mRNAs encoding the Arp2/3 components are also localized to sites of actin branching and cytoskeletal rearrangement in mammalian cells [20,21,22,23,24,25], suggesting that this may be a common mechanism for localization of mRNAs important for actin polarization. Localization and Dynamics as mRNA-Specific Programs Both the localization pattern and dynamics of BBC1 and ARG3 are distinct. The differences could be due to the different interactions with motor systems, local cellular structures, ribo- somes, and RNPs. We suspect that differences in mRNP localization and dynamics are encoded in each mRNA by an mRNA-specific arrangement of RBP binding sites, which may also program interactions with the translation apparatus and mRNA decay machinery [5,43,46,120]. Previous studies of ASH1 localization in yeast have indicated that the actomyosin protein, Myo4, binds the ASH1 transcript indirectly through the RBP, She2, and the adapter and RBP, She3 [8,9,10,11,12,13,14], as well as the RBPs Khd1 and Puf6, which act jointly to mediate specific transport and to repress translation until the mRNA has reached the appropriate cellular location [41,42,125,126]. Disruption of SHE2, SHE3, PUF6, or KHD1 impairs proper ASH1 localization [6,41,125,127]. The precedent set by the ASH1 system suggests that the specific constellation of RBPs and ribosomes that associate with each mRNA may have distinct and important roles in mRNA localization. The full molecular system that executes the localization program of AP mRNAs remains to be defined, although we suspect that one or more RBPs may act as sequence-specific adapters between Myo3 and the AP mRNAs. The definition of such a molecular system will be a critical step toward understanding how the localization pattern and dynamics of the AP mRNAs are encoded and executed and how they contribute to fitness. In the model we favor for AP mRNA localization, the non- processive motor, Myo3, promotes asymmetric localization of AP mRNAs using a ‘‘parkour-like’’ mechanism that uses actin filaments it encounters by diffusion as platforms for saltatory, directed movements, to introduce a directional bias to the diffusion of AP mRNPs. We hypothesize that the ‘‘parkour’’ mechanism could be a simple and widespread mechanism by which non- processive motors can localize mRNAs and other cargoes to diverse cellular sites. Indeed, the abundance of non-processive motors in other organisms combined with the diversity of mRNAs that might localize to distinct locations within the crowded and heterogeneous cellular environment might necessitate such a simple and adaptable means of achieving asymmetry without the traffic jams and queueing that could impair the efficiency of processive motors. New analytical tools will need to be developed in order to detect and verify what will likely be subtle biases in path for non-processive, motor-associated mRNAs and other asymmet- rically localized cellular components. Materials and Methods RNA immunoaffinity purifications Formaldehyde crosslinking of motors. 250 ml cultures of S. cerevisiae strains containing 9Myc- or GFP-tagged motor proteins were grown to mid-log phase in YPD medium then incubated with 1% formaldehyde (v/v) for 20 min before collection by centrifugation. Cell pellets were washed with 25 mls and then 10 mls of cold Tris-buffered saline (20 mM Tris-HCl [pH 7.5], 150 mM NaCl). Pellets were then frozen in LN2 and stored at 280uC. For lysate preparation, pellets were resuspended in 450 ml of lysis buffer (50 mM HEPES-KOH Actin Patch mRNA Localization and Dynamics [pH 7.5], 140 mM NaCl, 0.1% Nonidet-P40, 1 mM EDTA, 0.1% sodium deoxycholic acid, 1 mM PMSF, 1 mM DTT, 0.1 U/ul SUPERase-In [Applied Biosystems], and 12.5 mg/ml each of pepstatin, leupeptin, aprotinin, antipain, and chymostatin) and mechanically disrupted with glass beads in a mini-beadbeater- 8 (Biospec Products). The insoluble pellet was recovered and suspended in 1.8 ml of lysis buffer then sonicated using a Branson Sonifier with settings at 50% duty cycle and at the microtip maximal power setting for three minutes with rests on ice to prevent heating of the sample. The sonicated extract was cleared by centrifugation at 4,000 g for 1 min and the soluble portion was transferred to a new tube; 50 ml of the extract was removed for reference RNA isolation. 100 ml of pan-mouse IgG Dynal magnetic beads (Invitrogen) was pre-blocked with 5 mg/ml BSA in cold phosphate-buffered saline (PBS) and pre-incubated with either 10 ml of 9E11 monoclonal anti-c-myc antibody (Lab Vision) or JL-8 (anti-GFP) Living Colors A.v. monoclonal antibody (Clontech) overnight. Beads were washed with 5 mg/ml BSA in PBS and pre-equilibrated in lysis buffer before addition to the lysate (,1.6 ml). Bead/lysate suspensions were incubated for 4 h at 4uC with gentle rotation. Beads were collected with a magnet and washed two times with 1 ml of lysis buffer, then two times with 1 ml of lysis buffer with increased sodium chloride concentration (360 mM NaCl final). Protein/RNA complexes were eluted from the beads by the addition of 100 ml of elution buffer (50 mM Tris- HCl [pH 8.0], 10 mM EDTA, 1% SDS, 0.04 U/ml SUPERase- In, and 10 mM DTT) and incubation at 70uC for 45 min with shaking. 50 ml of the reference extract was combined with 150 ml of elution buffer and also incubated at 70uC for 45 min with shaking. RNA was purified using the RNeasy Mini Kit (Qiagen) with on-column DNAse digestion following manufacturer’s instructions. Latrunculin B purification of motors. Immuno- purifications of 9Myc- or GFP-tagged motor proteins from cultures treated with latrunculin B were carried out in a manner similar to that described for crosslinking cells with some modifications. 250 ml cultures were grown to mid-log phase, harvested by centrifugation, and washed with 25 mls and then 10 mls of cold Tris-buffered saline. Pellets were placed on ice and resuspended in 600 ml of lysis buffer with the addition of 2 mg/ml latrunculin B, then mechanically disrupted by a beadbeater. Lysates were cleared by centrifugation at 4,000 g for 1 min at 4uC, the volume was brought up to 1.4 ml using lysis buffer, and 200 ml was removed for reference RNA isolation. 100 ml of pan-mouse IgG Dynal magnetic beads pre-incubated with antibody (see above) was combined with the remaining lysate and incubated for 4 h at 4uC with gentle rotation. Washes were performed as described above. Protein/RNA complexes were eluted with elution buffer (see above), incubation at 70uC for 10 min, and vigorous vortexing. Immunopurified and total lysate RNA were extracted with phenol:chloroform:isoamyl alcohol (25:24:1; Invitrogen) twice and chloroform once using 2.0 ml Phase Lock Heavy tubes (Eppendorf) followed by ethanol precipitation in the presence of 7.5 mg Glycoblue (Ambion) as carrier. Microarray sample preparation, hybridization, washing, and scanning The entire RNA eluate (,1 mg) from immunopurifications and 1 mg of total RNA purified from lysates were amplified using the amino allyl messageamp II RNA amplification kit (Applied Biosystems) following manufacturer’s instructions with minor modifications. 20 ng of doping control RNAs (Stanford Functional Genomics Facility) were spiked into the labeling reactions to aid in microarray scanning. The entire product of IP-enriched RNA PLoS ONE | www.plosone.org 14 February 2012 | Volume 7 | Issue 2 | e31912