Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Washington University School of Medicine

When cooled below approximately 7 degrees C, recently endocytosed vesicles in the motor terminals of the garter snake fail to shed their clathrin coats. Perhaps as a result, the terminals complete only about one-half of the compensatory endocytosis expected after a given period of stimulation. Upon return to room temperature (RT), endocytosis resumes immediately and is complete within minutes. This "delayed" endocytosis following release from cold block provides an opportunity to study clathrin-dependent endocytotic mechanisms in temporal isolation from those events, such as Ca2+ entry and consequent exocytosis, that are normally associated with the activation of nerve terminals. We have taken advantage of clathrin decoating blockade to examine the rate, temperature dependence and extracellular Ca2+ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35 degrees C. Moreover, the rate of endocytosis varied significantly with change in [Ca2+]o; the rate at 7.2 mM (single exponential time constant, approximately 3 s) was approximately double that at 0 mM (single exponential time constant, approximately 7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca2+]o, potentially regulated by changes in the milieu of the synaptic cleft during neural activity.

"Delayed" endocytosis is regulated by extracellular Ca2+ in snake motor boutons.

Snake nerve-muscle preparations are well-suited for study of both motor innervation patterns at the systems level and NMJ function at the cellular level. Their small size ( approximately 100 myofibers) and thinness (one fiber) allows access to all NMJs in one muscle. Snake NMJs are of three types, two twitch subtypes and a single tonic type. Properties of the NMJs supplied by a particular motor neuron, and of the motor unit fibers they innervate, are precisely regulated by the motor neuron in a manner consistent with the Henneman Size Principle. Unlike its amphibian or mammalian cousins, the snake NMJ comprises approximately 50 (twitch) or approximately 20 (tonic) individual one-bouton synapses, similar to synapses found in the central nervous system. Each bouton releases a few quanta per stimulus. Larger fibers, which require more synaptic current to initiate contraction, receive nerve terminals that contain more boutons and express receptor patches with higher sensitivity to transmitter. Quantal analysis suggests that transmitter release sites in one bouton do not behave independently; rather, they may cooperate to reduce fluctuations and enhance reliability. After release, two mechanisms coexist for retrieval and reprocessing of spent vesicles-one involving clathrin-mediated endocytosis, the other macropinocytosis. Unanswered questions include how each mechanism is regulated in a use-dependent manner.

The nerve-muscle synapse of the garter snake.

In nerve terminals, endocytosis in compensation for transmitter exocytosis was once thought 'housekeeping'. Now, several lines of research utilizing optical endocytic probes have coalesced into a new functional model. The model comprises two distinct endocytosis and vesicle-processing paradigms that are differentially regulated in response to demand for transmitter. Under some circumstances, such as recovery from short-term depression, it appears that endocytosis, and not exocytosis, is rate-limiting for transmitter release and is therefore a principal determinant of synaptic strength.

Endocytosis and synaptic plasticity: might the tail wag the dog?

At the snake neuromuscular junction, low temperature (LT, 5-7 degrees C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca(2+)](i) directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca(2+)](i). With brief stimulation at LT, [Ca(2+)](i) transiently increased but returned to baseline ( approximately 63 nm) in < 8 min. After 15 min at LT, [Ca(2+)](i) was altered by incubating preparations in the Ca(2+) ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca(2+)](i) increased; the rate at 300 nm Ca(2+) was approximately double that under basal conditions. Thus the rate of CME - isolated from stimulation, transmitter release, and other forms of endocytosis - is directly influenced by intraterminal Ca(2+).

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+.

