Biology subjects

Casler, J. C.

Publications and source records attributed to Casler, J. C..

4 recordsLinked to original sources

bioRxiv2021

Clathrin adaptors mediate two sequential pathwaysof intra-Golgi recycling

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

cell biology↗

bioRxiv2020

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block exit 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 transport out of 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.

cell biology↗

bioRxiv2020

Deuteration improves small-molecule fluorophores

Fluorescence microscopy relies on dyes that absorb short-wavelength photons and emit longer-wavelength light. In addition to this fluorescence process, dyes can undergo other photochemical reactions that result in spectral shifts and irreversible photobleaching. Increases in brightness, chromostability, and photostability of fluorescent dyes are therefore crucial for advancing the frontier of bioimaging. Here, we describe a general approach to improve small-molecule fluorophores using deuteration. Incorporating deuterium into the alkylamino substituents of rhodamines and other dyes improves fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching. These compounds are easily synthesized and show improved performance in cellular imaging experiments.

cell biology↗

bioRxiv2020

A microscopy-based kinetic analysis of yeast vacuolar protein sorting

The yeast Saccharomyces cerevisiae is amenable to studying membrane traffic by live-cell fluorescence microscopy. We used this system to explore two aspects of cargo protein traffic through prevacuolar endosome (PVE) compartments to the vacuole. First, at what point during Golgi maturation does a biosynthetic vacuolar cargo depart from the maturing cisternae? To address this question, we modified a regulatable fluorescent secretory cargo by adding a vacuolar targeting signal. Traffic of the vacuolar cargo requires the GGA clathrin adaptors, which arrive during the early-to-late Golgi transition. Accordingly, the vacuolar cargo begins to exit the Golgi near the midpoint of maturation, significantly before exit of a secretory cargo. Second, how are cargoes delivered from PVE compartments to the vacuole? To address this question, we tracked biosynthetic and endocytic cargoes after they had accumulated in PVE compartments. The results imply that stable PVE compartments repeatedly deliver material to the vacuole by a kiss-and-run mechanism.

cell biology↗