Figure S5.
N-terminally truncated Feo::GFP weakly dimerizes and causes nuclear separation defects, and full-length Feo::GFP protein partially rescues nuclear delivery to the cortex of feo RNAi embryos. Related to Fig. 5. (A) Coomassie-stained 15% SDS-PAGE of heterologous expressed and purified full-length sFeo::GFP-His6 (left) and an N-terminally truncated sFeo::GFP-His6 construct missing the putative dimerization and Klp3A-binding domain (right). The expected molecular mass is at the top, and the measured mass is on the right of the bands (details in Materials and methods). The lower bands are contaminants that were not separated by gel filtration and are of bacterial origin as determined by mass spectrometry. (B) Western blot of a 4–12% gradient SDS-PAGE (left) next to a native-PAGE (right) with sFeoFL::GFP-His6 and sFeoN::GFP-His6, using mouse anti-GFP antibody. Both protein constructs migrate according to expected molecular mass of a monomer in the SDS-PAGE gel. In the native-PAGE gel, the full-length Feo migrates as expected from a dimer. The slight deviation from the expected mass of a dimer (212 kD) can be explained by the rod-like structure of the protein, as was shown for the human homologue Prc1 (Subramanian et al., 2010). Conversely, the truncated construct migrates like a weak dimer, causing a wide distribution ranging between 100 and 200 kD. (C) Time-lapse fluorescence images of a (control) blastoderm embryo expressing H2Av::RFP (magenta) after injection of sFeoN::GFP-His6 protein (green) and Alexa Fluor 647–Tubulin (gray). During the cycle just after injection (left), the localization of sFeoN::GFP-His6 at the spindle midzone was not detectable (arrowhead). However, the effect of protein addition manifests in nuclear separation defects, and in the subsequent division (middle), spindles are observed in unnatural proximity, leading to spindle fusion (arrow). In telophase of the same cycle (right), sFeoN::GFP-His6 is detected at the spindle midzone (arrowheads). This delayed localization after injection is not observed for sFeoFL::GFP-His6 (Fig. 5 B). Time is in min:s. Scale bar, 10 µm. (D) The position (circle) of every nucleus arriving at the embryo cortex after the last preblastoderm division, relative to the axial and lateral borders of the embryo, for each condition: feo (35467) mock-injected (buffer), feo (35467) rescued by protein injection, and control (mcherry) mock-injected. The green dashed rectangle represents the area of the embryo bounded by the length and width of the visible embryo in the confocal stacks, with the anterior end at the coordinate origin. The cyan cross represents the location of the 2D centroid defined from the position of all nuclei. The nuclei in interphase of the first division at the cortex are marked in black, and nuclei that have progressed to metaphase/anaphase are marked in magenta.

N-terminally truncated Feo::GFP weakly dimerizes and causes nuclear separation defects, and full-length Feo::GFP protein partially rescues nuclear delivery to the cortex of feo RNAi embryos. Related to Fig. 5. (A) Coomassie-stained 15% SDS-PAGE of heterologous expressed and purified full-length sFeo::GFP-His6 (left) and an N-terminally truncated sFeo::GFP-His6 construct missing the putative dimerization and Klp3A-binding domain (right). The expected molecular mass is at the top, and the measured mass is on the right of the bands (details in Materials and methods). The lower bands are contaminants that were not separated by gel filtration and are of bacterial origin as determined by mass spectrometry. (B) Western blot of a 4–12% gradient SDS-PAGE (left) next to a native-PAGE (right) with sFeoFL::GFP-His6 and sFeoN::GFP-His6, using mouse anti-GFP antibody. Both protein constructs migrate according to expected molecular mass of a monomer in the SDS-PAGE gel. In the native-PAGE gel, the full-length Feo migrates as expected from a dimer. The slight deviation from the expected mass of a dimer (212 kD) can be explained by the rod-like structure of the protein, as was shown for the human homologue Prc1 (Subramanian et al., 2010). Conversely, the truncated construct migrates like a weak dimer, causing a wide distribution ranging between 100 and 200 kD. (C) Time-lapse fluorescence images of a (control) blastoderm embryo expressing H2Av::RFP (magenta) after injection of sFeoN::GFP-His6 protein (green) and Alexa Fluor 647–Tubulin (gray). During the cycle just after injection (left), the localization of sFeoN::GFP-His6 at the spindle midzone was not detectable (arrowhead). However, the effect of protein addition manifests in nuclear separation defects, and in the subsequent division (middle), spindles are observed in unnatural proximity, leading to spindle fusion (arrow). In telophase of the same cycle (right), sFeoN::GFP-His6 is detected at the spindle midzone (arrowheads). This delayed localization after injection is not observed for sFeoFL::GFP-His6 (Fig. 5 B). Time is in min:s. Scale bar, 10 µm. (D) The position (circle) of every nucleus arriving at the embryo cortex after the last preblastoderm division, relative to the axial and lateral borders of the embryo, for each condition: feo (35467) mock-injected (buffer), feo (35467) rescued by protein injection, and control (mcherry) mock-injected. The green dashed rectangle represents the area of the embryo bounded by the length and width of the visible embryo in the confocal stacks, with the anterior end at the coordinate origin. The cyan cross represents the location of the 2D centroid defined from the position of all nuclei. The nuclei in interphase of the first division at the cortex are marked in black, and nuclei that have progressed to metaphase/anaphase are marked in magenta.

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