Breznau EB , Murt M , Blasius TL , Verhey KJ , Miller AL .

R01 GM052932 NIGMS NIH HHS , R00 GM089765 NIGMS NIH HHS , R01 GM070862 NIGMS NIH HHS , R01 GM112794 NIGMS NIH HHS , T32 GM007315 NIGMS NIH HHS

	Fig. 1. MgcRacGAP localizes to microtubule plus ends at the equatorial cortex as cytokinesis initiates. (A) Still images from a single z-plane time-lapse movie of a X. laevis embryo co-injected with Mgc MO+Mgc–3×GFP and a probe for MTs (2×mChe–EMTB). Mgc–3×GFP (green) localizes in the nucleus of interphase cells, at overlapping central spindle MTs, as well as at individual MTs at the equatorial cortex prior to furrowing. A FIRE look-up table (LUT) plugin was applied to the Mgc–3×GFP channel to highlight Mgc localization, and enlarged regions are shown on the right (arrows, overlapping central spindle MTs; arrowheads, individual MTs at equatorial cortex). (B) Still images from a single z-plane time-lapse movie of a X. laevis embryo co-injected with Mgc MO+Mgc– 3×GFP and 2×mChe–EMTB. The dashed boxes in B indicate regions that are enlarged in B′ and B″. The frames in B′ show Mgc–3×GFP decorating equatorial astral MTs (arrow) and Mgc–3×GFP puncta at MT plus ends (arrowheads). The frames in B″ show directed movement of an Mgc–3×GFP puncta (arrowheads), apparently along astral MTs, as Mgc–3×GFP forms clusters at the equatorial cell cortex.
	Fig. 2. MgcRacGAP tracks with EB3 at growing microtubule plus ends. (A) Amino acid sequence alignment surrounding the SxIP motif (yellow box) of five known EB1-binding proteins and the SxIP motif in the C-terminus of X. laevis Mgc. Basic arginine (R) and lysine (K) residues are shown in blue. Stop codons are indicated by *. (B) Mgc domain diagram. Endogenous Mgc is knocked down with MOs that target the 5′UTR; MgcWT and MgcSxNN constructs do not contain the endogenous 5′ UTR sequence, so they are not targeted by the MOs. The location of the putative SxIP motif in Mgc (red circle) and the SxNN mutation (blue square) are indicated. (C) TIRF microscopy image of a Cos-7 cell transfected with EB3–mChe (red) and MgcWT–mNeon (green). Scale bar: 20 µm. (C′) An enlarged view of the boxed region in C showing that EB3–mChe and MgcWT–mNeon accumulate on the plus ends of microtubules (yellow arrowheads). Scale bar: 5 µm. (D) Representative kymographs following an EB3–mChe comet in cells co-expressing EB3–mChe and MgcWT–mNeon. Scale bar: 4.3 µm (distance), 1 sec (time). (E) TIRF microscopy image of a Cos-7 cell expressing EB3–mChe (red) and MgcSxNN–mNeon (green). Scale bar: 20 µm. (E′) An enlarged view of the boxed region in E showing EB3–mChe comets (yellow arrowheads) and the strongly reduced corresponding signal in the MgcSxNN–mNeon channel. Scale bar: 5 µm. (F) Representative kymographs following an EB3–mChe comet in cells co-expressing EB3–mChe and MgcSxNN–mNeon. The total time represented is 6.5 s; scale bar: 4.3 µm (distance). (G–I) Quantification of (G) Mgc–mNeon intensity at EB3 comets. MgcWT=26 kymographs, 9 cells; MgcSxNN=25 kymographs, 8 cells. (H) EB3–mChe run duration on MT plus ends. Control=35 EB3 comets, 7 cells; MgcWT=50 EB3 comets, 10 cells; MgcSxNN=50 EB3 comets, 10 cells. (I) Velocity of EB3–mChe comets. Control=15 EB3 comets, 7 cells; MgcWT=17 EB3 comets, 9 cells; MgcSxNN=17 EB3 comets, 9 cells. Mean±s.e.m. (unpaired Student’s t-test). All n values represent cumulative values from three independent experiments. ns, not significant.
