Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

(D) Schematic representation of a C. elegans embryo during anaphase spindle pole rocking indicating where kymographs are positioned in the embryo. (E) Representativekymographs of eGFP::DHC-1 localization at the posterior pole and cortex during anaphase spindle rocking. Graphs show relative cortical eGFP::DHC-1intensity as quantified by generating line scans of both top and bottom cortex intensities and subsequent normalization over adjacent cytoplasmic values.Bars: (A and B) 1 um; (E) 5 um.

Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

(A) Quantification of cortical eGFP::LIN-5 during centration, earlymetaphase, late metaphase, and anaphase as shown in representative images in top panels. Cortical intensity values are normalized over either cytoplasmic traces from below the cortex (first row of graphs) or over a single mean cytoplasmic value (second row). Dotted lines at y = 100% indicate a 1:1cortex/cytoplasm ratio. Nonnormalized cytoplasmic intensities indicating a gradient of eGFP::LIN-5 during all mitotic stages are also shown (third row).(B) Individual nonnormalized cortical traces of embryos expressing eGFP::LIN-5 and mCherry::DHC-1 during late metaphase and anaphase. Three representative embryos from the dataset used to quantify cortical enrichment and the cross-correlation index in Fig. 5 C are shown in both stages. Bars, 5 um.

Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

(A) SDCM images showingeGFP::DHC-1 localization in control and lin-5(RNAi) metaphase embryos. Graphs show relative cortical over cytoplasmic eGFP::DHC-1 intensities as measured along the cortex (far right, green line) and normalized to a cytoplasmic line (red). Dotted lines at y = 100% indicate a 1:1 cortex/cytoplasm ratio.Mean (green) and individual (gray) traces are shown. n = 12 embryos and 24 cortexes for control, and n = 5 embryos and 10 cortexes for lin-5(RNAi).(B) SDCM images of anaphase embryos of indicated genotypes. Graphs show relative cortical over cytoplasmic eGFP::DHC-1 intensities, quantified andshown as described in A. n = 6 embryos per condition. (C) SDCM images show mCherry::DHC-1 and eGFP::LIN-5 localization. Mean (thick lines) andindividual (thin lines) intensity plots are shown in left graphs. Right graphs show cross-correlation between cortical LIN-5 and DHC-1 plots expressed asmeans +- SEM. n = 10 embryos and 20 cortexes.

Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

Figure 1. Endogenous tagging of dhc-1. (A) Dynein tagging strategies. Colors indicate dhc-1 exons (blue), mcherry (red), egfp (green), and linkers (orange). Cartoons illustrate dynein heavy chain with FP fused to the C (top) or N terminus (bottom). (B) Brood size and embryonic lethality of dhc-1 knock-instrains shown as means +- SD. n = 4 animals each. (C) Widefield image showing mCherry::DHC-1 expression in an adult with a zoom of the germline (reddashed box) below. Bars: (top) 50 um; (bottom) 10 um. (D) SDCM images of eGFP::DHC-1 localization in the one-cell embryo. Bar, 5 um.

Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

Figure S4. EBP-2 is required for proper spindle midzone establishment, and cortical dynein patches follow oscillatory spindle movements. (A) Schematicrepresentation (left) of approach to measure MT (right) density along the mitotic spindle. (B) Montages of representative SDCM videos of one-cell embryosexpressing either GFP::TBB-2 or TBA-2::YFP (MTs) in different WT or ebp mutant backgrounds as indicated in each image. Montages are generated fromseven consecutive frames from time-lapse videos acquired at a rate of one frame per 5 s, and all were acquired with the same laser power and exposuretime of 500 ms. (C) Quantification of MT abundance along the horizontal axis of the spindle in different genetic backgrounds during early anaphase asindicated in the graph, represented as mean (n = 7) intensity relative to the maximum value of all datasets. Spindle length is in pixels. (D) Schematic representation of a C. elegans embryo during anaphase spindle pole rocking indicating where kymographs are positioned in the embryo. (E) Representativekymographs of eGFP::DHC-1 localization at the posterior pole and cortex during anaphase spindle rocking. Graphs show relative cortical eGFP::DHC-1intensity as quantified by generating line scans of both top and bottom cortex intensities and subsequent normalization over adjacent cytoplasmic values.Bars: (A and B) 1 um; (E) 5 um.

Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

Figure S5. Extended analysis of cortical LIN-5 and dynein localization during mitosis. (A) Quantification of cortical eGFP::LIN-5 during centration, earlymetaphase, late metaphase, and anaphase as shown in representative images in top panels. Cortical intensity values are normalized over either cytoplasmic traces from below the cortex (first row of graphs) or over a single mean cytoplasmic value (second row). Dotted lines at y = 100% indicate a 1:1cortex/cytoplasm ratio. Nonnormalized cytoplasmic intensities indicating a gradient of eGFP::LIN-5 during all mitotic stages are also shown (third row).(B) Individual nonnormalized cortical traces of embryos expressing eGFP::LIN-5 and mCherry::DHC-1 during late metaphase and anaphase. Three representative embryos from the dataset used to quantify cortical enrichment and the cross-correlation index in Fig. 5 C are shown in both stages. Bars, 5 um.(C) Quantification of cytoplasmic eGFP::LIN-5 and mCherry::DHC-1 during late metaphase and anaphase as shown in representative images in B. Nonnormalized cytoplasmic intensities are plotted as individual (thin) and mean (thick) plots. (D) Quantification of cytoplasmic eGFP::LIN-5 and eGFP::DHC-1FRAP. Curves are expressed as means +- SEM; egfp::lin-5, n = 7; egfp::dhc-1, n = 30.

Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

Figure 5. Cortical dynein depends on recruitment from the cytoplasm by LIN-5 but not on EBP-2-mediated plus end tracking. (A) SDCM images showingeGFP::DHC-1 localization in control and lin-5(RNAi) metaphase embryos. Graphs show relative cortical over cytoplasmic eGFP::DHC-1 intensities as measured along the cortex (far right, green line) and normalized to a cytoplasmic line (red). Dotted lines at y = 100% indicate a 1:1 cortex/cytoplasm ratio.Mean (green) and individual (gray) traces are shown. n = 12 embryos and 24 cortexes for control, and n = 5 embryos and 10 cortexes for lin-5(RNAi).(B) SDCM images of anaphase embryos of indicated genotypes. Graphs show relative cortical over cytoplasmic eGFP::DHC-1 intensities, quantified andshown as described in A. n = 6 embryos per condition. (C) SDCM images show mCherry::DHC-1 and eGFP::LIN-5 localization. Mean (thick lines) andindividual (thin lines) intensity plots are shown in left graphs. Right graphs show cross-correlation between cortical LIN-5 and DHC-1 plots expressed asmeans +- SEM. n = 10 embryos and 20 cortexes. (D) SDCM images showing dynein or LIN-5 localization in perm-1(RNAi) or perm-1(RNAi); lin-5(RNAi)embryos treated with 1 uM nocodazole (noc.). Graphs indicate fluorescence intensity profiles as measured in dashed boxes shown in superimposed panels.The red arrow points to mitotic spindle remnant. Plots are mean (n = 10) intensities relative to cytoplasmic values +- SD. Bars, 5 um.

Picture from Schmidt R et al. (2017) J Cell Biol "Two populations of cytoplasmic dynein contribute to spindle positioning in C. ...."

(C) Widefield image showing mCherry::DHC-1 expression in an adult with a zoom of the germline (reddashed box) below. Bars: (top) 50 um; (bottom) 10 um. (D) SDCM images of eGFP::DHC-1 localization in the one-cell embryo. Bar, 5 um.

Picture from Inoue T et al. (2004) Proc Natl Acad Sci U S A "Type II platelet-activating factor-acetylhydrolase is essential for ...."

(I and J) paf-2::GFP expression in embryos (lateral view, dorsal is up). Nomarski micrographs (I) and corresponding paf-2::GFP expression (J). paf-2 reporter gene constructs are expressed in epidermal cells (J, arrowheads) and intestinal cells (J, arrow) at the beginning of elongation. Anterior is to the left. (Bar, 10 um.)

Picture from Solari F et al. (1999) Development "The Caenorhabditis elegans genes egl-27 and egr-1 are similar to MTA1, a member of ...."

(J,K) egl-27::gfp expression in 1.5-fold embryo (J) and L3 hermaphrodite (K). All somatic cells appear to express the reporter gene.