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. 2020 Sep 9;9:e60619. doi: 10.7554/eLife.60619

Figure 1. SEP-Nfasc186 accumulates at the AIS and the cell surface of the soma and axon terminus before the formation of the AIS.

(A) Immunostaining of cortical neurons at DIV 7 shows that SEP-Nfasc186 is delivered to the AIS where it colocalises with ßIV-Spectrin. Location of the cell body is shown by asterisks. Scale bar, 10 µm. (B) Quantitation of signal intensity shows comparable enrichment of SEP-Nfasc186 relative to either the soma or distal axon when compared to endogenous Neurofascin irrespective of expression in WT or Neurofascin-null neurons. n = 3, ≥41 cells; one-way ANOVA; ns = not significant. (C) Live imaging before AIS formation at DIV 3 shows SEP-Nfasc186 at the surface of the soma and axon terminus (arrows). KHC560-halo identifies the axon terminus. Dashed lines outline the axon. Scale bar, 10 µm. (D) Line scan of top panel in (C) showing the SEP-Nfasc186 signal intensity in the cell body, axon and terminal relative to background.

Figure 1.

Figure 1—figure supplement 1. SEP-Nfasc186 expressed in Neurofascin-null neurons and endogenous Neurofascin in WT cells accumulate at the cell surface of the soma and axon terminus before the formation of the AIS.

Figure 1—figure supplement 1.

(A) SEP-Nfasc186 was transfected at DIV 2 in Neurofascin-null cortical neurons. The expression at DIV 3 shows enrichment at the cell surface membrane of the soma and axon terminal (arrows). Scale bar, 10 µm. (B) Line scan of A showing SEP-Nfasc186 signal intensity in the cell body, axon and terminal relative to background. (C) The surface staining of neuronal Neurofascin (Nfasc) using an antibody directed to the extracellular domain of Nfasc186 at DIV 2 shows increased signal intensity at the soma and axon terminal (inset), Phalloidin staining identifies the cell body and the axon terminal (inset of axon terminal). Scale bar 5 µm.