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Evidence for a Clathrin-independent mode of endocytosis at a continuously active sensory synapse.

Fuchs M, Brandstätter JH, Regus-Leidig H - Front Cell Neurosci (2014)

Bottom Line: Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses.To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy.These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70.

View Article: PubMed Central - PubMed

Affiliation: Animal Physiology, Department of Biology, Friedrich-Alexander-University Erlangen-Nuremberg Erlangen, Germany.

ABSTRACT
Synaptic vesicle exocytosis at chemical synapses is followed by compensatory endocytosis. Multiple pathways including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kiss-and-run retrieval have been reported to contribute to vesicle recycling. Particularly at the continuously active ribbon synapses of retinal photoreceptor and bipolar cells, compensatory endocytosis plays an essential role to provide ongoing vesicle supply. Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses. To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy. In mouse ON bipolar cell terminals, Clathrin-mediated endocytosis seemed to be the dominant mode of endocytosis at all adaptation states analyzed. In contrast, in mouse photoreceptor terminals in addition to Clathrin-coated pits, clusters of membranously connected electron-dense vesicles appeared during prolonged darkness. These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70. We hypothesize that rod and cone photoreceptors possess an additional Clathrin-independent mode of vesicle retrieval supporting the continuous synaptic vesicle supply during prolonged high activity.

No MeSH data available.


Related in: MedlinePlus

Ultrastructural analysis of clusters of electron-dense endocytotic vesicles in 3 h dark adapted rod photoreceptor terminals. (A) Electron micrograph of outer plexiform layer (OPL), showing clusters of electron dense endocytotic vesicles (CEV; encircled with dashed lines) in three neighboring rod photoreceptor terminals of a 3 h dark adapted C57BL/6JRj retina. (B) Representative electron micrograph of a rod photoreceptor terminal in the 3 h dark adapted retina. Arrows point to single vesicles of the CEV emanating from the plasma membrane at the postsynaptic invaginations. The high magnification inset shows the membranous connection between individual vesicles of a CEV. (C) 3D-reconstruction of a 3 h dark adapted rod photoreceptor terminal. The horseshoe-shaped synaptic ribbon (black) bends around the postsynaptic invaginations of bipolar and horizontal cells. Ribbon-tethered vesicles are shown in blue, Clathrin-coated pits (CCP) in red, and CEV in green. (D) Electron micrograph of a 3 h dark adapted rod photoreceptor terminal showing CEV in close association with vesicles tethered to the synaptic ribbon. Arrowheads point to synaptic ribbons. HC, horizontal cell; BC, bipolar cell. Scale bar in A–D: 200 nm, inset in B: 50 nm.
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Figure 7: Ultrastructural analysis of clusters of electron-dense endocytotic vesicles in 3 h dark adapted rod photoreceptor terminals. (A) Electron micrograph of outer plexiform layer (OPL), showing clusters of electron dense endocytotic vesicles (CEV; encircled with dashed lines) in three neighboring rod photoreceptor terminals of a 3 h dark adapted C57BL/6JRj retina. (B) Representative electron micrograph of a rod photoreceptor terminal in the 3 h dark adapted retina. Arrows point to single vesicles of the CEV emanating from the plasma membrane at the postsynaptic invaginations. The high magnification inset shows the membranous connection between individual vesicles of a CEV. (C) 3D-reconstruction of a 3 h dark adapted rod photoreceptor terminal. The horseshoe-shaped synaptic ribbon (black) bends around the postsynaptic invaginations of bipolar and horizontal cells. Ribbon-tethered vesicles are shown in blue, Clathrin-coated pits (CCP) in red, and CEV in green. (D) Electron micrograph of a 3 h dark adapted rod photoreceptor terminal showing CEV in close association with vesicles tethered to the synaptic ribbon. Arrowheads point to synaptic ribbons. HC, horizontal cell; BC, bipolar cell. Scale bar in A–D: 200 nm, inset in B: 50 nm.

Mentions: To analyze the presynaptic localization of the punctate immunoreactivity for Dynamin3, Endophilin1, and Synaptojanin1, we first triple labeled cryostat sections of 3 h dark adapted BL/6 retinae for Calbindin (green; labels horizontal cells and their invaginations into photoreceptor terminals), Dynamin3 (red), and Piccolino, a ribbon-specific Piccolo variant labeling the synaptic ribbon (blue; Regus-Leidig et al., 2013; Figure 6A). The merge of the three stainings shows that Dynamin3 puncta localize in the periactive zone close to the horseshoe shaped synaptic ribbons as well as the tips of horizontal cell processes (Figure 6A; arrows). To characterize this putative endocytotic hot spot at the ultrastructural level, we next performed pre-embedding immunoelectron microscopy on 3 h dark adapted BL/6 retinae with antibodies against Dynamin3, Endophilin1, Synaptojanin1 (Figures 6B–E), and Clathrin (data not shown). We also tried post-embedding immunoelectron microscopy, which unfortunately did not satisfactorily work in our hands. In rod photoreceptor terminals, immunoreactivity for Dynamin3, Endophilin1, Synaptojanin1 concentrated in the vicinity of the postsynaptic invaginations and the synaptic ribbon (Figures 6B–E; dashed circles). As expected, Clathrin LC immunoreactivity was diffusely distributed throughout the photoreceptor synaptic terminals, confirming the light microscopical absence of Clathrin LC in these endocytotic hot spots (data not shown). Sometimes, presumptive vesicles were visible in the labeled region (Figure 6C), but most of the times the intense staining obscured any underlying structures. Therefore, we analyzed dark adapted BL/6 retinae with conventional electron microscopy for the presence of conspicuous vesicle clusters in the vicinity of the synaptic ribbon (Figure 7).


