Actin- and Dynamin-Dependent Maturation of Bulk Endocytosis Restores Neurotransmission following Synaptic Depletion

Abstract

Bulk endocytosis contributes to the maintenance of neurotransmission at the amphibian neuromuscular junction by regenerating synaptic vesicles. How nerve terminals internalize adequate portions of the presynaptic membrane when bulk endocytosis is initiated before the end of a sustained stimulation is unknown. A maturation process, occurring at the end of the stimulation, is hypothesised to precisely restore the pools of synaptic vesicles. Using confocal time-lapse microscopy of FM1-43-labeled nerve terminals at the amphibian neuromuscular junction, we confirm that bulk endocytosis is initiated during a sustained tetanic stimulation and reveal that shortly after the end of the stimulation, nerve terminals undergo a maturation process. This includes a transient bulging of the plasma membrane, followed by the development of large intraterminal FM1-43-positive donut-like structures comprising large bulk membrane cisternae surrounded by recycling vesicles. The degree of bulging increased with stimulation frequency and the plasmalemma surface retrieved following the transient bulging correlated with the surface membrane internalized in bulk cisternae and recycling vesicles. Dyngo-4a, a potent dynamin inhibitor, did not block the initiation, but prevented the maturation of bulk endocytosis. In contrast, cytochalasin D, an inhibitor of actin polymerization, hindered both the initiation and maturation processes. Both inhibitors hampered the functional recovery of neurotransmission after synaptic depletion. Our data confirm that initiation of bulk endocytosis occurs during stimulation and demonstrates that a delayed maturation process controlled by actin and dynamin underpins the coupling between exocytosis and bulk endocytosis.

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“…Roles for actin and dynamin in bulk endocytosis in neurons and neuroendocrine cells have been demonstrated (Dillon and Goda, 2005;Newton et al, 2006;Nguyen et al, 2012). Here, we used the actin polymerization inhibitor cytochalasin D (4 µM) and the dynamin inhibitor dynasore (80 µM) to assess the effect of inhibiting actin and dynamin on the initiation of endocytosis and on the development of large granules and endosomes.…”

Bulk endocytosis at neuronal synapses

“…F, Bulk endosomes revealed as large donut-shaped structures by fluorescence imaging of FM1-43 dye (a) and ultrastructural analysis following photoconversion of FM1-43 (b). Note the large number of vesicles containing photoconverted material in close proximity to the delimiting membrane of the bulk cisternae (inset) [30]. with calcineurin emerging as a candidate to fulfil this role.…”

“…Syndapin, a F-BAR domain-containing protein that is capable of sensing membrane curvature and inducing tabulation [47], may play an active role in driving membrane curvature upon initiation of ADBE. The essential nature of dynamin in ADBE is further demonstrated by the fact that application of the potent inhibitors of dynamin, dynasore [48] and Dyngo4a TM [49], results in the complete block of ADBE both in primary cultured neurons [40] and at the neuromuscular junction [30].…”

“…The actin cytoskeleton is important in the process of macropinocytosis, the uptake of extracellular fluid in non-neuronal cells [51]. In neuronal cells, actin dynamics are essential for ADBE in both the amphibian neuromuscular junction [30,31] and goldfish retinal bipolar neurons [29]. The involvement of syndapin I in ADBE provides another line of evidence implicating actin in this process.…”

“…Recently, Nguyen et al demonstrated that both dynamin and actin are differentially required for the development and maturation of ADBE at the amphibian neuromuscular junction [30]. This maturation process was characterized by a transient bulging of the plasma membrane prior to formation of bulk endosomes.…”