	Fig. 3. The MgcRacGAP SxIP motif is necessary for successful cytokinesis and proper astral microtubule structure. (A) Still images from time-lapse movies of X. laevis embryos at NF stage 10-11. Embryos were co-injected with Mgc MO and either MgcWT or MgcSxNN along with a probe for DNA (mChe–H2B) and a probe for F-actin (mChe–UtrCH). Red asterisks indicate multinucleate cells. (B) Still images from time-lapse movies of NF stage 13-14 X. laevis embryos injected as described in A. Red asterisks indicate multinucleate cells. (C) Quantification of the percent of multinucleate cells in MO+MgcWT or MO+MgcSxNN embryos at NF stage 10-11 or NF stage 13-14. Stage 10-11 – MO+MgcWT=230 cells, 13 embryos, 5 experiments; MO+MgcSxNN=203 cells, 12 embryos, 5 experiments; stage 13-14 – MO+MgcWT=109 cells, 3 embryos, 1 experiment; MO+MgcSxNN=60 cells, 4 embryos, 1 experiment. (D) Still images from time-lapse movies of NF stage 10-11 X. laevis embryos injected as described in A showing symmetric (MO+MgcWT) and asymmetric (MO+MgcSxNN) divisions. Quantification of the percent of asymmetric cell divisions in MO+MgcWT or MO+MgcSxNN embryos is indicated below each panel. (E) Images of fixed NF stage 10-11 X. laevis embryos that had been co-injected with a lineage tracer (mChe–membrane, not shown) along with MO alone, MO+MgcWT, or MO+MgcSxNN, then fixed and stained with anti-α-tubulin antibodies to reveal MTs, and DAPI to label the DNA. (F) Schematic of the astral MT intensity quantification strategy. Intensity measurements were taken from the boxed regions and averaged for each cell. (G) Quantification of astral MT intensity, normalized to the cytosol background. Note that in order to observe astral MTs, the spindle MTs sometimes had to be saturated, but the MTs used for astral MT quantification were not saturated. Control=7 cells, 6 embryos; MO=7 cells, 7 embryos; MO+MgcWT=7 cells, 6 embryos; MO+MgcSxNN=8 cells, 6 embryos. Mean±s.e.m. (unpaired Student’s t-test). Scale bars: 25 µm (A,B); 10 µm (D,E). All n values represent cumulative values from three independent experiments, unless otherwise indicated. Maximum projection images are shown and were used for quantification.
	Fig. 4. Mutation of the MgcRacGAP SxIP motif disrupts equatorial accumulation of the Centralspindlin complex. (A) Still images from live movies of NF stage 10-11 X. laevis embryos that had been co-injected with MO+MgcWT–3×GFP or MO+MgcSxNN–3×GFP (gray), a probe for MTs (2×mChe–EMTB, red) and a probe for DNA (mChe–H2B, red). (B) Still images from live movies of NF stage 10-11 X. laevis embryos that had been co-injected with MO+MgcWT or MO+MgcSxNN, along with MKLP1–3×GFP (gray), 2×mChe–EMTB (red) and mChe–H2B (red). (C) Schematic of contractile ring (CR) and polar cortex intensity quantification strategy. Quantification was performed at 25% cleavage furrow ingression. (D) Quantification of the Mgc–3×GFP intensity at the contractile ring. MO+MgcWT–3×GFP, 9 cells; MO+MgcSxNN–3×GFP, 10 cells. (E) Quantification of the Mgc–3×GFP intensity along the polar cortex of dividing cells. MO+MgcWT, 9 cells; MO+MgcSxNN, 10 cells. (F) Quantification of the MKLP1–3×GFP intensity at the contractile ring. MO+MgcWT, 8 cells; MO+MgcSxNN, 8 cells. (G) Quantification of the MKLP1–3×GFP intensity along the polar cortex of dividing cells. MO+MgcWT, 8 cells; MO+MgcSxNN, 8 cells. Scale bars: 10 µm. Mean±s.e.m. (unpaired Student’s t-test). All n values represent cumulative values from three or more independent experiments. Maximum projection images are shown and were used for quantification.
