Capu and Spire Assemble a Cytoplasmic Actin Mesh
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1 Developmental Cell 13 Supplemental Data Capu and Spire Assemble a Cytoplasmic Actin Mesh that Maintains Microtubule Organization in the Drosophila Oocyte Katja Dahlgaard, Alexandre A.S.F. Raposo, Teresa Niccoli, and Daniel St Johnston Table S1. Frequency of Cytoplasmic Seething and Streaming at Stage 9 No. oocytes with No. oocytes with cytoplasmic cytoplasmic seething streaming wt 3 0 capu G7 /Df(2l)ed SZ capu G7 /Df(2l)ed SZ1 ; GFP-Capu 13 0 capu G7 /Df(2l)ed SZ1 ; GFP-CapuΔN 10 0 capu G7 /Df(2l)ed SZ1 ; GFP-SpireD 0 5 spire RP 0 10 spire RP ; GFP-Capu 5 0 spire RP ; GFP-CapuΔN 7 0 spire RP /spire 2F ; GFP-CapuΔN 8 1 spire RP /Df(2L)Exel spire RP /Df(2L)Exel 6046 ; GFP-SpireD 5 0 spire RP /Df(2L)Exel 6046 ; GFP-SpireC 0 3 chic chic 1320 /chic chic 1320 /chic 221 ; GFP-Capu 0 10 chic 1320 /chic 221 ; GFP-CapuΔN 0 12 Table showing the number of egg chambers of various genotypes that show either the slow random movements in the oocyte cytoplasm called seething (See Figure 1I), or unidirectional fast movements of premature streaming (See Figure 1J). UASp Capu and Spire constructs were expressed under the control of noa-gal4-vp16.
2 Table S2. Microtubule Appearance in Stage 9 and 10A Oocytes Stage9 Stage 10A Cortical arrays N Cortical arrays N w 0% 60 0% 17 capu G7 /Df(2l)ed SZ1 100% % 40 capu G7 /Df(2l)ed SZ1 ; GFP-Capu 0% 58 0% 13 capu G7 /Df(2l)ed SZ1 ; GFP-CapuΔN 0% 65 0% 24 capu G7 /Df(2l)ed SZ1 ; GFP-Spir-D 100% % 12 spire RP 99% % 15 spire RP ; Capu 48% % 17 spire RP ; GFP-Capu 12% % 32 spire RP ; GFP-CapuΔN 1% 69 40% 15 spire RP /spire 2F ; GFP-Spir-D 0% 32 0% 10 capu RK FRT nlsgfp/ capu G7 95% % 8 capu RK khc 17 / capu G7 khc 17 0% 8 0% 2 Table showing the percentage of egg chambers with dense arrays of microtubules along the oocyte cortex (See Figure 1O) instead of the normal anterior to posterior gradient of microtubules (See Figure 1M). N indicates the number of egg chambers analysed for each genotype. UASp Capu and Spire constructs were expressed under the control of noa-gal4-vp16. Table S3. oskar mrna Localisation in Stage 9-10B Egg Chambers Posterior Posterior & Diffuse Diffuse N W 100% 0% 0% 71 capu G7 /Df(2l)ed SZ1 0% 0% 100% 56 capu G7 /Df(2l)ed SZ1 ; GFP-CapuΔN 100% 0% 0% 106 spire RP 0% 2% 98% 101 spire RP ; GFP-Capu 21% 70% 9% 133 spire RP ; GFP-CapuΔN 53% 21% 25% 255 spire 2F /spire RP ; GFP-Spir-D 100% 0% 0% 26 Table showing the distribution of oskar mrna localisation phenotypes, as visualised by oskar in situ hybridisation, in stage 9-10B egg chambers of various genotypes. The localisation patterns were classified as: Diffuse localisation (See Figure 1N); Posterior and diffuse (See Figure S2B) and Posterior localisation (See Figure 1P). N indicates the number of egg chambers analysed for each genotype. UASp Capu and Spire constructs were expressed under the control of noa-gal4-vp16.
