Figure 4. Gravin-Aurora A-Plk1 scaffold is preferentially sequestered at mother spindle poles.

(A) Spinning disc confocal micrograph (maximum projection) of a metaphase wild-type MEF depicts asymmetric enrichment of p766-Gravin (red) at one spindle pole. Counterstaining with tubulin (MTs, green) and DAPI (DNA, blue) are shown. Composite image is shown. Bar, 5 μm. (B) Ground state depletion microscopy (GSDIM) was performed on prometaphase HEK293 cells (top). Cells were immunostained for a mother spindle pole marker, Cenexin (green) and p-Gravin (red). Quantification of these signals is shown below micrographs. Integrated intensity profiles for (top) cenexin and (bottom) p-Gravin at the mother spindle pole (left). Intensity profiles for both proteins at the daughter spindle pole are also presented (right). Scale bar, 1 μm. (C) Relative GSD signals for p766-Gravin, Aurora A, Plk1, and Cenexin at the mother spindle pole (red). Centrobin (gray) was used as a daughter spindle pole marker. Cell numbers used in each calculation are indicated on graph (n = 3 experiments ± SEM). (D) GSDIM micrographs showing the distribution of Plk1 at spindle poles in (top, left) control and (top, right) Gravin-depleted HEK293 cells. Densitometric analyses depict the asymmetric distribution of Plk1 at mother and daughter spindle poles in (bottom, left) control and (bottom, right) Gravin knockdown cells. (E) Amalgamated data are shown in graph. Cell numbers used in each calculation are indicated on graph (n = 3 experiments ± SEM). (F) SIM maximum projection of (top) wild-type and (bottom) Gravin null MEFs at metaphase. Immunostaining for tubulin (green), centrobin (red), and pericentrin (blue) are presented. The daughter spindle pole was decorated with centrobin (red) and marked on the micrograph with D, whereas the mother spindle pole is denoted with M. Composite images are included. Dashed line (white) depicts path of line-scan used to determine which pole contained the most centrobin (see Figure 4—figure supplement 4I and J). Scale bar, 2 μm. (G) Comparison of astral microtubule (MT) length (μm) protruding from the mother (red; n = 64) and daughter spindle poles (gray, n = 62) in wildtype MEFs (n = 5 cells, ±SEM, ***p < 0.0001). (H–I) Quantitation of astral microtubule (MT) length protruding from the mother (red; n = 34) and daughter spindle poles (gray, n = 35) in Gravin null MEFs (n = 5 cells, ±SEM, ns depicts not significant).

DOI: http://dx.doi.org/10.7554/eLife.09384.008

Figure 4—figure supplement 1. Super-resolution microscopy identifies p-Gravin, Plk1, and Aurora A location and codistribution in mitotic cells.

(A and B) Improved resolution with GSD microscopy. (A) An HEK293 cell in metaphase stained for p-Gravin (red) and Cenexin (green) was imaged by widefield microscopy using the Leica oil-immersion HC PL APO 160×/1.43 NA super-resolution objective on a Leica GSD/TIRF microscope. White box outlines the area in which ground state depletion (GSD) was performed for part (B). Bar, 20 μm. (B) Comparative analysis of the same cell by GSD microscopy. Mother and daughter spindle poles are indicated (white boxes). Analyses of these regions are presented in Figure 4B,C. (C) GSDIM (top) shows cells immunostained for a mother spindle pole marker, Plk1 (green) and Aurora A (red). Quantification of these signals is shown below micrographs. Integrated intensity profiles for (top) Plk1 and (bottom) Aurora A at the mother spindle pole (left). Intensity profiles for both proteins at the daughter spindle pole are also presented (right). Scale bar, 1 μm. (D) GSD microscopy showing the distribution of Centrobin (green) and pT766-Gravin (red) at both spindle poles of an HEK293 cell. Bar, 1 μm. An intensity profile for each signal is included. (E) GSD microscopy showing the distribution of Centrobin (green) and Aurora A (red) at both spindle poles of an HEK293 cell. Bar, 1 μm. Intensity profiles of each signal are included. (F) A PLA (red) was used in U2OS cells stably expressing a mother centriole marker (myc-centriolin)¹ to determine which spindle pole predominates for Aurora A/p766-Gravin interaction. The interaction sites are identified by PLA puncta (red), and centriolin (blue), and tubulin (green) are shown. Bar, 10 μm. (G) Quantification of the relative amount of PLA signal at the mother spindle pole compared to the daughter spindle pole is shown for (light gray) Aurora A/p766-Gravin interaction and (dark gray) Plk1/p766-Gravin interaction (total number of cells are indicated, ±SEM, n = 3 experiments). (H) HEK293 cells that stably express control or Gravin shRNA were synchronized in mitosis. Lanes 1 and 2 were loaded with mitotic lysates, whereas lanes 3 and 4 were loaded with control interphase lysates. Immunoblots were probed for (top) Gravin, (upper mid) Aurora A, (lower mid) Plk1, and (bottom) GAPDH as a loading control. (I, J) Line plot analysis of (I, blue line) pericentrin and (J, red line) centrobin showing the integrated fluorescent intensity of each protein (points along a line) across the mitotic spindle (as depicted in Figure 4F). (K) The number of astral microtubule at the mother (red bars) and daughter (gray bars) spindle poles were counted in wild-type and Gravin null MEFs, and cells rescued with the murine Gravin T766A mutant that is unable to interact with Plk1 (n = 5 cells ± SEM).

DOI: http://dx.doi.org/10.7554/eLife.09384.009