Periodic Reporting for period 3 - NEUROSORTER (Uncovering the machinery for the sorting of newly synthesized proteins into the axon)
Período documentado: 2023-11-01 hasta 2025-04-30
Neuronal development and function rely on the polarized distribution of organelles and transmembrane proteins (cargoes) across their somatodendritic and axonal domains. However, it is unknown how organelle organization regulates the polarized sorting of transmembrane proteins to ensure proper neuronal function.
The classical model for sorting of newly synthesized transmembrane proteins to the plasma membrane (PM) follows the biosynthetic pathway via the rough endoplasmic reticulum (ER) and Golgi, which are restricted to the somatodendritic domain in neurons. It is unclear whether this classical secretion pathway is the main route for cargo sorting into the axon or whether an alternative route to the axon is used for most axonal cargoes.
Here, we aim to:
1) Identify the sorting routes for newly synthesized axonal proteins
2) Unravel the machinery required for Golgi-dependent and independent cargo sorting into the axon, and
3) Elucidate its impact on neuronal development and function
We are currently using high spatio-temporal resolution imaging and mass-spectrometry combined with novel strategies to control and track cargo secretion, as well as proximity-based labeling to identify key players in the newly identified machinery.
A broad spectrum of human diseases is associated to cargo Golgi-bypass. Neurons offer a unique advantage in spatial resolution to characterize this unconventional route, which could play a key role in human health and disease.
The classical model for sorting of newly synthesized transmembrane proteins to the plasma membrane (PM) follows the biosynthetic pathway via the rough endoplasmic reticulum (ER) and Golgi, which are restricted to the somatodendritic domain in neurons. It is unclear whether this classical secretion pathway is the main route for cargo sorting into the axon or whether an alternative route to the axon is used for most axonal cargoes.
Here, we aim to:
1) Identify the sorting routes for newly synthesized axonal proteins
2) Unravel the machinery required for Golgi-dependent and independent cargo sorting into the axon, and
3) Elucidate its impact on neuronal development and function
We are currently using high spatio-temporal resolution imaging and mass-spectrometry combined with novel strategies to control and track cargo secretion, as well as proximity-based labeling to identify key players in the newly identified machinery.
A broad spectrum of human diseases is associated to cargo Golgi-bypass. Neurons offer a unique advantage in spatial resolution to characterize this unconventional route, which could play a key role in human health and disease.
Within this NEUROSORTER project, we have found that axonal ER tubules are required for local axonal translation. In particular, we have found that ribosomes interact with the axonal ER, and this interaction is mediated by the axonally distributed ER protein P180, which binds ribosomes in a mRNA-dependent manner. In addition, external neuronal cues regulate ER-ribosome interaction and axonal translation, and ER-ribosome interaction is required for proper axonal development. We are currently investigating which exact mRNAs are translated in the axonal ER and the relevance for axonal remodeling during development upon external cues. Our results are consistent with our hypothesis of local secretion of proteins along the axon, and it explains how different transmembrane proteins mRNAs are translated along the axon. Part of this work has been deposited in BioRxiv, and soon will be submitted to a journal.
We are currently studying how these locally translated proteins can be sorted from the axonal ER into the plasma membrane.
For our NEUROSORTER project we have developed novel strategies/tools to visualize protein-protein interactions at the nanoscale resolution. We have generated a library of axonal and dendritic transmembrane cargoes to study their trafficking from the ER into their final destination. In addition, we have recently developed a system for the tracking of transmembrane protein secretion from the ER to the final destination with spatio-temporal mass spectrometry. With this powerful tool, the compartments involved in protein trafficking can be identified with high precision, even in highly complex polarized cells such as neurons.
We are currently studying how these locally translated proteins can be sorted from the axonal ER into the plasma membrane.
For our NEUROSORTER project we have developed novel strategies/tools to visualize protein-protein interactions at the nanoscale resolution. We have generated a library of axonal and dendritic transmembrane cargoes to study their trafficking from the ER into their final destination. In addition, we have recently developed a system for the tracking of transmembrane protein secretion from the ER to the final destination with spatio-temporal mass spectrometry. With this powerful tool, the compartments involved in protein trafficking can be identified with high precision, even in highly complex polarized cells such as neurons.
This is the first time that it has been shown that ER tubules can play a key role in local translation in mammalian cells, and we are excited to determine its contribution to neuronal development. It has been known for many years that ribosomes prefer to associate to flatten ER membrane for translation stability. Axonal ER are extremely curved, axonal ER tubules are even more narrow that ER tubules in other cells types, so how ribosomes can bind the highly curved ER membrane? We are trying to answer this and other questions regarding this ER tubule – ribosome interaction.
For long time has been questioned whether axonal translation existed, as most of the translation occurs in the soma of neurons. Recent evidence has further supported the model of axonal translation, which has a key role in axon development and function. However, most of these studies have investigated translation of cytosolic proteins. We have now identified a mechanism for the translation of transmembrane proteins along the axon, which could explain why mRNA for transmembrane proteins are present along the axon. Now more than even, we need to identify how these locally translated proteins can exit the ER in an unconventional manner, since the Golgi Apparatus is not present along the axon. We are excited that within this project we will be able to reveal the mechanism and relevance of unconventional protein secretion along the axon of neurons. We expect to also reveal remaining open questions regarding conventional secretion of proteins in polarized neurons. We hope at the end of this project we will get mechanistic insights in the polarized trafficking rules for different axonal and dendritic proteins.
For long time has been questioned whether axonal translation existed, as most of the translation occurs in the soma of neurons. Recent evidence has further supported the model of axonal translation, which has a key role in axon development and function. However, most of these studies have investigated translation of cytosolic proteins. We have now identified a mechanism for the translation of transmembrane proteins along the axon, which could explain why mRNA for transmembrane proteins are present along the axon. Now more than even, we need to identify how these locally translated proteins can exit the ER in an unconventional manner, since the Golgi Apparatus is not present along the axon. We are excited that within this project we will be able to reveal the mechanism and relevance of unconventional protein secretion along the axon of neurons. We expect to also reveal remaining open questions regarding conventional secretion of proteins in polarized neurons. We hope at the end of this project we will get mechanistic insights in the polarized trafficking rules for different axonal and dendritic proteins.