3 Table S4. The Effect of Latrunculin A on Ooplasmic Movement at Stage 9 Without Latrunculin A With Latrunculin A Streaming Seething Streaming Seething w capu G7 /Df(2l)ed SZ1 ;GFP-Capu capu G7 /Df(2l)ed SZ1 ;GFP-CapuΔN spire RP /spire 2F ;GFP-Spir-D 7 0 spire RP /spire 2F ;GFP-CapuΔN Table showing the number of control egg chambers or egg chambers treated with Latrunculin A of various genotypes that show either slow random movements of the oocyte cytoplasm (seething (see Figure 1I)), or the fast unidirectional movements of premature streaming (See Figure 1J). UASp Capu and Spire constructs were expressed under the control of noa-gal4-vp16. Table S5. The Effect of Latrunculin A on Microtubule Appearance at Stage 9 Without With Latrunculin A Latrunculin A Cortical N Cortical arrays N arrays w 0% % 55 capu G7 /Df(2l)ed SZ1 ;GFP-Capu 0% 54 97% 65 capu G7 /Df(2l)ed SZ1 ;GFP-CapuΔN 0% 53 0% 54 spire RP /spire 2F ;GFP-Spir-D 0% % 63 spire RP /spire 2F ;GFP-CapuΔN 52% 67 96% 56 Table giving the percentage of control stage 9 egg chambers or stage 9 egg chambers treated with Latrunculin A that contain dense arrays of microtubules along the oocyte cortex (See Figure 1O) instead of the normal anterior to posterior gradient of microtubules (See Figure 1M). N indicates the number of egg chambers analysed for each genotype. UASp Capu and Spire constructs were expressed under the control of noa-gal4-vp16.
4 Figure S1. oskar mrna Localises to the Posterior of Late Stage spire Mutant Oocytes (A-C ) Fluorescent in situ hybridisations to oskar mrna in spire RP /Df(2L)Exel 6046 egg chambers. (A-C) Shows the fluorescent signal from an in situ hybridization to oskar mrna, while (A -C ) show DIC images of the oocytes. (A and A ) Stage 9; (B and B ) Stage 10A; (C and C ) Stage 13. As oogenesis progresses, more oskar mrna accumulates at the posterior of the oocyte. (D) Table showing the distribution of oskar mrna at different stages of oogenesis in spire RP /Df(2L)Exel 6046 oocytes. The localisation patterns are classified as diffuse localisation as seen in (A); posterior and diffuse as in (B) and posterior as in (C).N indicates the number of egg chambers analysed at each stage.
5 Figure S2. Comparisons of the Actin Mesh in a Range of chic and capu Alleles (A-D, F-J ) Stage 9 egg chambers stained with TRITC-Phalloidin. (A -D ) show magnifications of the oocyte cytoplasm imaged 10 µm below the cortex. (A-D ) and (F-J ) show egg chambers stained, imaged and processed in parallel under identical conditions. The vertical lines show the standard deviations in mean pixel intensities. The F-actin forms a dense actin mesh in wildtype oocytes (A). The actin mesh appears slightly less dense in chic 1320 /+ (B), is almost gone in chic 1320 oocytes, (C), and is completely absent in chic 1320 /chic 221 oocytes (D).
6 (E) Bar diagram showing the relative average of mean pixel intensity of the F-actin staining in 30 µm by 30 µm regions of 3-5 stage 9 egg chambers of different chic genotypes. (F-J ) A similar graded effect on the actin mesh is observed in capu mutant combinations of increasing strength. (F) Wildtype; (G) Df(2L)ed SZ1 /+ ; (H) capu 2F ; (I) capu 2F /Df(2L)ed SZ1 ; (J) capu G7 /Df(2L)ed SZ1. (J ) shows the bright dots of residual F-actin staining in the oocyte cytoplasm in the absence of Capu.
7 Figure S3. The chic Mutant Phenotype Is Not Rescued by GFP-Capu, GFP- CapuΔN, or GFP-SpireD (A-E) Confocal images of s7age 8-9 egg chambers stained with TRITC-Phalloidin to label F-actin. (A - E ) show a magnification of the oocyte cytoplasm imaged 10 µm from the cortex. (A) wildtype egg chamber, (B) chic 1320 /chic 221, (C) chic 1320 /chic 221 ; GFP-Capu, (D) chic 1320 /chic 221 ; GFP-CapuΔN, (E) chic 1320 /chic 221 ; GFP-Spir-D.
8 Figure S4. The Actin Mesh in Untreated and Latrunculin A Treated Egg Chambers (A-J) Confocal images of stage 9 egg chambers stained with TRITC-Phalloidin to label F-actin, (Grey scale bar, 30 µm). (A -J ) show a magnification of the F-actin stained oocyte cytoplasm imaged 10 µm from the cortex (White scale bar, 10µm). (A,C,E,G, I) are images of untreated egg chambers, while (B,D,F,H) are images of egg chambers after 8 hours of treatment. (A-D) were processed in parallel as were (E) and (F), (G) and (H), and (I) and (J). (A-B ) wildtype; (C-D ) capu G7 /Df(2L)ed SZ1 ; GFP-Capu; (E-F ) capu G7 /Df(2L)ed SZ1 ; GFP-CapuΔN; (G-H ) spire 2F /spire RP ; GFP-Spir-D; (I-J ) spire 2F /spire RP ; GFP- CapuΔN.
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