【2017年整理】共聚焦显微镜spastin内质网蛋白定位.doc

PAGE 18 PAGE 16 SPG20 Spartin: Localization to the mitochondria and interaction with microtubule structure Key Words: Hereditary spastic paraplegia HSP, Spartin SPG20, Mitochondria, MIT Abbreviations list HSP Hereditary spastic paraplegia SPG, spastic paraplegia/paraparesis gene aa amino acid residual(s) ER endoplasmic reticulum MIT Microtubule Interacting and Trafficking TRS Troyer syndrome Abstract Hereditary spastic paraplegia (HSP) describes a diverse group of disorders characterized by length dependent axonal degeneration that causes progressive paraparesis primarily affecting the lower limbs. Troyer syndrome (TRS) is a rare autosomal recessive form of HSP where affected patients have characteristic spastic paraparesis due to retrograde axonal degeneration. TRS is caused by mutations in SPG20, one of which 1100 del A leads to a frameshift in exon 4 and premature termination of corresponding protein spartin (fs369-398x399). Normal spartin has 666 aa with a microtubule interacting and trafficking (MIT) domain at 36-114aa. However the function of this protein is unknown and there are no reported studies of its subcellular localisation. We cloned full-length spartin cDNA into a eukaryotic expression vector inframe with an enhanced fluorescent protein (ECFP-Spartin). We also constructed plasmids expressing prematurely truncated spartin (ECFP-Spartin1-398) or N terminal deleted spartin (ECFP-Spartin347-666) for comparison. The Spartin1- 398 has the same amino acid sequences to that reported in TRS patients. Confocal microscopy of a variety of transfected neuronal and non-neuronal cells revealed cytoplasmic expression that was primarily punctuate with a weak diffuse cytoplasmic distribution. We established that full-length spartin is located in mitochondria with superimposed organelle marker or immunocytochemistry staining. Colocalization measurement revealed that the ECFP-Spartin colocalized with mitochondria and tubulin. Analysis of cells expr