The Golgi complex is essential for many aspects of cellular function, including trafficking and sorting of membrane and secretory proteins and posttranslational modification by glycosylation. We observed reversible fragmentation of the Golgi complex in cultured hippocampal neurons cultured in hyperexcitable conditions. In addition, Golgi fragmentation was found in cultured neurons with hyperactivity due to prolonged blockade of GABA(A)-mediated inhibition or withdrawal of NMDA receptor antagonism. The interplay between neuronal hyperactivity and Golgi structure established in this study thus reveals a previously uncharacterized impact of neuronal activity on organelle structure. This finding may have important roles in protein processing and trafficking in the Golgi as well as effects on neuronal signaling.

The Golgi complex is essential for many aspects of cellular function, including trafficking and sorting of membrane and secretory proteins and posttranslational modification by glycosylation. We observed reversible fragmentation of the Golgi complex in cultured hippocampal neurons cultured in hyperexcitable conditions. In addition, Golgi fragmentation was found in cultured neurons with hyperactivity due to prolonged blockade of GABA(A)-mediated inhibition or withdrawal of NMDA receptor antagonism. The interplay between neuronal hyperactivity and Golgi structure established in this study thus reveals a previously uncharacterized impact of neuronal activity on organelle structure. This finding may have important roles in protein processing and trafficking in the Golgi as well as effects on neuronal signaling.

Key points

Kv1.2 and related voltage-gated potassium channels have a highly conserved N-linked glycosylation site in the first extracellular loop, with complex glycosylation in COS-7 cells similar to endogenous Kv1.2 glycosylation in hippocampal neurons. COS-7 cells expressing Kv1.2 show a crucial role of this N-linked glycosylation in the forward trafficking of Kv1.2 to the cell membrane. Although both wild-type and non-glycosylated mutant Kv1.2 channels that have reached the cell membrane are internalized at a comparable rate, mutant channels are degraded at a faster rate. Treatment of wild-type Kv1.2 channels on the cell surface with glycosidase to remove sialic acids also results in the faster degradation of internalized channels. Glycosylation of Kv1.2 is important with respect to facilitating trafficking to the cell membrane and enhancing the stability of channels that have reached the cell membrane.

Abstract

Studies in cultured hippocampal neurons and the COS-7 cell line demonstrate important roles for N-linked glycosylation of Kv1.2 channels in forward trafficking and protein degradation. Kv1.2 channels can contain complex N-linked glycans, which facilitate cell surface expression of the channels. Additionally, the protein stability of cell surface-expressed Kv1.2 channels is affected by glycosylation via differences in the degradation of internalized channels. The present study reveals the importance of N-linked complex glycosylation in boosting Kv1.2 channel density. Notably, sialic acids at the terminal sugar branches play an important role in dampening the degradation of Kv1.2 internalized from the cell membrane to promote its stability.

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.