Macroendocytosis and endosome processing in snake motor boutons

Area of Science:

• Neuroscience
• Cell Biology
• Synaptic Plasticity

Background:

• Nerve terminal membrane retrieval is crucial for sustained synaptic transmission.
• Two main endocytic pathways, macroendocytosis (ME) and clathrin-mediated endocytosis (CME), contribute to membrane recycling.
• The processing kinetics of endosomes formed by ME and their role in synaptic function remain incompletely understood.

Purpose of the Study:

• To investigate the dynamics of endosomes formed by macroendocytosis (ME) in active motor terminal boutons.
• To compare the processing of ME-derived endosomes with vesicles internalized via clathrin-mediated endocytosis (CME).
• To determine if endosome processing kinetics influence the delay in membrane reuse at the nerve-muscle synapse.

Main Methods:

• Utilized FM1-43 fluorescence imaging and electron microscopy with horseradish peroxidase (HRP) to visualize endosomes.
• Stimulated snake nerve-muscle synapses at low, intermediate, and high frequencies.
• Quantified endosome formation, dissipation, and fission rates, and correlated them with synaptic activity levels.

Main Results:

• Endosomes formed rapidly (1-2 s) via ME at all stimulation frequencies, concurrently with CME.
• Endosomes dissipated quickly (approx. 10 s) into vesicles, independent of activity level.
• Endosome processing kinetics did not account for the >3 min delay in membrane reuse but could limit vesicle availability at very high activity levels.
• ME and CME pathways exhibited similar processing dynamics across different stimulation frequencies, with no evidence of differential recruitment.

Conclusions:

• Macroendocytosis is a rapid endocytic process at nerve terminals that parallels CME in its kinetics.
• The processing of ME-derived endosomes is not the rate-limiting step for synaptic membrane reuse.
• Endosome processing may influence vesicle supply for exocytosis during intense neuronal activity.