Puf2 associates with Whi3 to control polarity. (A) puf2Δ cells grown for 20 h (30°C). Bar, 200 µm. (B) Whi3 (white) is localized in puf2Δ. Images are z-projected (>9 planes). Cells are outlined in gray. (C) Whi3-GFP and Puf2-6HA are localized in the same cell grown for 16 h (30°C; one focal plane). The inset shows 10× magnification. Pearson’s colocalization coefficient (from the top): 0.84, 0.61, and 0.60 (at hyphal tip, 5 × 5 µm²); 0.71, 0.56, and 0.50 (whole hypha). Costes p-value: 1. (D) Whi3-GFP is immunoprecipitated with GFP-trap in whole cell extract (WCE), WCE with RNasin, or WCE without RNasin (in RNase-free conditions). The untagged strain only expresses Puf2-6HA. Puf2-6HA was blotted with α-HA antibody. Molecular weights: Puf2-6HA, 122.3 kD; Whi3-GFP, 105.7 kD. (E) mRNAs bound to Puf2-6HA are RIPed with α-HA antibody, and cDNA generated from RT-PCR was identified by PCR. (F) BNI1 or SPA2 transcripts (yellow) are localized. Blue, DNA. (G) Overall mRNA density in each strain. P > 0.14 according to a KS test for both strains. (G–I) n > 38 for each strain. (H) Degree of mRNA clustering in each strain. *, P < 0.01 by KS test. (I) SPA2 (top) or BNI1 (bottom) mRNAs were counted and converted to a percentage. Error bars indicate SEM. Bars, 5 µm.