Surfaceome dynamics reveal proteostasis-independent reorganization of neuronal surface proteins during development and synaptic plasticity

Marc Van Oostrum,Benjamin Campbell,Charlotte Seng,Bernd Wollscheid,Shiva K Tyagarajan,Patrick G A Pedrioli,Csaba Földy,Jacqueline Hammer,Susanne Tom Dieck,Maik Müller

doi:10.1038/s41467-020-18494-6

Abstract

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization. While brain transcriptome, connectome and global proteome maps are being generated, system-wide analysis of temporal protein dynamics at the subcellular level are currently lacking. Here, we perform a temporally-resolved surfaceome analysis of primary neuron cultures and reveal dynamic surface protein clusters that reflect the functional requirements during distinct stages of neuronal development. Direct comparison of surface and total protein pools during development and homeostatic synaptic scaling demonstrates system-wide proteostasis-independent remodeling of the neuronal surface, illustrating widespread regulation on the level of surface trafficking. Finally, quantitative analysis of the neuronal surface during chemical long-term potentiation (cLTP) reveals fast externalization of diverse classes of surface proteins beyond the AMPA receptor, providing avenues to investigate the requirement of exocytosis for LTP. Our resource (neurosurfaceome.ethz.ch) highlights the importance of subcellular resolution for systems-level understanding of cellular processes.

Highlights

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization
We found that Gria[1] and Gria[2] had significantly increased surface abundance upon stimulation (Fig. 6c, d) indicating that the chemical long-term potentiation (cLTP) protocol triggers AMPA receptor exocytosis
In addition to AMPA receptor subunits, we identified 36 surface proteins that were significantly increased in abundance and four that were reduced in surface abundance after cLTP (Fig. 6g)

Summary

Introduction

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization. We perform a temporally-resolved surfaceome analysis of primary neuron cultures and reveal dynamic surface protein clusters that reflect the functional requirements during distinct stages of neuronal development. The plasma membrane proteome of primary neuronal cultures has been investigated using metabolic labeling and subsequent enrichment of glycoproteins[27,28] This approach enables quantitative comparison of the plasma membrane glycoproteome between different conditions, but cannot unambiguously define the acute surfaceome. After cell lysis and tryptic digestion, glycopeptides are enriched and released by peptide:N-glycosidase F (PNGase F) treatment This leaves a deamidation within the N-XS/T consensus sequence of formerly N-glycosylated peptides, enabling specific identification of extracellular N-glycosylation sites and quantification of protein abundance with subcellular resolution by MS32. The data indicates that localized proteotype maps provide functional insights into spatiotemporal controlled biological processes, such as synapse formation, which are typically masked in broad omics-style approaches

Methods

Results

Conclusion