downstream (0.8 kb) homologous sequences to the genomic locus of DIC. A DIC mutant strain (ro- 6
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1 A B C D ts Figure S1 Generation of DIC- mcherry expressing N.crassa strain. A. N. crassa colony morphology. When a cot1 (top, left panel) strain is grown at permissive temperature (25 C), it exhibits straight hyphal growth morphology. At restrictive ts ts temperature (top, right panel; 37 C) cot1 strains exhibit compact colony growth. cot1 suppression mediated by ropy mutations lead to curly hyphal growth morphologies at permissive temperatures (bottom, left panel) and enlarged colony morphology at restrictive temperatures (bottom, right panel). B. A schematic representation of the targeting DNA consisting of mcherry coding sequence flanked by upstream (1.8kb) and 7bp Δ 1695 downstream (0.8 kb) homologous sequences to the genomic locus of DIC. A DIC mutant strain (ro- 6 ) exhibiting ropy 7bp Δ 1695 hyphal growth morphology was used for transformation. Homologous recombination of the targeting DNA into the ro- 6 locus resulted in the rescue of the ropy phenotype and restoration of wildtype hyphal growth morphology. 7bp Δ 1695 C. Comparison of colony morphologies between wildtype, DIC mutant ro- 6 and wildtype DIC- mcherry strains. Note that the wildtype DIC- mcherry strain exhibits growth morphology is indistinguishable from the wildtype strain. 7bp Δ 1695 D. Bar graph showing radial growth rates of wildtype, DIC mutant ro- 6, WT - DIC- mcherry strains. Data are shown as mean ± S.D. 2 SI S. Sivagurunathan et al.
2 Figure S2 Protein levels in representative DHC mutant strains. Western blot analysis of DHC expression/protein levels in 17 of the 34 mutant strains, including at least one representative from every class. The class of each strain is indicated in parentheses after the designated strain name. S. Sivagurunathan et al. 3 SI
3 Figure S3 An 8- second time series showing photobleaching of bright hyphal tip dynein fluorescence in a wildtype strain. Bars: 10 µm. 4 SI S. Sivagurunathan et al.
4 Figure S4 Colocalization of dynein and dynactin in wildtype and DHC mutant strains. A. Epifluorescence images of growing hyphae in strains expressing DIC- mcherry and EGFP- dynactin p150. Left panels: dynein; middle panels: dynactin, right panels: overlay. B. Epifluorescence images of growing hyphae in strains expressing DIC- mcherry in dynactin p150 null background. Bars: 10 µm in all panels. S. Sivagurunathan et al. 5 SI
5 A B Figure S5 A. Dynein purification scheme for N.crassa. B. Representative silver- stained gel image showing the final elution fraction from the StrepTrap column. The arrow points to the DHC band and the bar indicates the band corresponding to the tagged DIC. 6 SI S. Sivagurunathan et al.
6 Files S1- S9 Supporting Movies Representative live cell movies of FM 4-64 labeled vesicle trafficking in hyphal tips from each of the strains analyzed. Each movie was of 30 seconds duration and is sped up six- fold. Each movie is 86 µm x 65 µm in size. Files S1- S9 are available for download at as compressed.avi files. S. Sivagurunathan et al. 7 SI
7 Table S1 Summary of vesicle transport dynamics. Wildtype DHC Δ p150 Δ Nkin mut. Class 1 Class 2 Class 3 Class 4 Class 5 Motility index Mean movements/ µm hyphal length Overall movements n Mean velocity (µm/sec ± S.D.) 1.64 ± ± 0.62* 1.34 ± 0.49* 1.35 ± 0.52* 1.51 ± 0.59* 1.24 ± 0.41* 1.33 ± 0.45* 1.33 ± 0.41* 1.31 ± 0.48* Mean distance (µm ± S.D.) 4.26 ± ± 1.71* 3.19 ± 1.44* 3.49 ± 2.07* 4.48 ± ± 1.64* 2.85 ± 1.15* 3.17 ± 1.33* 3.96 ± 2.33 Inward movements Percent of total movements 48.7 % 47.1 % 48.9 % 48.8 % 41.7 % 52.7 % 53.8 % 41.5 % 47.1 % Mean velocity (µm/sec ± S.D.) 1.60 ± ± 0.53* 1.27 ± 0.51* 1.36 ± 0.53* 1.43 ± 0.54* 1.23 ± 0.45* 1.29 ± 0.48* 1.24 ± 0.40* 1.28 ± 0.53* Mean distance (µm ± S.D.) 4.16 ± ± 1.27* 2.78 ± 1.12* 3.23 ± 1.88* 3.90 ± ± 1.38* 2.69 ± 1.02* 2.66 ± 0.96* 3.77 ± 2.40 Outward movements Percent of total movements 51.3 % 52.9 % 51.1 % 51.2 % 58.3 % 47.3 % 46.2 % 58.5 % 52.9 % Mean velocity (µm/sec ± S.D.) 1.67 ± ± ± 0.47* 1.35 ± 0.51* 1.56 ± ± 0.36* 1.38 ± 0.42* 1.40 ± 0.41* 1.35 ± 0.44* Mean distance (µm ± S.D.) 4.36 ± ± 1.98* 3.59 ± 1.59* 3.75 ± 2.22* 4.89 ± ± 1.83* 3.05 ± 1.27* 3.53 ± 1.45* 4.13 ± 2.26 Vesicle accumulation Hyphal tip vesicle accumulation Hyphal body vesicle accumulation 60.0 % 92.3 % 27.3 % 100 % 72.7 % 86.7 % 90.0 % 78.6 % 100 % 13.3 % 7.7 % 90.9 % 0 % 9.1 % 6.7 % 20.0 % 21.4 % 0 % * = statistically significant difference from wild- type value as determined by students t- test (P < 0.05). 8 SI S. Sivagurunathan et al.
