Abstract
One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca2+ and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca2+ levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca2+ mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca2+ stress levels.
Highlights
The secretion of proteins from eukaryotic cells is a tightly regulated vital process based on complex membrane trafficking pathways [1]
To visualize and analyze synchronized endoplasmic reticulum (ER)-to-Golgi transport in individual living HeLa cells, we imaged fusion constructs that consist of conditional aggregation domains (CADs) fused to cargo proteins of the secretory pathway and a green fluorescent protein (GFP) variant
To study ER-to-Golgi transport of a classical bulk-flow cargo, a fusion construct consisting of an ER-targeting signal sequence, GFP, and CADs, as well as human growth hormone at the C-terminus, was imaged
Summary
The secretion of proteins from eukaryotic cells is a tightly regulated vital process based on complex membrane trafficking pathways [1]. Up to one third of all proteins in eukaryotic cells enter the secretory pathway [3,4,5,6,7]. Cells 2020, 9, 2311 second [8]. Given these high rates of secretory activity and the overall importance of this process, it is not surprising that defects of the secretory pathway and membrane trafficking can impair cell functions and cause severe disease [1,7,9,10,11,12,13]. Protein secretion in cancer cells is crucial for malignancy [3,14,15,16,17,18,19]
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