Mical-L1 Is Required for Cargo Protein Delivery to the Cell Surface

This review appeared in the January 2022 edition of the Fluorescence Microscopy Journal Club — a monthly email brief highlighting leading-edge research and the latest discoveries supported by Bruker fluorescence microscopes.

Membrane trafficking distributes proteins and other macromolecules throughout the cell. It is a tightly regulated process and involves proteins and subcellular structures such as molecules that interact with CasL-like1 (MICAL-L1), endoplasmic reticulum (ER), Golgi apparatus (GA), and plasma membrane. In this paper by Sikora et al. published in Biology Open, the authors used multiple methods to investigate the role of MICAL-L1 protein in cargo delivery to the plasma membrane.

Confocal microscopy was used to map the location of MICAL-L1, which was found to be partially colocalized with markers of GA and recycling endosomes. The retention using selective hooks (RUSH) assay and RNA interference with shRNA revealed that MICAL-L1 was required for the delivery of a subset of cargo proteins to cell surface. In vitro experiments showed that MICAL-L1 promoted PACSIN3-dependent membrane tubulation. The mutation of two amino acid sites at the C terminus of MICAL-L1 reduced its association with phosphatidic acid and membrane tubule.

A Bruker Vutara super-resolution microscope was used to localize MICAL-L1 protein and two markers of the Golgi apparatus, GM130, and TGN46, within double-stained HeLa cells. MICAL-L1 protein and GM130 were labeled by antibodies conjugated with AlexaFluor647 and AlexaFluor568, respectively. The overlay of widefield fluorescence images of AF647-MICAL-L1 and AF568-GM130 demonstrated their close association with Golgi stacks. However, the localizations of these two proteins, resolved with Bruker's SRX software at the scale of a few tens of nanometers, showed they were located in different Golgi cisternae. They found the same result with the other marker, TGN46.

      _{KEY TERMS:}

• Membrane Trafficking; MICAL-L1 Protein; Plasma Membrane; Single-Molecule Localization Microscopy (SMLM); Widefield Fluorescence Imaging