Fig 2: Fat3 Kif5-ID mediates protein delivery to distal neuronal process.(A,E,I) Individual cultured hippocampal neurons expressing mCherry (magenta) and HA-tagged Fat3 constructs. (B,F,J) HA immunofluorescence (green) in the cell body and proximal dendrites of transfected cells where the panels ‘B’,’F’ and ‘J’ correspond to the boxed areas from ‘A’,’E’ and ‘I’ respectively. (C,D) High magnification images of the distal tips of neuronal processes from the two framed regions in ‘A’ demonstrating the enrichment of HA-Fat3 in these locations. (G,H,K,L) High magnification images of the distal tips of neuronal processes from framed regions in ‘E’ and ‘I’. Hippocampal neurons expressing HA-Fat3 (+32) (E-H) or HA-Fat3 (Y/E) (I-L) variants in which the Kif5-ID is mutated fail to deliver HA-tagged proteins to the distal tips of neuronal processes. Arrows show the distal tip of a neuronal process. (M) Immunofluorescence intensity of HA relative to mCherry along the most distal 10um of the neuronal process, averaged from 11–16 different processes for each condition. Error bars represent standard error of the mean. Scale bars: 100μm for ‘A’,’E’ and ‘I’, and 10μm for remainder.

Fig 3: Alternative splicing of Fat3 mRNA disrupts a conserved Kif5-ID.(A) Fat3 is a single-pass transmembrane protein with 34 extracellular cadherin repeats and Laminin-G and EGF-like motifs. Numbered arrows indicate regions of the Fat3 protein modified by alternative splicing. (B) Conventional PCR products amplified from E11.5 mouse head cDNA using PCR primer pairs flanking each splicing junction predicted from Fat3 NCBI reference sequence NM_001080814.1. Arrows indicate amplification products resulting from the insertion of alternative exons. (C) Schematic of the Fat3 cDNA showing the position of each of the amplified products in (B) relative to the 25 Fat3 exons and the three alternative exons 5.1, 24.1 and 24.2. The length of cDNA encoded by each exon is presented in basepairs, and exon lengths are not drawn to scale. Exon 23 is shaded maroon and encodes the Fat3 transmembrane domain. (D) Primary amino acid sequence encoded by nucleotide sequences at the splice junction between mouse Fat3 exons 24 and 25 includes a conserved DNXYH motif (gray shading) that has been described in mouse Fat1 [14]. (E) This motif is disrupted by the insertion of 32 amino acids encoded by alternative exons 24.1 and 24.2.

Fig 4: Effects of caspase-cleaved tau on kinesin 1 and dynein expression in immortalized cortical neurons. Panels (A,B) show representative western blot images of CN 1.4 cells transfected with GFP, GFP-T4 (normal tau), and GFP-T4C3 (truncated tau) showing the relative kinesin 1 (KIF5) or dynein expression levels. Also, Panels (A′,B′) show a quantitative analysis of the relative expression levels of kinesin 1 (A′) and dynein (B′) in immortalized cells transfected with GFP and GFP-tau (s) forms. Data are mean ± SE, n = 3. Total RNA was extracted from immortalized cortical neurons transfected with GFP, GFP-T4 (full-length tau), and GFP-T4C3 (truncated tau), and mRNA expression levels of kinesin (A″) and dynein (B″) were measured by RT-PCR (Jara et al., 2018). Data represent mean ± SE, n = 4.

Fig 5: A Conserved Kif5-ID mediates the apical delivery of P75NTR.(A) Primary amino acid alignments of Fat3, Fat1 and P75NTR protein sequences from a collection of mammalian species suggest conservation of the Fat3 Kif5-ID (bracketed sequences). Amino acids that are highly conserved between proteins and species are red, and conservative amino acid substitutions are blue. Gray shading indicates motifs in Fat3 and P75NTR that have been experimentally tested. (B) Pairs of individual MDCK cells co-transfected P75-GFP (green) and P75-RFP (magenta) show colocalization at the apical cell surface. (C) Individual MDCK cells transfected with P75-GFP ΔDGGLYS in which a portion of the conserved Kif5-ID is deleted show redistribution of GFP labeling on the basolateral surface (arrowheads). (D) MDCK cells transfected with P75-GFP (Y/E) (green) show a mild redistribution of GFP labeling to the basolateral surface (arrowheads). (B-D) For each panel, MDCK cell profiles are orthogonal views reconstructed from Z-stacks obtained by structured illumination microscopy, and E-cadherin immunolabeling (grayscale) marks the basolateral cell surfaces.