ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Abstract

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms. [Media: see text] [Media: see text] [Media: see text]

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome

“…Because of technical difficulties in applying the RUSH system to neurons (i.e., there was some "leakage" from ER prior to release of the ER-bound cargo), we used a different system to release bound fluorescent cargo from ER in order to test the role of Golgi satellites in secretory trafficking in neurons. The system used a modified version of a regulatable fluorescent secretory cargo called ESCargo (Casler et al 2020). ESCargo forms homo-aggregates in the ER lumen when expressed but can be released into the secretory pathway upon addition of a membrane-permeable ligand that dissociates the aggregates (Casler et al 2020).…”

“…The system used a modified version of a regulatable fluorescent secretory cargo called ESCargo (Casler et al 2020). ESCargo forms homo-aggregates in the ER lumen when expressed but can be released into the secretory pathway upon addition of a membrane-permeable ligand that dissociates the aggregates (Casler et al 2020). In neurons expressing ESCargo and Man-GFP, ER aggregates in the soma, dendrites and axons could be seen prior to release in nicotine-treated cells (Figure 3B).…”

“…Other cDNAs generously provided include: St3-GFP from Dr. Sakari Kellokumpu, (University of Oulu, Oulu, Finland), GalNAc T2-mCherry from Dr. Dibbyendu Bhattacharya (Tata Memorial Center, Mumbai, India), GalT-3xGFP and mCherry-Sec23 from Dr. Jennifer Lippincott-Schwartz (HHMI, Janelia Research Campus), GM130-GFP from Dr. Christine Suetterlin (University of California, Irvine, CA), pDsRed-ER (Takara Bio, CA), RUSH-GPI-mApple, Rush-GPI-Halo, and Ensconsin-mCherry (Addgene, MA). For imaging ER bulk flow trafficking to Golgi, a bicistronic plasmid encoding a modified ESCargo (Casler et al 2020) and ManII-GFP was generated in the laboratory of Benjamin Glick (University of Chicago, Chicago, IL). The M. musculus ManIIA1gene was obtained from BioBasic (Toronto, Canada), PCR amplified and inserted into the ESCargo plasmid using in-fusion cloning (Takara Bio, CA).…”

CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae

“… 2007 , Casler et al . 2020 ). In this work, the secretion of α-amylase was guided by the pre-pro-α-factor signal peptide.…”

Acute manipulation and real-time visualization of membrane trafficking and exocytosis inDrosophila

“…Classical methods for synchronizing secretory protein trafficking either rely on temperature blocks (Griffiths et al, 1985) or drugs (e.g., Brefeldin A;Lippincott-Schwartz et al, 1989) to reversibly arrest intracellular transport, or employ special conditionally mis-folded or aggregated proteins that are retained in the endoplasmic reticulum (ER). Release of these proteins from the ER is achieved by shifting cells to permissive temperature (Kreis and Lodish, 1986;Lafay, 1974), by adding a small-molecule ligand (Casler et al, 2020;Rollins et al, 2000), or by illumination with UV light (Chen et al, 2013). Although these approaches have revealed fundamental insights into the organization and dynamics of the secretory apparatus, they are limited to special proteins (e.g., the conditional thermosensitive mutant viral glycoprotein VSVGtsO45; Kreis and Lodish, 1986;Presley et al, 1997;Scales et al, 1997) and require treatments using nonphysiological temperatures, drugs, or potentially damaging doses of UV light.…”