Hahn I , Voelzmann A , Parkin J , Fülle JB , Slater PG , Lowery LA , Sanchez-Soriano N , Prokop A .

Wellcome Trust , P40 OD018537 NIH HHS , R01 MH109651 NIMH NIH HHS, BB/I002448/1 Biotechnology and Biological Sciences Research Council , BB/P020151/1 Biotechnology and Biological Sciences Research Council , BB/L000717/1 Biotechnology and Biological Sciences Research Council , BB/M007553/1 Biotechnology and Biological Sciences Research Council , BB/M007456/1 Biotechnology and Biological Sciences Research Council , BB/R018960/1 Biotechnology and Biological Sciences Research Council

	Fig 1. Eb1, Msps and Tau share the same combination of axonal loss-of-function phenotypes in Drosophila primary neurons.A-H) Images of representative examples of embryonic primary neurons pre-cultured up to 6 days (to deplete maternal gene product; see Drosophila Primary Cell Culture Preparation) and either immuno-stained for Eb1 (top) or for tubulin (bottom); neurons were either wild-type controls (ctrl) or carried the mutant alleles msps1, tauKO or Eb104524 in homozygosis (from left to right); asterisks indicate cell bodies, black arrow heads the axon tips, white arrow heads point at areas of MT curling, dashed squares in A-D are shown as 3.5-fold magnified close-ups below each image with black arrows pointing at Eb1 comets; the axonal outline in D is indicated by a dotted line; scale bar in A represents 15 μm in all images. I-N) Quantification of different parameters (as indicated above each graph) obtained from pre-cultured embryonic primary neurons with the same genotypes as shown in A-H. Data were normalised to parallel controls (dashed horizontal line) and are shown as median ± 95% confidence interval (I-M) or mean ± SEM (N); data points in each plot, taken from at least two experimental repeats consisting of 3 replicates each; large open circles in graphs indicate median/mean of independent biological repeats. P-values obtained with Kruskall-Wallis ANOVA test for the different genotypes are indicated in each graph. For raw data see S1 Data.
	Fig 2. Eb1, tau and msps interact genetically.A-B”) Axon length, MT curling and Eb1 amount (as indicated on the right), for primary neurons displaying heterozygous (A-A”, larval cultures) and homozygous (B-B’, embryonic 6d pre-cultures’) mutant conditions, alone or in combination. Data were normalised to parallel controls (dashed horizontal lines) and are shown as scatter dot plots with median ± 95% confidence interval (A, A”,B, B”) or bar chart with mean ± SEM (A’, B’) of at least two independent repeats with 3 replicates each; large open circles in graphs indicate median/mean of independent biological repeats. P-values above data points/bars were obtained with Kruskall-Wallis ANOVA tests; used alleles: mspsA, tauKO, Eb104524. C) The graph compares Eb1 amounts at MT plus-ends with the degree of MT curling (MT disorganisation index/MDI) for a range of genetic conditions used in this work: green dots show data from pre-cultured embryonic neurons (B’ vs. B”), purple dots show comparable data obtained from larval primary neurons (A’ vs. A”); in addition, green/purple dots contain data from Fig 1J and 1N and S1B and S1D Fig; black dots show similar data obtained from primary Xenopus neurons (Fig 3G and 3H); r and p-value determined via non-parametric Spearman correlation analysis; see further detail of these correlations in S4 Fig. For raw data see S2 Data.
	Fig 3. Eb1, Msps and Tau functionally interact in the fly brain and in frog primary neurons.A,B) Medulla region of adult brains at 26–27 days after eclosure, all carrying the GMR31F10-Gal4 driver and UAS-GFP-α-tubulin84B (GMR-tub) which together stain MTs in a subset of lamina neuron axons that terminate in the medulla; the further genetic background is either wild-type (A) or triple-heterozygous (Eb104524/+mspsA/+ tauKO/KO; B); white/black arrows indicate axonal swellings without/with MT curling; rectangles outlined by red dashed lines are shown as 2.5 fold magnified insets where white arrow heads point at disorganised curling MTs. C,D) Quantitative analyses of specimens shown in A and B: relative number of total swellings per axon (C) and of swellings with MT curling per axon (D); bars show mean ± SEM; P values from Kruskal–Wallis one-way tests are given above each column, merged sample numbers (i.e. individual axon bundles) from at least two experimental repeats at the bottom of each bar. E-E””) Primary Xenopus neurons stained for tubulin (tub): asterisks indicate cell bodies, white arrows indicate unbundled MTs, white arrowheads unbundled areas with MT curling. F-F”’) Xenopus neurons labelled with MACF43::GFP (MACF43): black arrows point at comets (visible as black dots). In E-F”’, black-stippled squares in overview images are shown as 2.5 fold magnified close-ups below; ↓ behind gene symbols indicates 50% knock-down thus approximating heterozygous conditions. G,H) Quantification of specimens shown in E-F””with respect to MT curling (G) and comet amount of MACF43::GFP (H); data were normalised to parallel controls (dashed horizontal lines) and are shown as mean ± SEM (G) or median ± 95% confidence interval (H); merged sample numbers from at least two experimental repeats are shown at the bottom, P-values obtained with Kruskall-Wallis ANOVA tests above data points/bars. The scale bar in A represents 15 μm in A,B and 20 μm in E-F””. For raw data see S3 Data.