Evidence for a Clathrin-independent mode of endocytosis at a continuously active sensory synapse.

Fuchs M, Brandstätter JH, Regus-Leidig H - Front Cell Neurosci (2014)

Ultrastructural analysis of clusters of electron-dense endocytotic vesicles in 3 h dark adapted rod photoreceptor terminals. (A) Electron micrograph of outer plexiform layer (OPL), showing clusters of electron dense endocytotic vesicles (CEV; encircled with dashed lines) in three neighboring rod photoreceptor terminals of a 3 h dark adapted C57BL/6JRj retina. (B) Representative electron micrograph of a rod photoreceptor terminal in the 3 h dark adapted retina. Arrows point to single vesicles of the CEV emanating from the plasma membrane at the postsynaptic invaginations. The high magnification inset shows the membranous connection between individual vesicles of a CEV. (C) 3D-reconstruction of a 3 h dark adapted rod photoreceptor terminal. The horseshoe-shaped synaptic ribbon (black) bends around the postsynaptic invaginations of bipolar and horizontal cells. Ribbon-tethered vesicles are shown in blue, Clathrin-coated pits (CCP) in red, and CEV in green. (D) Electron micrograph of a 3 h dark adapted rod photoreceptor terminal showing CEV in close association with vesicles tethered to the synaptic ribbon. Arrowheads point to synaptic ribbons. HC, horizontal cell; BC, bipolar cell. Scale bar in A–D: 200 nm, inset in B: 50 nm.
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Figure 7: Ultrastructural analysis of clusters of electron-dense endocytotic vesicles in 3 h dark adapted rod photoreceptor terminals. (A) Electron micrograph of outer plexiform layer (OPL), showing clusters of electron dense endocytotic vesicles (CEV; encircled with dashed lines) in three neighboring rod photoreceptor terminals of a 3 h dark adapted C57BL/6JRj retina. (B) Representative electron micrograph of a rod photoreceptor terminal in the 3 h dark adapted retina. Arrows point to single vesicles of the CEV emanating from the plasma membrane at the postsynaptic invaginations. The high magnification inset shows the membranous connection between individual vesicles of a CEV. (C) 3D-reconstruction of a 3 h dark adapted rod photoreceptor terminal. The horseshoe-shaped synaptic ribbon (black) bends around the postsynaptic invaginations of bipolar and horizontal cells. Ribbon-tethered vesicles are shown in blue, Clathrin-coated pits (CCP) in red, and CEV in green. (D) Electron micrograph of a 3 h dark adapted rod photoreceptor terminal showing CEV in close association with vesicles tethered to the synaptic ribbon. Arrowheads point to synaptic ribbons. HC, horizontal cell; BC, bipolar cell. Scale bar in A–D: 200 nm, inset in B: 50 nm.
Mentions: To analyze the presynaptic localization of the punctate immunoreactivity for Dynamin3, Endophilin1, and Synaptojanin1, we first triple labeled cryostat sections of 3 h dark adapted BL/6 retinae for Calbindin (green; labels horizontal cells and their invaginations into photoreceptor terminals), Dynamin3 (red), and Piccolino, a ribbon-specific Piccolo variant labeling the synaptic ribbon (blue; Regus-Leidig et al., 2013; Figure 6A). The merge of the three stainings shows that Dynamin3 puncta localize in the periactive zone close to the horseshoe shaped synaptic ribbons as well as the tips of horizontal cell processes (Figure 6A; arrows). To characterize this putative endocytotic hot spot at the ultrastructural level, we next performed pre-embedding immunoelectron microscopy on 3 h dark adapted BL/6 retinae with antibodies against Dynamin3, Endophilin1, Synaptojanin1 (Figures 6B–E), and Clathrin (data not shown). We also tried post-embedding immunoelectron microscopy, which unfortunately did not satisfactorily work in our hands. In rod photoreceptor terminals, immunoreactivity for Dynamin3, Endophilin1, Synaptojanin1 concentrated in the vicinity of the postsynaptic invaginations and the synaptic ribbon (Figures 6B–E; dashed circles). As expected, Clathrin LC immunoreactivity was diffusely distributed throughout the photoreceptor synaptic terminals, confirming the light microscopical absence of Clathrin LC in these endocytotic hot spots (data not shown). Sometimes, presumptive vesicles were visible in the labeled region (Figure 6C), but most of the times the intense staining obscured any underlying structures. Therefore, we analyzed dark adapted BL/6 retinae with conventional electron microscopy for the presence of conspicuous vesicle clusters in the vicinity of the synaptic ribbon (Figure 7).

Bottom Line: Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses.To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy.These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70.

View Article: PubMed Central - PubMed

Affiliation: Animal Physiology, Department of Biology, Friedrich-Alexander-University Erlangen-Nuremberg Erlangen, Germany.

ABSTRACT
Synaptic vesicle exocytosis at chemical synapses is followed by compensatory endocytosis. Multiple pathways including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kiss-and-run retrieval have been reported to contribute to vesicle recycling. Particularly at the continuously active ribbon synapses of retinal photoreceptor and bipolar cells, compensatory endocytosis plays an essential role to provide ongoing vesicle supply. Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses. To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy. In mouse ON bipolar cell terminals, Clathrin-mediated endocytosis seemed to be the dominant mode of endocytosis at all adaptation states analyzed. In contrast, in mouse photoreceptor terminals in addition to Clathrin-coated pits, clusters of membranously connected electron-dense vesicles appeared during prolonged darkness. These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70. We hypothesize that rod and cone photoreceptors possess an additional Clathrin-independent mode of vesicle retrieval supporting the continuous synaptic vesicle supply during prolonged high activity.

No MeSH data available.


Related in: MedlinePlus