Microtubules

Poor characterization of the regulatory links between mitotic kinases and division-specific trafficking patterns represents a fundamental gap in our understanding of the interplay between cell division and signaling

Poor characterization of the regulatory links between mitotic kinases and division-specific trafficking patterns represents a fundamental gap in our understanding of the interplay between cell division and signaling. We have begun to address this gap by studying cranial-cardiac progenitor specification in the invertebrate chordate, (Type A, also referred to as embryos, the heart is derived from a set of 4 precardiac founder cells. heart progenitor induction in embryos cotransfected as indicated. Data were obtained from 3 impartial trials, > 17 per trial. Scale bars are indicated in micrometers. Significance indicated; n.s., not significant. Significance was decided using one-way ANOVA followed by Tukey multiple comparison test. Error bars represent SEM. Numerical values for all those graphs can be found in S6 Data. ATM, Anterior Tail Muscle Cell; FGFR, Fibroblast Growth Factor Receptor; SEM, standard error of mean; TVC, Trunk ventral cell/Cranial-cardiac progenitor.(PDF) pbio.3001029.s001.pdf (666K) GUID:?EC65F2D8-55E9-453A-BC06-D6DEADF39965 S2 Fig: Stage-specific quantitation of mitotic FGFR trafficking patterns (related to Fig 2). (A-A) Graphical summary of whole cell (A) and regional FGFR::VENUS/ CLIP::RAB7 colocalization (A-A; Manders overlap) during founder cell division (data shown correspond to data presented in Fig 2D). > 6 for each mitotic stage. Regional overlap was measured in 3 concentric regions, plasma membrane, peripheral cytoplasm, and deep cytoplasm (Fig 1A-A; Methods). Lack of any significant change (> 0.05) is indicated by no change in lettering (a for all those columns). Significance was decided using one-way ANOVA followed by Tukey multiple comparison test. Numerical values for all those graphs can be found in S7 Data. Error bars represent SEM. FGFR, Fibroblast Growth Factor Receptor; SEM, standard error of mean.(PDF) pbio.3001029.s002.pdf (121K) GUID:?5B06AA3A-8F72-445E-8EA2-3612E02FE5F8 S3 Fig: Inhibition of CDK1 does not impact endosomal maturation or slow recycling of FGF receptors during mitotic entry (related to Figs ?Figs22 and ?and33). (A-B) Masked/thresholded transverse sections of founder cells electroporated with and and treated as indicated. For clarity, panels showing only colocalized FGFR::VENUS/CLIP::RAB11 puncta are provided (OVERLAP; Manders overlap; MOC) (A and B). (C-E) Graphical summary of whole cell (C) and regional FGFR::VENUS/ CLIP::RAB11 colocalization (D-E; Manders overlap) in founder cells treated as indicated. (F-H) Graphical summary of whole cell (F) and regional FGFR::VENUS/ CLIP::RAB4 colocalization (G-H; Manders overlap) in founder cells treated as indicated. Data were obtained from 2 impartial trials, > 14. Scale bars are indicated in micrometers. Significance indicated by = 0.264). Data were obtained from 2 impartial trials, > 7. (D) Graphical summary of mitotic arrest observed for founder cells electroporated. Data were obtained from 3 impartial trials, > 22 per trial. (E-F) Representative micrographs of late tailbud embryos showing cranial-cardiac progenitor induction (indicated by overlap of and reporter expression along with migration into the head/trunk region) versus noninduced precardiac founder lineage cells (indicated by LY2119620 LY2119620 reporter expression alone along with lack of migration) in embryos coelectroporated with either or as indicated [20,40,41,21]. Note that prolongation of CDK1 activity appears to disrupt induction. This may be due to failure of transgenic cells to properly exit mitosis or it may reflect observed FGFR internalization. (G-H) Graphical summary of mitotic arrest and heart progenitor induction in embryos cotransfected as indicated. Data were obtained from 3 impartial trials, > 8 per trial. Arrested transgenic embryos (A-C) were fixed and analyzed at Hotta Stage 16 [22], approximately 1 hour after control cells (alone or in combination with and treated with vehicle (DMSO) or Roscovitine (14 mol/L) as indicated. alone also resulted in a modest, but LY2119620 not significant, increase in plasma membrane-associated FGFR::VENUS. Because phalloidin staining obscures FGFR::VENUS localization, red dashed lines were used to indicate phalloidin-stained cell membranes (A-B). Some regions are labeled with an a or b to denote significant changes (< 0.05) that occurred within this region across stages. Other regions are labeled n.s. to denote that no significant changes occurred for the indicated stages. Significance was decided using one-way ANOVA followed by Tukey multiple comparison test. (C) Quantification of the FGFR::VENUS enrichment in the plasma membrane-associated region of founder cells electroporated and treated as indicated. Significance was decided using one-way ANOVA followed by Tukey multiple comparison test. Numerical values for all those graphs can be found in S10 Data. Scale bars are indicated in micrometers. CDK1, Cyclin-dependent Kinase 1; FGF, Fibroblast Growth Factor.(PDF) pbio.3001029.s005.pdf (215K) GUID:?A4ED5718-5D9B-484A-8C44-319FFA879FB9 S6 Fig: RAB4 phosphomutants impact TVC induction (related to Fig 4). (A-D) Representative micrographs of late tailbud embryos showing induced cranial-cardiac progenitors (TVCs, arrowheads point to cells showing overlapping and Rabbit Polyclonal to SGK269 reporter expression) versus noninduced anterior muscle lineage cells (ATMs, arrows point to cells showing reporter expression alone) in.