Fig 6. Shot-mediated guidance mechanistically links Eb1 at MT plus-ends to bundle organisation.

A) Schematic representation of Shot constructs (CH, actin-binding calponin-homology domains; EF, EF-hand motifs; GRD, MT-binding Gas2-related domain; Ctail, unstructured MT-binding domain containing Eb1-binding SxIP motifs in blue); in Shot-3MtLS*::GFP the SxIP motifs are mutated (orange lines). B) Fixed primary neurons at 18HIV obtained from late larval CNSs stained for GFP (green) and tubulin (magenta), which are either wild-type (top) or Eb1^04524/+msps^A/+ tau^KO/+ triple-heterozygous (indicated on right) and express GFP or either of the constructs shown in D; scale bar 10μm. C) Quantification of MT curling of neurons as shown in B. D,E) MT curling in shot^3/3 Eb1^04524/04524 double-mutant neurons is not enhanced over single mutant conditions assessed in fixed embryonic primary neurons at 6HIV (D) or 12HIV following 6 day pre-culture (E). In all graphs data were normalised to parallel controls (dashed horizontal lines) and are shown as mean ± SEM from at least two independent repeats with 3 experimental replicates each; large open circles in graphs indicate median/mean of independent biological repeats. P-values obtained with Kruskall-Wallis ANOVA tests are shown above bars. F,F’) Model derived from previous work [22], proposing that the spectraplakin Shot cross-links Eb1 at MT plus-ends with cortical F-actin, thus guiding MT extension in parallel to the axonal surface; yellow dots represent GTP-tubulins providing high affinity sites for Eb1-binding. For raw data see S6 Data.