Activity-dependent bulk endocytosis proteome reveals a key presynaptic role for the monomeric GTPase Rab11

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• Activity-dependent bulk endocytosis (ADBE) is the primary mechanism for synaptic vesicle recycling during intense neuronal activity.
• Understanding ADBE's physiological role is limited by the absence of identified, unique molecular components.
• This study addresses this gap by characterizing the proteome of bulk endosomes, key organelles in ADBE.

Purpose of the Study:

• To identify and characterize the molecular constituents of bulk endosomes involved in ADBE.
• To uncover novel proteins and pathways regulating presynaptic function during high-frequency neuronal activity.
• To identify potential regulators of ADBE and synaptic vesicle trafficking.

Main Methods:

• Proteomic analysis of purified bulk endosomes using two independent enrichment strategies: subcellular fractionation and magnetic nanoparticle isolation.
• Bioinformatic and network analyses to identify enriched protein categories and central molecular hubs within the ADBE proteome.
• Functional investigation of specific Rab GTPases, particularly Rab11, in regulating ADBE and clathrin-mediated endocytosis.

Main Results:

• Two independent methods yielded a 77% overlap in identified proteins, defining the core ADBE proteome.
• The ADBE proteome is significantly enriched in cell adhesion, cytoskeletal, and signaling molecules, alongside known synaptic and trafficking proteins.
• Network analysis highlighted Rab GTPases as a central hub; subsequent experiments confirmed Rab11 facilitates ADBE and accelerates clathrin-mediated endocytosis.

Conclusions:

• The identified ADBE proteome provides a valuable resource for future research into presynaptic function and synaptic vesicle dynamics.
• Rab11 is identified as a novel regulator influencing both activity-dependent bulk endocytosis and clathrin-mediated endocytosis.
• These findings advance our understanding of the molecular mechanisms governing synaptic plasticity and neurotransmission during sustained neuronal firing.