共聚焦显微镜spastin内质网蛋白定位

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1、24SPG20 20050820 Spartin: Localization to the mitochondria and interaction with microtubule structure Key Words: Hereditary spastic paraplegia HSP, Spartin SPG20, Mitochondria, MIT Abbreviations listHSP Hereditary spastic paraplegia SPG, spastic paraplegia/paraparesis geneaa amino acid residual(s)ER

2、 endoplasmic reticulumMIT Microtubule Interacting and TraffickingTRS Troyer syndrome AbstractHereditary spastic paraplegia (HSP) describes a diverse group of disorders characterized by length dependent axonal degeneration that causes progressive paraparesis primarily affecting the lower limbs. Troye

3、r 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 pr

4、otein 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 ex

5、pression 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 repor

6、ted 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 organel

7、le marker or immunocytochemistry staining. Colocalization measurement revealed that the ECFP-Spartin colocalized with mitochondria and tubulin. Analysis of cells expressing either ECFP-Spartin1-398 or ECFP-Spartin347-666 revealed that mitochondrial expression is dependent on sequences in the C termi

8、nal region. Bioinfomatic analysis found transmembrane fragments and internal mitochondrial targeting signals, which are mainly located at the C-terminal half of the spartin protein sequences. Using acceptor quenching FRET analysis of HeLa cells co-expressing EYFP alpha tubulin and ECFP-spartin and i

9、ts derivatives, we examined the interaction of these tagged spartin with the microtubule network. The interaction between ECFP-Spartin and microtubule structure is stronger (Ef =8.007 5.927) than that the interaction observed with prematurely truncated ECFP-Spartin1-398 (Ef =2.8074.306), the interac

10、tion between N terminal deleted ECFP-spartin347-666 and EYFP-tubulin with the microtubule was lost (Ef 0). These results indicate that spartin is another microtubule interacting protein and that sequences in the N terminal region, which contains an MIT domain, are important in mediating this interac

11、tion. Axonal degeneration happened in TRS patients may be the result of energy supply and/or axonal transportation disorder due to the failure of mutant spartin1-398 entering into mitochondria and/or the presence of truncated protein in the cytoplasm interfering with the interaction of normal sparti

12、n with microtubule networks.IntroductionHereditary spastic paraplegia (HSP) describes a genetically and clinically diverse group of inherited neurodegenerative disorders that are characterised by progressive spastic paraparesis primarily affecting the lower limbs. To date at least 28 HSP causing loc

13、i have been mapped, designated SPG1 to SPG28 in order of discovery (Bouslam 2005). Pathologically, HSP is characterized by degeneration of the longest axons in the body, the corticospinal tracts and to a lesser extent the fasiculis gravis. The length of these neurons, which in some cases can reach u

14、p to 1 metre, creates a cellular environment that is highly dependent on intracellular transport mechanisms. Troyer syndrome (TRS) is a complicated form of autosomal recessive (AR) HSP where affected patients have in addition to progressive spastic paraplegia other symptoms including pseudobulbar pa

15、lsy and distal amyotrophy, together with mild developmental delay and subtle skeletal abnormalities. A mutation in the SPG20 gene on chromosome 13q23, which encodes spartin has been identified in a family with Troyer syndrome (Patel 2002). To date there have been no studies reporting the function or

16、 cellular localisation of this protein and little is known about the pathogenesis of this disease. Our knowledge of the general mechanisms involved in the pathogenesis of HSP is advancing rapidly with recent publications of cellular localisation and protein interaction studies. Several of the causat

17、ive proteins in HSP have been found to be closely associated with membrane-bound organelles such as the association of spastin (SPG4) with endosomes (Reid et al., 2005) and the endoplasmic reticulum) (personal communication), and the association of both paraplegin (SPG7) and heat shock protein 60 (S

18、PG13) with mitochondria (Ferreirinha 2004, Atorino 2003, Lindholm 2004). Some of these membrane-bound organelles such as endosomes are known to associate with microtubules. In the case of SPG7 linked HSP, animal model studies show that mice with mutant paraplegin had mitochondrial morphological abno

19、rmalities in synaptic terminals and in distal regions of axons before the axon degeneration (Ferreirinha 2004, Atorino 2003). All these indicate that impaired cellular traffic of the membrane-bound organelles could be the cause of axonal degeneration. Spartin is found widely expressed both within th

20、e nervous system and also in non-neuronal tissues (Patel 2002). There are no reports on its sub-cellular localization and few clues to its function. The N- terminal region of spartin contains an MIT (microtubule interacting and trafficking) domain, a domain that is present in a number of proteins in

21、cluding VPS4 and SKD1 that have a well defined role in intracellular protein trafficking (Ciccarelli et al., 2003). Interestingly this MIT domain is also present in spastin, a protein which when mutated causes the most common form of HSP. Studies exploring the function of spastin have indicated that

22、 the protein interacts transiently with microtubules (Errico et al., 2002). Recently spastin was reported interacting with an endosomal protein, CHMP1B, which has a role in membrane traffic (Reid et al., 2005). We have further identified that spastin localizes to the endoplasmic reticulum, an active

