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. 2019 Dec 9;219(1):e201902088. doi: 10.1083/jcb.201902088

Figure S2.

Figure S2.

Multiple domains of TRIM67 are required to fully rescue growth cone response to netrin-1. (A) TRIM67 constructs used in structure–function assays. The RING domain of TRIM proteins contain zinc binding pockets necessary for E3 ubiquitin ligation activity and can mediate oligomerization of TRIM proteins (Freemont, 1993; Koliopoulos et al., 2016; Meroni and Diez-Roux, 2005); we therefore made both a RING-deletion construct (TRIM67ΔRING) and one containing mutations at cysteines 7 and 10 to abolish zinc binding in the RING domain and thus any ligase activity (TRIM67-LD). The coiled-coil (CC) domains of TRIM proteins mediate homo- and heterodimerization with other members of the same TRIM class (Sanchez et al., 2014; Short et al., 2002). In our previous investigation of TRIM9, the CC domain also interacted with the filopodial tip-localized actin polymerase VASP (Menon et al., 2015); therefore, we generated a construct of TRIM67 lacking the coiled-coil domain (TRIM67ΔCC). Finally, we generated a construct possessing only the three N-terminal tripartite motif domains (TRIM67-N). All constructs possessed an N-terminal myc tag. (B) ICC of Trim67−/− growth cones stained for actin, β-III-tubulin, and the indicated myc-TRIM67 construct, showing expression and distribution of each construct. (C–E) Individual data points and box-and-whisker plots showing the data spread in the interquartile range (box) and minimum and maximum (whiskers) of growth cone area (C), filopodial density (n [cells] = 96 myc media, 88 myc netrin, 121 TRIM67 media, 114 TRIM67 netrin, 65 LD media, 52 LD netrin, 68 ΔRING media, 68 ΔRING netrin, 45 ΔCC media, 52 ΔCC netrin, 52 NH2 media, 65 NH2 netrin; D), and filopodia length in neurons expressing each rescue construct (E). All statistical comparisons in red are to myc-expressing, untreated growth cones. n (cells) = 128 myc media, 118 myc netrin, 121 TRIM67 media, 119 TRIM67 netrin, 64 LD media, 50 LD netrin, 68 ΔRING media, 68 ΔRING netrin, 45 ΔCC media, 50 ΔCC netrin, 52 NH2 media, 64 NH2 netrin. n (cultures) = 3. *, P < 0.05; ***, P < 0.005. Box plots are minimum, Q1, Q2, Q3, maximum.