Studying Cargo Transport Using RudLOV

Background

Approximately 30% of the cellular proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-side of the Golgi apparatus. These proteins, referred to as cargoes, traverse the Golgi apparatus and arrive at the trans-Golgi network (TGN), a cargo-sorting station located on the trans-side of the Golgi apparatus [1,2]. In the TGN, the cargo is sorted and transported to its destination or secretion. However, the mechanisms underlying the intracellular cargo transport and sorting remain unclear. Recent advances in optical microscopy, combined with methods for the synchronized release of cargo proteins fused to fluorescent proteins or tags, have enabled the real-time imaging of their journey from sequestered organelles to their destinations [3,4]. Direct observation of cargo protein transport and sorting is crucial for elucidating the mechanisms underlying these processes.

Several methods have been developed to synchronize cargo trafficking. In mammalian cells, the retention using the selective hooks (RUSH) system developed by Boncompain et al. [5] is widely used. The RUSH system is based on two key components: cargo fused to a streptavidin-binding peptide (SBP) and streptavidin fused to an organelle retention signal as a hook. The interactions between these proteins retain cargo within the organelle. The addition of biotin triggers the synchronized release of cargo proteins because it has a higher affinity for streptavidin than that of SBP. Although the RUSH system is easy to use and useful for cells that are accessible to externally applied biotin, the administration of biotin is not feasible for some cells within organisms. Furthermore, the time from biotin administration to the initiation of cargo release varies between cells. Optical cargo release methods have also been reported. In one approach, multiple UVR8-fused cargo proteins are sequestered in the ER, and a brief pulse of UV light (~310 nm) triggers cargo exit from the ER [6]. The zapalog-mediated ER trap (zapERtrap) uses UV light (405 nm) to trigger the breakdown of zapalog, which is the ER trap mechanism, allowing cargo release from the ER [7]. Both systems enable the precise control of illumination at the single-cell or subcellular level using the same microscope employed for subsequent live imaging, providing exceptional spatial and temporal accuracy for cargo release. Although these optical cargo-releasing methods are advantageous, UV light can potentially damage cells, and zapERtrap requires a continuous supply of relatively expensive zapalogs.

We recently developed a new optically synchronized cargo release method called retention using the dark state of LOV2 (RudLOV) [8], which is based on LOVTRAP (LOV2 Trap and Release of Protein) [9]. LOVTRAP is an optogenetic system consisting of LOV2 and Zdk1 tags, capable of repeatedly and reversibly controlling protein binding with precise kinetics. LOV2 is the photosensor domain of Avena sativa phototropin with an absorption maximum of 450 nm that utilizes the endogenous cofactor flavin mononucleotide (FMN) as a chromophore. Zdk1, an engineered aptamer that selectively binds to the dark state of LOV2, was developed by mRNA display screening of a library based on the Z-domain of protein A. LOVTRAP uses LOV2 as a trap for Zdk1-tagged proteins, which can be reversibly released by blue light illumination. LOV2 in the light state is spontaneously restored to the dark state, with a half-life of approximately 9 s in the case of wild-type Avena sativa LOV2.

In RudLOV, the cargo is fused to Zdk1, which is captured in the dark by LOV2 fused to ER-localized Sec61β. Mild blue light illumination (445 or 488 nm) induces a conformational change in LOV2 (-10 ms [10]), releasing Zdk1-fused cargo, which then synchronously exits the ER. The release of cargo from the ER occurs simultaneously in all illuminated cells (Figure 1F, G in Tago et al. [8]). The LOV2 conformation spontaneously reverts to the dark form within 55–81 s and recaptures the cargo with Zdk1 [11]. This mechanism allows control of the amount of cargo released by adjusting the strength and duration of illumination. We successfully demonstrated the laser power- and duration-dependent release of cargo, as well as cargo release in a single cell and even in a single Golgi stack (Figure 2A, B in Tago et al. [8]).

Live imaging requires the detection of fluorescent proteins fused to the cargo. Because both the transport trigger and cargo detection use illumination, careful selection of laser and filter sets to control illumination and/or detection is crucial for the successful implementation of RudLOV. Here, we present a step-by-step protocol for using RudLOV.