Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

Dean, Camin; Dunning, F. Mark; Liu, Huisheng; Bomba-Warczak, Ewa; Martens, Henrik; Bharat, Vinita; Ahmed, Saheeb; Chapman, Edwin R.

doi:10.1091/mbc.e11-08-0707

Cited by 74 publications

(88 citation statements)

References 61 publications

(93 reference statements)

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“…Syt XI is the most abundant Syt isoform in astrocytes and is expressed more abundantly in the astrocytes as compared with the neurons; 27,28,30 however, its subcellular localization in astrocytes is not known. 41,42 Use of western blot analysis confirmed expression of Syt XI protein in primary astrocytes (Figure 4c). Using cell fractionation we found that endogenous Syt XI is enriched in the lysosome fractions (Figure 4d), demonstrating its localization on astrocyte lysosomes.…”

Section: Syt XI Regulates Lysosomal Exocytosis In Astrocytesmentioning

confidence: 75%

“…28,[48][49][50] Similarly, Syt XI-containing vesicles undergo depolarization-triggered exocytosis in neurons. 42 Similar to neuronal lysosomes, 51 Syt XI-containing exocytic vesicles in neurons are acidic in nature and localize to dendrites. 42 To test whether, similar to Syt IV, the C2B domain of Syt XI helps trigger exocytosis, we mutated the three putative Ca 2+ -binding aspartate (D) residues in the C2B domain of Syt XI to asparagines (N) (SytXIrDN).…”

Section: Discussionmentioning

confidence: 99%

“…42 Similar to neuronal lysosomes, 51 Syt XI-containing exocytic vesicles in neurons are acidic in nature and localize to dendrites. 42 To test whether, similar to Syt IV, the C2B domain of Syt XI helps trigger exocytosis, we mutated the three putative Ca 2+ -binding aspartate (D) residues in the C2B domain of Syt XI to asparagines (N) (SytXIrDN). This mutant protein rescued the knockdown of endogenous Syt XI as efficiently as WT Syt XI, indicating that Syt XI-regulated lysosome exocytosis does not require Ca 2+ binding to C2 domains.…”

Section: Discussionmentioning

confidence: 99%

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Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

et al. 2015

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Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca 2+ -triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca 2+ -regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte -Syt XI -suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

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Section: Syt XI Regulates Lysosomal Exocytosis In Astrocytesmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

et al. 2015

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“…We used live-cell imaging by total internal reflection fluorescence (TIRF) microscopy and complementary biochemical assays to determine whether sublytic MACs stimulate lysosome exocytosis in the RPE. We transfected primary RPE with Syt7 tagged with pHluorin, whose fluorescence is quenched in acidic lysosomal pH and relieved upon exposure to neutral pH after exocytosis (30). Individual exocytic events after addition of 10% NHS were detected as bright flashes of unquenched pHluorin in control cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Protective responses to sublytic complement in the retinal pigment epithelium

Tan

Toops

Lakkaraju

2016

Proc. Natl. Acad. Sci. U.S.A.

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The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4 −/− Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4 −/− mice, indicating that they could be effective therapeutic approaches for macular degenerations.he complement system is an important regulator of immunity and inflammation. Complement activation by the classical, alternative, or lectin pathways results in the assembly of C5b-9 membrane attack complexes (MACs), which form membrane pores and cause target cell lysis. Within the eye, a low level of constant complement activity is necessary for immune surveillance, and several membrane-bound and soluble regulators prevent excessive complement activation (1). An imbalance between complement activation and inhibition in the retina is implicated in the pathogenesis of age-related macular degeneration (AMD), which causes central vision loss in more than 30 million people globally (reviewed in refs. 2-4).Evidence suggests that the retinal pigment epithelium (RPE), which helps maintain ocular immune privilege, is the first line of defense against unregulated complement activation in the retina (5). The complement cascade is activated by insults that disturb RPE homeostasis, including defective clearance of phagocytosed photoreceptor outer segments (6) and abnormal accumulation of vitamin A dimers (7-9) or oxidized lipids (10) in the RPE. Studies also show that exposure to sublytic levels of MAC alters RPE barrier integrity, causes...

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“…These include certain SNAREs (Kennedy et al, 2010;Lau et al, 2010;Suh et al, 2010), synaptotagmins (Dean et al, 2012), Rab-GTPases (Brown et al, 2005;Park et al, 2006), components of the exocyst complex (Gerges et al, 2006), and actin-based molecular motors (Wang et al, 2008). Acidification of vesicles is also an important determinant of their biogenesis and function (Weisz, 2003), yet the mechanisms controlling pH homeostasis of postsynaptic endosomes are not well understood.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Recruitment of Endosomal Na⁺/H⁺Exchanger NHE6 into Dendritic Spines of Hippocampal Pyramidal Neurons during NMDA Receptor-Dependent Long-Term Potentiation

Deane

Ilie

Sizdahkhani³

et al. 2013

J. Neurosci.

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Postsynaptic endosomal trafficking has emerged as a principal regulatory mechanism of structural and functional plasticity of glutamatergic synapses. Recycling endosomes perform activity-dependent transport of AMPA receptors (AMPARs) and lipids to the postsynaptic membrane, activities that are known to contribute to long-term synaptic potentiation and hypothesized to subserve learning and memory processes in the brain. Recently, genetic defects in a widely expressed vesicular pH-regulating transporter, the Na ϩ /H ϩ exchanger NHE6 isoform, have been implicated in neurodevelopmental disorders including severe X-linked mental retardation and autism. However, little information is available regarding the cellular properties of this transporter in the CNS. Here, we show by quantitative light microscopy that the protein abundance of NHE6 is developmentally regulated in area CA1 of the mouse hippocampus. Within pyramidal neurons, NHE6 was found to localize to discrete puncta throughout the soma and neurites, with noticeable accumulation at dendritic spines and presynaptic terminals. Dual immunolabeling of dendritic spines revealed that NHE6 partially colocalizes with typical markers of early and recycling endosomes as well as with the AMPAR subunit GluA1. Significantly, NHE6-containing vesicles exhibited enhanced translocation to dendritic spine heads during NMDA receptor (NMDAR)-dependent long-term potentiation. These data suggest that NHE6 may play a unique, previously unrecognized, role at glutamatergic synapses that are important for learning and memory.

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Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

Cited by 74 publications

References 61 publications

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

Protective responses to sublytic complement in the retinal pigment epithelium

Enhanced Recruitment of Endosomal Na⁺/H⁺Exchanger NHE6 into Dendritic Spines of Hippocampal Pyramidal Neurons during NMDA Receptor-Dependent Long-Term Potentiation

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Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

Cited by 74 publications

References 61 publications

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

Protective responses to sublytic complement in the retinal pigment epithelium

Enhanced Recruitment of Endosomal Na+/H+Exchanger NHE6 into Dendritic Spines of Hippocampal Pyramidal Neurons during NMDA Receptor-Dependent Long-Term Potentiation

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Enhanced Recruitment of Endosomal Na⁺/H⁺Exchanger NHE6 into Dendritic Spines of Hippocampal Pyramidal Neurons during NMDA Receptor-Dependent Long-Term Potentiation