that ZDHHC14 is required for PSD93 targeting to the AIS in hippocampal neurons.

Figure 3—figure supplement 3.

Hippocampal neurons transduced with lentivirus to express GFP alone (A and C; ‘Control’), or GFP together with Zdhhc14 shRNA #1 (B; ‘14 sh#1’) or Zdhhc14 shRNA #2 (D; ‘14 sh#2’) were fixed and immunostained with antibodies against PSD93 (left column and green in merged images on right), AnkG (AIS marker, middle column, and magenta in merged images on right), and GFP (marker of infected neurons, blue in merged images, right column). Arrowheads indicate AISs in image. Scale bar: 10 μm.