8 Table S2 Summary of dynein mislocalization phenotypes. Mislocalization phenotype Domain Mutation Class 1 (Distal - long linear tracks) AAA1 (Turn p- loop) AAA1 (H4) AAA2 (PS1 Insert) AAA2 (PS I Insert) AAA3 (β3/walker B) AAA5 (Strut C1) G1946R R2056H S2333R L2335P E2675Q aa Δ (RRSNLI) Class 2 (Apical - long linear tracks) Class 3 (Comet tails) Class 4 (Aggregates) Class 5 (Disperse) AAA1(Sensor 1 (β4)) AAA3 (β4 before sensor 1 ) MT Stalk (H8 - CC1) AAA6 (H1) Tail Tail Linker Subdomain 3 (H11 ) MT Stalk (H8 - CC1) MT- binding domain (H1) o)mtbd MT- Stalk (CC2) MT- binding domain (H7) MT- stalk ( H9 - CC2) N2050S C2722R aa Δ (SLEIQAALE) V4049D Y110S W1308G R1672S D3224P T3323P L3332P R3396G aa ins. C- terminus (H1) L4333* AAA1 (H3) MT- BS (H8 stalk CC1) AAA5 (Strut C1) AAA1 (H0) AAA1 (AAA1 H1 and B2) AAA1 (H4/ β4 R finger) AAA1 (H8) AAA3 (β4 before sensor 1) AAA5 (H0) AAA6 (H5) AAA6 (H7) AAA6 (between H8- H9) AAA6 (between h11 and h12) S2009W G3215D aa Δ (QLEKKLL) L1933P G1961R K2065E T2166P V2719D I3591P G4146A E4168K I4232N AAA6 (H12) W4311* AAA6 ( between H12- H13) D4296E; aa Δ (LVV) P4316S AAA- AAA domains of dynein, MT microtubule, H helix, β β strand, CC- coiled- coil, aa - amino acid, Δ - deletion, ins - insertion, * - nonsense mutation. S. Sivagurunathan et al. 9 SI
9 Table S3 Primary Neurospora crassa strains used in this study. Genotype Source Description mat a FGSC WT strain (FGSC # 4200) mat A FGSC WT strain (FGSC # 2489) mat a; kin- 1 FGSC Nkin mutant strain (FGSC # 9939) mat a; ro- 1::hygr This study DHC Δ mutant strain mat a; ro- 3::hygr FGSC Dynactin p150 Δ mutant strain (FGSC # 14726) rid(rip1) mat A his- 3 + ::Pccg- 1- Bml + - sgfp + mat A his- 3 + ::Pccg- 1- hh1 + - sgfp + FGSC FGSC Tubulin- GFP strain (FGSC # 9520) Histone h1- GFP strain (FGSC # 9518) mat a ro- 6 7bp Δ 1695 This study DIC mutant strain mat A ro- 6 + ::mcherry + This study WT DIC- mcherry strain mat A ro- 6 + ::mcherry + ; kin- 1 This study Nkin mutant - DIC- mcherry strain mat A ro- 6 + ::mcherry + ; Δ ro- 3 This study Dynactin p150 Δ mutant - DIC- mcherry strain mat A; ro- 3 + ::egfp + This study WT EGFP- Dynactin p150 strain mat A ro- 6 + ::hs + This study WT DIC- 6xHis- Strep- tag II strain 10 SI S. Sivagurunathan et al.
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