At a variety of fast chemical synapses, spent synaptic vesicles are recycled via a large 'reserve' vesicle pool at high stimulus frequencies, and via fast 'local cycling' near release sites (e.g. 'kiss and run' transmitter release) at low stimulus frequencies. We have investigated recycling at the snake neuromuscular junction (NMJ), specifically seeking evidence for local cycling. Activity-dependent staining and destaining of the endocytic probe FM1-43 were directly compared to transmitter release over a range of stimulus frequencies. We found a fixed proportionality between staining/destaining and summed endplate potentials (EPPs) representing total transmitter release. There was no direct dependence of staining or destaining on stimulus frequency, as would be expected if local cycling (and consequent altered FM1-43 retention) were more prevalent at one frequency than another. In other experiments the drug vesamicol was used to abolish refilling of vesicles with transmitter, thereby blocking EPPs contributed by recycled vesicles. Control and vesamicol-treated NMJs had identical quantal content for the first 10 min of 1 Hz stimulation. Afterwards EPP amplitudes at vesamicol-treated NMJs declined at a rate consistent with use of a large pool containing approximately 130,000 vesicles. Finally, calibrated paired stimulations show that regenerated vesicles have poorer than random probability of re-release. Our findings are inconsistent with local cycling and suggest that the snake motor terminal utilizes exclusively a single large vesicle pool.

Vesicles in snake motor terminals comprise one functional pool and utilize a single recycling strategy at all stimulus frequencies.

We have examined the processing of endosomes formed by macroendocytosis (ME), or bulk membrane retrieval, in active motor terminal boutons at the snake nerve-muscle synapse. Endocytic probes were imaged at light (FM1-43) and electron (horseradish peroxidase (HRP)) levels over stimulus frequencies representing low, intermediate and high levels of use. Endosomes formed rapidly (1-2 s) at all frequencies, concomitant with clathrin-mediated vesicular endocytosis (CME). Endosomes dissipated rapidly into vesicles (approximately 10 s). The dissipation rate was not influenced by activity. Many endosomes split into clusters of 2-20 smaller endosomes of varying size. Vesicles budded from these smaller endosomes, from large endosomes that had not undergone fission into smaller ones, and from precursor membrane infoldings that had not yet internalized. In snake, exocytosed vesicular membrane is not competent for reuse until after a delay (> 3 min). We found that time required for endosome processing is not responsible for this delay. Endosome processing might, however, limit availability of some vesicles for release at very high levels of use. Generally, endosome processing paralleled that of vesicles internalized directly from the plasma membrane via CME, regardless of stimulus frequency. There was no evidence for differential recruitment of ME versus CME depending upon level of use.

Macroendocytosis and endosome processing in snake motor boutons.

Activity at the vertebrate nerve-muscle synapse creates large macroendosomes (MEs) via bulk membrane infolding. Visualized with the endocytic probe FM1-43, most (94%) of the ∼25 MEs/terminal created by brief (30-Hz, 18-second) stimulation dissipate rapidly (∼1 minute) into vesicles. Others, however, remain for hours. Here we study these "late" MEs by using 4D live imaging over a period of ∼1 hour after stimulation. We find that some (51/398 or 13%) disappear spontaneously via exocytosis, releasing their contents into the extracellular milieu. Others (at least 15/1,960 or 1%) fuse or closely associate with a second class of endosomes that take up acidophilic dyes (acidic endosomes [AEs]). AEs are plentiful (∼47/terminal) and exist independent of stimulation. Unlike MEs, which exhibit Brownian motion, AEs exhibit directed motion (average, 83 nm/sec) on microtubules within and among terminal boutons. AEs populate the axon as well, where movement is predominantly retrograde. They share biochemical and immunohistochemical markers (e.g., lysosomal-associated membrane protein [LAMP-1]) with lysosomes. Fusion/association of MEs with AEs suggests a sorting/degradation pathway in nerve terminals wherein the role of AEs is similar to that of lysosomes. Based on our data, we propose that MEs serve as sorting endosomes. Thus their contents, which include plasma membrane proteins, vesicle proteins, and extracellular levels of Ca(2+) , can be targeted either toward the reformation and budding of synaptic vesicles, toward secretion via exocytosis, or toward a degradation process that utilizes AEs either for lysis within the terminal or for transport toward the cell body.

Robert S. Wilkinson

Department of Cell Biology and Physiology

Washington University School of Medicine

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Robert S. Wilkinson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology and Physiology

Washington University School of Medicine

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Department of Cell Biology and Physiology