(A) N-terminal TPR domain (amino acids 42–398), central linker region (399–466), and 'GoLoco domain' containing three GoLoco motifs (467–658). Serine 436 (S436; black ball).
(B) Spindle angle assay: a vector perpendicular to the Ed crescent (red dashed line) and along the spindle axis (black dashed line) indicates spindle angle.
(C–K) Pins domains were fused in frame to Echinoid tagged with a FLAG epitope (Ed-FLAG) and transfected into S2 cells. Cells were stained for FLAG (red) and α-tubulin (green). FL, Pins full length; FL+Gαi, PinsFL co-transfected with Gαi (Gαi is colocalized with Pins, not shown); FL(R-F)+Gαi, PinsFL with a mutated Gαi binding domain co-transfected with Gαi (Gαi is delocalized, not shown); TPR, Pins TPR domain alone; GoLoco, Pins GoLoco domain alone; Linker, Pins Linker domain alone; ΔLinker, Pins full length protein lacking the linker domain; TPR+Linker, Pins TPR and Linker domains. Scale bar 3 µm.
(L) Quantification. Mean spindle angle and standard deviation is shown (sampling error is estimated at ± 3°; see methods). The Pins TPR, GoLoco, and ΔLinker proteins show no spindle orientation (30–60° spindle angles; Pins FL, FL(R-F)+Gαi, Linker proteins show partial spindle orientation (~15–30° spindle angles); Pins FL+Gαi and TPR+Linker proteins show good spindle orientation (~0–15° spindle angles). *, highly significant compared to Ed alone 'none' (p < 0.01); #, significant spindle orientation compared to Pins FL (p < 0.05), other proteins showed no difference from Ed alone (p > 0.05).

(A) mud RNAi reduces Pins^TPR+LINKER spindle orientation to that of Pins^LINKER alone. (B) The Pins N226F TPR domain mutation abolishes Mud-Pins interaction in a FRET assay. (C) The Pins^{TPR(N226F)+LINKER} mutant has spindle orientation equal to that of Pins^LINKER alone. (D) lis-1 RNAi reduces Pins^TPR+LINKER spindle orientation to that of Pins^LINKER alone. (E) dlc RNAi reduces Pins^TPR+LINKER spindle orientation to that of Pins^LINKER alone. (F) Western blots show RNAi knockdown of Lis-1 and Dlc; detection of α-tubulin demonstrates equal loading of lysates. Scale bar 3 µm for A–E.
(G) Quantification of spindle orientation (mean spindle angle ± standard deviation). *, significantly better spindle orientation compared to Pins^LINKER (p < 0.01).
(H–K) The Pins^TPR domain is sufficient to recruit Mud to the cortex. Transfected GFP:Mud (residues 1825–2016, the Pins-binding domain) is recruited to the cortex by Pins^TPR+LINKER (I) or Pins^TPR (J) but not by Ed alone (H) or Pins^LINKER (K). Scale bar 3 µm for H–K.

(A) Interphase S2 cell transfected with Ed:FLAG:Pins^TPR+LINKER stained for the centriolar marker Sas-4 showing no centriole anchoring to the crescent.
(B) Prophase S2 cell transfected with Ed:FLAG:Pins^TPR+LINKER stained for the centrosomal marker Cnn showing centrosome anchoring to the crescent.
(C) Quantification of mean centrosome (Cnn) or centriole (Sas4) angle ± standard deviation. *, significant compared to Ed alone control (p < 0.01).
(D) Western blot demonstrates partial RNAi knockdown of Aurora-A; alpha-tubulin blot demonstrates equal loading of lysates.
(E) Aurora-A phosphorylates Pins on residue 436 in the Linker domain. Recombinant Aurora-A kinase and [γ-32P]-ATP were incubated alone or in the presence of wild-type or S436A versions of full-length or TPR+Linker Pins.
(F–K) Aurora-A phosphorylation of Pins S436 is required for Dlg recruitment and spindle orientation. Scale bar 3 µm for C–H.
(L) Quantification of spindle orientation (mean spindle angle ± standard deviation). The number of cells scored for each genotype are 26, 34, 28, 26, 8, 22, and 19 (from top to bottom). *, highly significant versus Ed alone (p < 0.01); #, significantly different from Pins^TPR+LINKER (p < 0.05 when assayed by either Cnn or Sas-4).
(M) Pins^LINKER/Aurora-A pathway is required in vivo for neuroblast spindle orientation. Larval neuroblasts in a pins62 homozygous mutant expressing the unphosphorylatable Pins^FL(S436A) protein show defective spindle orientation (left panel; note alignment to edge of crescent), whereas the phosphomimetic Pins^FL(S436D) protein show spindle pole alignment to the center of the Pins crescent (right panel). Pins (red), α-tubulin (green).
(N) A model for Pins-mediated spindle orientation. See text for details.