	Fig. 5. The MgcRacGAP SxIP motif contributes to limiting and focusing RhoA-GTP and F-actin at the cell equator. (A) Still images from time-lapse movies of NF stage 10-11 X. laevis embryos that had been co-injected with MO+MgcWT or MO+MgcSxNN along with a probe for active RhoA (GFP–rGBD), a probe for F-actin (mChe–UtrCH) and a probe for DNA (mChe–H2B). Yellow arrowheads highlight RhoA-GTP accumulation along the equatorial cell cortex during early anaphase. (B) Same cells as in A showing the F-actin accumulation during early anaphase. (C) Schematic of the analysis approach for fitting a Gaussian curve to the intensity plot data to determine FWHM (breadth). The intensity of RhoA-GTP at the equatorial cortex just after anaphase onset and the intensity of F-actin accumulation during early anaphase (at 25% ingression), were measured. The FWHM is graphed as a percentage of cell width. (D) FWHM quantification of the breadth of RhoA-GTP accumulation at the equatorial cortex. MO+MgcWT=6 cells; MO+MgcSxNN=9 cells. (E) RhoA-GTP intensity quantification at the equatorial cortex. MO+MgcWT=6 cells; MO+MgcSxNN=9 cells. (F) FWHM quantification of F-actin contractile ring breadth. MO+MgcWT=8 cells; MO+MgcSxNN=6 cells. (G) F-actin contractile ring intensity quantification. MO+MgcWT=8 cells; MO+MgcSxNN=6 cells. Scale bars: 10 µm. Mean±s.e.m. (unpaired Student’s t-test). ns, not significant. All n values represent cumulative values from three or more independent experiments. Maximum projection images are shown and were used for quantification.
	Fig. 6. The MgcRacGAP SxIP motif is necessary for proper adherens junction structure. (A,C,E) Fixed maximum projection en face and side-view images of mosaic NF stage 10-11 X. laevis embryos. The mosaic injection strategy allows for approximately half of the embryo to express the indicated knockdown and rescue constructs along with GFP-membrane or mChe-membrane as a lineage tracer, while the other half of the embryo serves as an internal control and is not injected. The dotted white line in the merged en face image represents the boundary between the internal control and injected region of mosaic embryos. Antibodies against GFP and one of Mgc (A), β-catenin (C; β-cat) or E-cadherin (E; E-cad) were applied to the embryos after fixation. Side views show the localization of the indicated protein along the apicobasal axis of cell–cell junctions (red arrowheads, internal control regions; green arrowheads, injected regions). (A) Mgc (red) and GFP-membrane (green). (B) Quantification of the normalized Mgc intensity at bicellular junctions from the apical to basal surfaces. Control, 13 cells; MO+MgcWT, 14 cells; MO+MgcSxNN, 19 cells. (C) β-catenin (red) and GFP-membrane (green). (D) Quantification of the normalized β-catenin intensity along the bicellular junctions from the apical to basal surfaces. Control, 17 cells; MO+MgcWT, 16 cells; MO+MgcSxNN, 19 cells. (E) E-cadherin (pseudo-colored red) and mChe-membrane (pseudo-colored green). (F) Quantification of the normalized E-cadherin intensity along the bicellular junctions from the apical to basal surfaces. Control, 19 cells; MO, 7 cells; MO+MgcWT, 15 cells; MO+MgcSxNN, 19 cells. Error bars represent s.e.m. (unpaired Student’s t-test). ns, not significant. Scale bars: 20 µm. All n values represent cumulative values from three independent experiments.
	Fig. 7. Model of MgcRacGAP tethering to MT plus ends. We propose that MKLP1 transports Centralspindlin toward MT plus ends, and the Mgc SxIP motif mediates tethering of Centralspindlin to MT plus ends via EB proteins in order to facilitate proper accumulation of Mgc at equatorial astral MTs during cytokinesis and at epithelial cell–cell junctions.

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