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. 2002 Dec 24;99(26):17143-8.
doi: 10.1073/pnas.222657399. Epub 2002 Dec 12.

Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice

Warren T Kim  1 , Sunghoe Chang, Laurie Daniell, Ottavio Cremona, Gilbert Di Paolo, Pietro De Camilli
Affiliations

Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice

Warren T Kim et al. Proc Natl Acad Sci U S A. .

Abstract

Synaptojanin 1 is a polyphosphoinositide phosphatase implicated in synaptic vesicle recycling. We used FM1-43 imaging and electron microscopy in cultured cortical neurons from control and synaptojanin 1 knockout mice to study how the absence of this protein affects specific steps of the synaptic vesicle cycle. Exoendocytosis after a moderate stimulus was unchanged. However, during prolonged stimulation, the regeneration of fusion-competent synaptic vesicles was severely impaired. In stimulated nerve terminals, there was a persistent accumulation of clathrin-coated vesicles and a backup of newly reformed vesicles in the cytomatrix-rich area around the synaptic vesicle cluster. These findings demonstrate that synaptojanin 1 function is needed for the progression of recycling vesicles to the functional synaptic vesicle pool.

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Figures

Fig 1.
Fig 1.
Overall synaptic vesicle turnover is intact in synaptojanin 1-deficient mice, but the functional recycling pool is 40% smaller. A plot of FM1-43 uptake ΔF (see text) as a function of AP number shows that knockout and control neurons have similarly shaped loading curves, both reaching maximal FM1-43 uptake with 600 APs. However, the absolute amount of trapped fluorescence in knockout synapses is 40% less than in control synapses.
Fig 2.
Fig 2.
Endocytosis kinetics of synaptic vesicle membrane are unchanged after moderate stimulation (100 APs at 10 Hz). A plot of FM1-43 uptake as a function of dye pulse delay shows that the half time (t1/2) for endocytosis is 15 s in both knockout and control neurons, and the overall shape of the curves is identical.
Fig 3.
Fig 3.
Repriming kinetics reveal a delay and impairment of synaptic vesicle reformation during prolonged stimulation. A plot of the ratio (ΔFo − ΔFΔt)/ΔFo (see text) gives the fraction of fluorescence depleted by the APs fired during the chase period Δt. During continuous stimulation, knockout neurons exhibit a minimum repriming interval of 60 s compared with 15 s in control. In addition, only 20% of the retrieved membrane is releasable during the sustained stimulus in the knockout compared with 60% in the control.
Fig 4.
Fig 4.
Electron microscopy of synaptojanin 1-deficient synapses shows a persistent, stimulus-dependent increase in clathrin-coated vesicles. (a–e) Electron micrographs show tracer-labeled (HRP and HRP-WGA as indicated) recycling vesicles in control (a and c) and knockout (b, d, and e) nerve terminals after a 900-AP train (10 Hz) and 10-min recovery period. In control nerve terminals, labeled vesicles are randomly intermixed in the cluster of synaptic vesicles. In knockout nerve terminals, there is an accumulation of labeled and unlabeled clathrin-coated vesicles (circles in b) that are often segregated at the periphery of the synaptic vesicle cluster. Note the row of clathrin-coated vesicles in e. (f) Morphometry of total clathrin-coated vesicles (labeled plus unlabeled) showing the persistent accumulation of clathrin-coated vesicles after stimulation. (g) HRP-WGA-labeled vesicles make up a large fraction of the accumulated clathrin-coated vesicles in knockout neurons, confirming that they are truly endocytic. (h) Fluid-phase HRP-labeled clathrin-coated vesicles decrease after a 10-min recovery period, showing that at least some of the vesicles progress to downstream recycling steps.
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