(A) The Echinoid (Ed) induced cortical polarity in S2 cells. Cortical polarization of Ed:GFP fusion protein (green); there is also cytoplasmic vesicle staining (asterisk); Cherry:Miranda protein (red) shows less cortical enrichment. Right: quantification of cortical pixel intensity (vesicle staining removed from plot).
(B) Induced functional cortical polarity in S2 cells. Polarized Ed:aPKC can exclude Cherry:Miranda (Mira) from the cortex, thereby inducing Cherry:Mira cortical polarization. Right: quantification of cortical pixel intensity. Scale bar 3 µm.
(C) Ed and Ed:aPKC are enriched at the site of cell-cell contact. Pixel intensity was measured for left (L), right (R), and contacting (C) cortical domains and fold enrichment at the contact site was calculated (C/L+R). Red line indicates a ratio of 1.0 (no enrichment or exclusion). n = 17 (Ed) and 18 (Ed:aPKC).
(D) aPKC excludes Miranda from the cortex. Cherry:Mira pixel intensity levels were measured as described in panel C. Red line indicates a ratio of 1.

S2 cells were transfected with Ed:GFP alone or the indicated Ed:Pins domains (green) and immunostained for endogenous Dlg or Tubulin (red). RNAi knockdown was performed for the indicated genes.
(A–E) Pins^LINKER recruits Dlg to the cortex.
(F–L) Pins^LINKER requires Dlg and Khc-73 for spindle orientation. The mitotic marker PH3 is shown in F. Western blot analysis shows RNAi knockdown of Dlg; detection of α-tubulin demonstrates equal loading of lysates (K). Scale bar 3 µm for A–J.
(L) Quantification of spindle orientation (mean spindle angle ± standard deviation). *, significantly better spindle orientation compared to Ed alone control (p < 0.01); other proteins showed no difference from Ed alone control (p > 0.05).

To focus on the mechanism of spindle alignment, only Ed:Pins cells that started metaphase with misaligned spindles (>15°) were analyzed.
(A–C) Top row: A–C correspond to supplemental movie 1–supplemental movie 3. Time is in minutes; arrowheads, spindle pole-to-cortex distance. Each line is a different cell; red line, cell shown in still frames and supplemental movies. Bottom row: quantification of spindle orientation angle to crescent center or crescent edge (Y axis) over time (X axis). (A) Ed:GFP control cells have slowly drifting spindles (n=4). (B) Ed:Pins^TPR+LINKER cells have dynamic spindle orientation (n=5). (C) Ed:Pins^LINKER cells have poor spindle orientation to the crescent center (top tracing), but good spindle orientation to the crescent edge (bottom tracings; n = 8). Scale bar 2 µm.
(D) Ed:Pins^TPR+LINKER cells have the greatest maximum spindle pole velocity.
(E) Ed:Pins^TPR+LINKER cells have the closest spindle pole-to-cortex distance during the 10 min interval prior to anaphase onset.
(F) Ed:Pins^LINKER cells show close spindle pole to edge of the crescent association, whereas Ed:Pins^TPR+LINKER cells show close spindle pole to center of the crescent association.