Optogenetic control of organelle transport using a photocaged chemical inducer of dimerization

Cell polarity, growth and signaling require organelle transport by cytoskeletal motor proteins that are precisely regulated in time and space. Probing these complex, dynamic processes requires experimental techniques with comparable temporal and spatial precision. Inducible dimerization offers the ability to recruit motor proteins to organelles in living cells. Approaches include rapamycin-induced dimerization of motors and cargo-bound binding partners [1] or the recent application of the TULIP light-inducible dimerization system [2,3]. In the latter system, motor recruitment is activated by blue light, and relaxes to an OFF state in the dark within seconds. While rapid relaxation is desirable for some applications, many experiments require sustained motor recruitment. Here, we use a photocaged chemical dimerizer to achieve sustained, spatially-defined motor recruitment to individual organelles with a single pulse of light. We demonstrate the general applicability of the system by recruiting microtubule plus end-directed kinesin-1 and minus end-directed dynein motors to peroxisomes and mitochondria in HeLa cells and primary neurons, leading to alterations in organelle transport on timescales from <10 seconds to >10 minutes after photoactivation. We recently developed a photoactivatable chemical dimerizer, cTMP–Htag, a synthetic small molecule comprising a Halotag ligand linked to photocaged trimethoprim (TMP). This molecule is designed to heterodimerize Halotag (Halo) and Escherichia coli DHFR (eDHFR) fusion proteins [4]. Here we use light to recruit eDHFR-tagged molecular motors or motor effectors to specific organelles. cTMP–Htag is cell permeable and covalently binds the Halotag protein, which we Correspondence localized to the cytosolic surface of either peroxisomes or mitochondria [1,4]. While photocaged, TMP does not bind eDHFR. Uncaging with a pulse of ~400 nm light recruits eDHFR-fusions to the organelle surface (Figure 1A). Photoactivation is spatially restricted to the illuminated organelle since uncaged TMP remains covalently tethered to the Halotag anchor. TMP–eDHFR binding is noncovalent, so individual motor– eDHFR proteins may bind and release, but at steady state the interaction sustains robust motor recruitment. Dimerization can be reversed within minutes by addition of free TMP [4]. We tested three constructs: the constitutively active motor domain of kinesin-1 (amino acids 1–560, K560); an amino-terminal fragment of kinesin light chain 1 (KLC1), which binds and recruits kinesin heavy chain; and an amino-terminal fragment of Bicaudal D (BICD), a motor effector that binds and recruits dynein. To localize Halotag protein, we used the peroxisometargeting sequence from human PEX3 or the mitochondrial outer membrane targeting sequence (Mito) from Listeria monocytogenes ActA (Figure 1A). HeLa cells expressing PEX3–GFP– Halo, together with either KLC1– mCherry–eDHFR or BICD-–mCherry– eDHFR, were treated with cTMP–Htag. Before uncaging, peroxisomes localized uniformly (Figure 1B), with motor or effector constructs diffuse throughout the cytosol. In response to a 500 ms widefield pulse of 387 ± 5 nm light, the motor and effector constructs relocalized to peroxisomes within 30 seconds (Figure S1A,B) and transported them to the periphery or to the center of the cell, respectively, as predicted for kinesinor dynein-driven motility (Figure 1B, Movie S1). Recruiting K560 or BICD to mitochondria induced transport as well as a striking increase in elongated mitochondria within 5–20 seconds (Figure S1C,D). KLC1 recruitment relocalized mitochondria more slowly, over ~10 minutes, without pronounced morphological changes (Figure S1E). These observations highlight the organelle-specific and motor/effector-specific regulation of intracellular transport [5]. The power of optogenetics is its potential for localized control on subcellular length scales. Using 405 nm light, we photoactivated defined regions