	Fig 4. Eb1 and Msps depend on each other for MT plus-end localisation.A-A”) Primary neurons at 6HIV co-expressing Eb1::mCherry (magenta, Eb1) and MspsFL::GFP (green, Msps) and imaged live; asterisks indicate somata, scale bar represents 10μm, dashed boxes indicate the positions of the 3.5-fold magnified close-ups shown at the bottom with arrowheads pointing at the position of Msps::GFP accumulation (same in C,C’). B,B’) Kymograph of live movies (as in A-A”) with the dashed line on the left representing a straightened version of the dashed lines shown in A’ and A” (i.e. the length of the axon; proximal at the top) and the x-axis indicating time; arrowheads point at trajectories of Msps and Eb1 which are almost identical. C) Primary neurons expressing Msps::GFP and imaged live, either displaying wild-type background (ctrl) or being homozygous mutant for Eb104524 (Eb1-/-); white arrowheads point at Msps::GFP comets which are much smaller in the mutant neurons. D) Schematic representations of MspsFL::GFP and MspsΔCTD::GFP. D’,D”) Graphs displaying axon length and MT curling (as indicated) for pre-cultured embryonic primary neurons expressing GFP or Msps::GFP constructs via the elav-Gal4 driver, either in wild-type or mspsA/1 mutant background; data were normalised to parallel controls (dashed horizontal lines) and are shown as median ± 95% confidence interval (D’) or mean ± SEM (D”) from at least two experimental repeats; large open circles in graphs indicate median/mean of independent biological repeats. P-values obtained with Kruskall-Wallis ANOVA tests are shown above data points/bars. E) Model view of the results shown here and in Fig 1; note that yellow circles represent GTP-tubulin which mediates the binding of Eb1; for further explanations see main text and Discussion. For raw data see S4 Data.
	Fig 5. Tau promotes Eb1 pools at MT plus-ends by outcompeting Eb1’s association with the MT lattice.A-A”) Example of an embryonic neuron at 6 HIV imaged live with curling MTs to illustrate Tau binding (green) along the MT lattice, separated from Eb1 comets (magenta); asterisks indicate the soma, the scale bar represents 10 μm, dashed boxes indicate the positions of the 4-fold magnified close-ups shown at the bottom, white arrowheads point at Eb1 comets (same in B-D). B-D) Primary neurons at 6 HIV stained for Eb1 which are either wild-type (B) or mutant for tauKO/Df (C) or expressing Eb1::GFP driven by elav-Gal4 in tauKO/Df mutant background (D); white arrowheads indicate Eb1 comets, red arrowheads Eb1 lattice localisation. E-I) Different parameters (as indicated) of control (ctrl) or tauKO/Df (tau-/-) mutant neurons at 6 HIV without/with elav-Gal4-driven expression of Eb1::GFP or Shot-Ctail::GFP (as indicated); data were normalised to parallel controls (dashed horizontal lines) and are shown as scatter dot plots with mean ± SEM (I) or median ± 95% confidence interval (E-H) from at least two independent repeats with 3 experimental replicates; large open circles in graphs indicate median/mean of independent biological repeats. P-values obtained with Kruskal-Wallis ANOVA and Dunn’s posthoc test as shown above data points/bars. J) Model view of the results shown here; note that yellow circles represent GTP-tubulin which provides higher affinity for Eb1 binding; for further explanations see main text and Discussion. For raw data see S5 Data.
	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 Eb104524/+mspsA/+ tauKO/+ 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 shot3/3 Eb104524/04524 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.
	Fig 7. A mechanistic model consistent with all reported data.A) In wild-type (WT) neurons, the three factors bind (dark grey arrows) to MTs at different locations: Msps binds to the very tip of MT plus-ends, Eb1 forms plus-end comets (bright yellow) by binding with higher affinity to GTP-tubulin (GTP-cap, curly bracket) but lagging behind the front, and Tau localises along the MT lattice primarily composed of GDP-tubulin (green). Tau outcompetes (red T-bar) low-affinity binding of Eb1 along the lattice, thus maintaining more Eb1 at MT plus-ends. Msps enhances (orange arrow) MT polymerisation (brown), thus sustaining a prominent GTP-cap that Eb1 can bind to. Eb1 stabilises MT plus-ends (green arrow) by promoting sheet formation of protofilaments at the plus-end (short Y-shaped tip), thus improving conditions for Msps binding. Eb1 also recruits the C-terminus of Shot which binds cortical actin via its N-terminus, thus establishing cross-linkage that can guide (blue arrow) extending MT plus-ends into parallel bundles. B-E) Illustrations explaining the changes triggered by the loss of the different factors (stippled X); any affected processes are shown as stippled lines with reduced thickness and reduced font sizes of accompanying texts. B) Upon loss of Eb1, the plus-end sheet structure is weakened (larger Y shape), thus reducing Msps binding and, in turn, reducing polymerisation; Shot detaches, thus abolishing guidance. C) Loss of Msps abolishes enhanced polymerisation so that the GTP-cap shrinks and less Eb1 binds, thus also weakening Shot binding and MT guidance. D) Upon loss of Tau, more Eb1 can bind to the MT lattice, thus reducing the amounts available for plus-end association; this causes a modest Eb1 depletion phenotypes with consequences similar to B, but less pronounced. E) Upon loss of Shot, the localisations and functions of the other three proteins are unaffected, but MT guidance is abolished.

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