An optogenetic assay shows that asymmetries in membrane potential can direct polarity.

<p>(A) Optogenetic assay to generate asymmetries in membrane potential and assess for effect on polarity. Schematic representation of the experimental setup: a yellow laser (λ = 535 nm) is used to photoactivate Halorhodopsin (Halo) in selected regions of <i>rsr1</i>Δ cells. θ is the final angle of shmoo or bud emergence with respect to the direction of the photoactivated region. (B) <i>rsr1</i>Δ (left) and Halorhodopsin-GFP-expressing <i>rsr1</i>Δ (right) cells in the presence of α-factor (αF) and retinal are continuously photoactivated from time 0 to 20 min at the indicated yellow region. After 2 h, shmoos grow and polarity orientation can be quantified with respect to the photoactivated region. White arrowheads indicate sites of shmoo formation. (C) Quantification of optogenetic experiments: radial histogram of polarized growth orientation with respect to photoactivation angle in <i>rsr1</i>Δ and <i>rsr1</i>Δ + Halorhodopsin-GFP cells treated with α-factor. (D) Average orientation of polarized growth in budding and shmooing cells after 2 h of growth following local photoactivation for a population of <i>rsr1</i>Δ, <i>rsr1</i>Δ Hxt3-GFP, and <i>rsr1</i>Δ + Halorhodopsin-GFP cells (<i>n</i>>70 cells gathered from four independent datasets for all conditions and <i>n</i> = 166 cells gathered from seven independent experiments for <i>rsr1</i>Δ + Halorhodopsin-GFP + α-factor). **Student's <i>t</i> test, <i>p</i><0.05. Error bars represent standard deviations.