Daphne L. Che

Optogenetic Control of Molecular Motors and Organelle Distributions in Cells

Intracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival, and apoptosis. Here, we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon exposure to blue light, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven toward the cell periphery upon light-induced recruitment of kinesin, or toward the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible, and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells.

The Dual Characteristics of Light-Induced Cryptochrome 2, Homo-oligomerization and Heterodimerization, for Optogenetic Manipulation in Mammalian Cells

The photoreceptor cryptochrome 2 (CRY2) has become a powerful optogenetic tool that allows light-inducible manipulation of various signaling pathways and cellular processes in mammalian cells with high spatiotemporal precision and ease of application. However, it has also been shown that the behavior of CRY2 under blue light is complex, as the photoexcited CRY2 can both undergo homo-oligomerization and heterodimerization by binding to its dimerization partner CIB1. To better understand the light-induced CRY2 activities in mammalian cells, this article systematically characterizes CRY2 homo-oligomerization in different cellular compartments, as well as how CRY2 homo-oligomerization and heterodimerization activities affect each other. Quantitative analysis reveals that membrane-bound CRY2 has drastically enhanced oligomerization activity compared to that of its cytoplasmic form. While CRY2 homo-oligomerization and CRY2-CIB1 heterodimerization could happen concomitantly, the presence of certain CIB1 fusion proteins can suppress CRY2 homo-oligomerization. However, the homo-oligomerization of cytoplasmic CRY2 can be significantly intensified by its recruitment to the membrane via interaction with the membrane-bound CIB1. These results contribute to the understanding of the light-inducible CRY2-CRY2 and CRY2-CIB1 interaction systems and can be used as a guide to establish new strategies utilizing the dual optogenetic characteristics of CRY2 to probe cellular processes.

Nanoparticle-assisted optical tethering of endosomes reveals the cooperative function of dyneins in retrograde axonal transport

Dynein-dependent transport of organelles from the axon terminals to the cell bodies is essential to the survival and function of neurons. However, quantitative knowledge of dyneins on axonal organelles and their collective function during this long-distance transport is lacking because current technologies to do such measurements are not applicable to neurons. Here, we report a new method termed nanoparticle-assisted optical tethering of endosomes (NOTE) that made it possible to study the cooperative mechanics of dyneins on retrograde axonal endosomes in live neurons. In this method, the opposing force from an elastic tether causes the endosomes to gradually stall under load and detach with a recoil velocity proportional to the dynein forces. These recoil velocities reveal that the axonal endosomes, despite their small size, can recruit up to 7 dyneins that function as independent mechanical units stochastically sharing load, which is vital for robust retrograde axonal transport. This study shows that NOTE, which relies on controlled generation of reactive oxygen species, is a viable method to manipulate small cellular cargos that are beyond the reach of current technology.

A close look at axonal transport: Cargos slow down when crossing stationary organelles

The bidirectional transport of cargos along the thin axon is fundamental for the structure, function and survival of neurons. Defective axonal transport has been linked to the mechanism of neurodegenerative diseases. In this paper, we study the effect of the local axonal environment to cargo transport behavior in neurons. Using dual-color fluorescence imaging in microfluidic neuronal devices, we quantify the transport dynamics of cargos when crossing stationary organelles such as non-moving endosomes and stationary mitochondria in the axon. We show that the axonal cargos tend to slow down, or pause transiently within the vicinity of stationary organelles. The slow-down effect is observed in both retrograde and anterograde transport directions of three different cargos (TrkA, lysosomes and TrkB). Our results agree with the hypothesis that bulky axonal structures can pose as steric hindrance for axonal transport. However, the results do not rule out the possibility that cellular mechanisms causing stationary organelles are also responsible for the delay in moving cargos at the same locations.

Motor Coordination in Long-Distance Transport in Axons

Retrograde transport of nerve growth factor signaling endosomes by microtubular motors, from the axon terminals to cell bodies, is vital for the survival of neurons. The robustness of this fast long-distance axonal transport and biased directionality could be attributed to the cooperative mechanics of multiple motors and/or intracellular regulation mechanisms. Here, we present a comprehensive motion analysis of retrograde nerve growth factor (NGF)-endosome trajectories in axons to show that cooperative motor mechanics and intracellular motor regulation are both important factors determining the endosome directionality. We used quantum dot (QD) to fluorescently label NGF and acquired trajectories of retrograde QD-NGF-endosomes with < 20 nm accuracy at 32 Hz, using pseudo-total internal reflection fluorescence imaging. Using a combination of transient motion analysis and Bayesian parsing, we segregated the trajectories into sustained periods of retrograde (dynein-driven) motion, constrained pauses and brief anterograde reversals. Mean square displacement analysis and the temperature dependence of transient reversals confirm that motors of opposite polarities (dyneins and kinesins) are both active on the endosomes during retrograde transport. Stochastic multi-motor model simulations show that the biased directionality as well as several statistical metrics of NGF-endosome transport can only be simulated reasonably by assuming that the microtubule-binding affinity of kinesin is down-regulated. Specifically, the simulations suggest that the NGF-endosomes are driven on average by 4-7 active dyneins and 1-3 down-regulated kinesins. These observations are corroborated by the dynamics of endosomes detaching under load in axons; showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. We discuss the ramifications of our results for intracellular transport regulation, in conjunction with recent studies on cellular cargo in a wide range of motility (bidirectional to unidirectional) regimes.