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Genetic analysis of stoned B/stonin 2 function in vivo

Başlık çevirisi mevcut değil.

  1. Tez No: 401192
  2. Yazar: MUHAMMED KASIM DİRİL
  3. Danışmanlar: PROF. DR. VOLKER HAUCKE
  4. Tez Türü: Doktora
  5. Konular: Tıbbi Biyoloji, Medical Biology
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2005
  8. Dil: İngilizce
  9. Üniversite: Georg-August-Universität Göttingen
  10. Enstitü: Yurtdışı Enstitü
  11. Ana Bilim Dalı: Belirtilmemiş.
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 144

Özet

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Özet (Çeviri)

Clathrin-mediated endocytosis at presynaptic nerve terminals is a regulated process tightly coupled to the exocytotic fusion event that requires clathrin, dynamin, AP2 as well as synaptotagmin 1. Stonin 2 is a brain-enriched protein which localizes to axonal vesicle clusters in neurons and clathin/AP2-coated pits in astroglial cells. Its Drosophila orthologue stoned B has a putative role in synaptic vesicle recycling, especially sorting and endocytosis of synaptotagmins. Mammalian stonin 2 interacts with the heterotetrameric clathrin adaptor AP2, via three novel WVXF motifs, and with other endocytic accessory proteins eps15 and intersectin 1 by virtue of its two NPF motifs in its amino-terminal domain. The carboxy-terminal μHD binds to synaptotagmin 1 and is required for proper localization of stonin 2 to synapses in neurons and to coated-pits in astroglial cells. We have recently developed an in vivo interaction assay, to test the interactions between stonins and synaptotagmin family members (1-11). Predominantly cytosolic stonin 2, translocates to the plasmalemma and colocalizes with AP2-coated pits when co-expressed together with synaptotagmins 1, 2, and 9. Transgenic expression of stonin 2 in a stable HEK293 cell line expressing lumenally FLAG-tagged synaptotagmin 1, stimulates endocytic internalization of plasma membrane-stranded synaptotagmin 1 more than 20-fold. Stonin 2 thus physically links synaptotagmin 1 and AP2 and this linkage is required for synaptotagmin 1 endocytosis. Disruption of the stonin 2-AP2 interaction by mutagenesis of WVXF motifs or RNAi-mediated silencing of AP2 expression, inhibits stonin 2-assisted synaptotagmin 1 uptake. In neuroendocrine PC12 cells, stonin 2 assists in targeting of internalized synaptotagmin 1 to SLMVs in addition to facilitating its endocytosis. For a better characterization of its functions, a stonin 2-deficient mouse strain was created by targeted disruption of the stonin 2 gene. Stonin 2 knock-out animals are viable and fertile. Preliminary analysis of synaptotagmin 1 distribution, endocytic recycling, number and morphology of synaptic vesicles in stonin 2 deficient nerve terminals did not display any abnormalities compared to wildtype littermates. Therefore refined experimental procedures seem to be required for characterization of stonin 2 deficiency in neurons and astrocytes. We propose that stonin 2 is an endocytic sorting adaptor for clathrin-mediated endocytosis of synaptotagmins in neurons and neurosecretory cells, as well as yet unidentified cargo molecule(s) in astroglial cells.

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