Actin Patch mRNA Localization and Dynamics Figure 1. Myo3 associates with RNAs encoding key regulators of actin nucleation at actin patches. (A) Identification of mRNAs associated with motor proteins in S. cerevisiae. Cells encoding motor proteins C-terminally tagged with 9Myc (shown) or EGFP were treated with latrunculin B (top) or fixed with formaldehyde (bottom). Cells were lysed and the motor proteins associated with mRNA cargoes were immunopurified using antibodies coupled to magnetic beads. Enriched mRNAs were eluted from the magnetic beads and, in the case of formaldehyde treatment, decrosslinked before labeling with cyanine dyes. Enriched mRNAs and mRNAs extracted from lysate were comparatively hybridized to yeast DNA oligonucleotide microarrays. (B) and (C) Distribution of the average log2 Cy5/Cy3 fluorescence ratios from four independent IPs and microarray hybridizations for identification of mRNAs associated with Myo3p in the presence of formaldehyde crosslinker (B) or after cells were incubated with latrunculin B (C). The insets show the mRNAs significantly enriched in Myo3p IPs after subtraction of mRNAs found in mock IPs. mRNAs encoding factors involved in endocytosis at actin patches are labeled in red. (D) Schematic of the steps of endocytic internalization at actin PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31912