23、 site for protein synthesis and transportation (Personal communication). Given the presence of the MIT domain in both spastin and spartin it is tempting to speculate that spartin may also interact with microtubules lending support to the hypothesis that defective cellular transport mechanisms are re

24、sponsible for the axonal degeneration seen in HSP.In an attempt to clarify the sub-cellular localization of spartin and to gain insight into its physiological function, we performed expression and cell localization experiments using both ECFP tagged full-length and mutant spartin together with organ

25、elle specific stains to identify with which cellular components spartin associated. Our results support the central role that microtubules appear to play in the general pathogenesis of HSP. We have established that spartin is located inside the mitochondria and this localisation is determined by seq

26、uences in the C-terminal region. Using fluorescence resonance energy transfer (FRET) examination we have also shown that spartin interacts with the microtubule network in the cytoplasm. Microtubule interaction is dependent on sequences in the N-terminal region of the protein while prematurely trunca

27、ted spartin lacking the C terminal region of spastin, which is the same to the mutation causing Troyer syndrome, has lost its localization to mitochondria while retaining the microtubule interaction activity. Axonal degeneration may be the result of the lost normal function by dislocation of the pro

28、tein from mitochondria and/or interference of the normal microtubule function by the cytoplasmic truncated spartin protein. Materials and MethodsPreparation of full-length and mutant spartin cDNA and construction of expression plasmidsHuman Normal Adult Brain cDNA (GenePool cDNA Cat No. D8030-01 Lot

29、 A707030 Invitrogen) was used as template to amplify full-length spastin cDNA via a nested PCR approach. Primer pairs with restrictive enzyme sites (Nde I and Hind III) were scheduled according to SWISS-PROT human spartin cDNA sequence (SPG20_HUMAN, Q8N0X7; Ensembl Gene ID ENSG00000133104). The expr

30、ession frame of full-length spastin cDNA was subcloned between the Nde I and Hind III restrictive enzyme sites of pDNR-a (BD bioscience, K1670-1) and transferred into mammalian expression vector pLP-ECFP-C1 (BD bioscience, 6343-1) through Cre-LoxP recombinase reaction. The resulting vector (pECFP-SP

31、G20) would express full-length spastin fused with ECFP (figure 1). PCR pairs were scheduled to prepare the area 2209 Xma I 3502 (Bcu I/Spe I) in 2 fragments with Del 2521A (Equal to D1110A at SPG20 ORF happened in Troyer patient) and Mun I sites at 2525. The two fragments were digested with Mun I an

32、d ligated. The ligation product and pLP-ECFP-SPG20 were further digested with Xma and Bcu I and ligated to produce plasmids pLP-ECFP-SPG20 Del 2521A. The protein product of the plasmid pLP-ECFP-SPG20 Del 2521A is ECFP-Spartin1-398. The mutation is the same to D1110A at SPG20 ORF, causing Troyer synd

33、rome (protein truncated by 268 aa, fs369-398x399) (Patel H). The pECFP-SPG20 vector was double-digested by Eco RI and Mun I, religated to produce vector expressing truncated spartin fused with ECFP. This resulted in ECFP-Spartin1-347, fs348-393x394. The pECFP-SPG20 vector was further treated by Acc

34、I and Eco RI double-digestion followed by Klenow Fill-in reaction to produce N-terminal 1346 deleted spastin tagged with ECFP (ECFP-Spartin347-666). A schematic representation of the proteins expressed by these vectors (pECFP-Spartin1-398, pECFP-Spartin1-347, pECFP-Spartin347-666) was shown in figur

35、e 1.Figure 1. Schematic representation of expressed full-length and mutant ECFP-spartin fusion proteins:NH2-COOH 1 666ECFP-Spastin ECFP- Spartin1-398 ECFP- Spartin1-347 ECFP-Spartin347-666 ECFP MIT domain Cell Culture and Transfection Lipofectamine 2000 (Invitrogen, 11668-027) was used for transfect

36、ion of the cells. The cells were then transfected at 50-90% confluence using 1.0-1.5mg of DNA and 2-3ml of Lipofectamine in 2ml Opti-MEM (Invitrogen, 51985-026) per well for 6-well plates. After 3-4 hours of transfection, the transfection medium was replaced with culture medium. In some experiments,

37、 the replacement culture medium was composed of reduced FCS and Ionomycin or ATRA to induce differentiation. Geneticin (G418 Sigma, A1720) was supplemented in sustained culture for selective continuous incubation of cells expressing the desired fusion protein. Where necessary, microtubules were stab

38、ilized with Taxol (Sigma, B5683).Immunocytochemistry, organelle tracker staining and Confocal MicroscopyOne to 4 days after transfection, the cells were rinsed with PBS and fixed with 4% paraformaldehyde in 1x PBS solution. Cells were permeabilized with 0.3%Triton X-100 in 1x PBS for 20 minutes and

39、washed in PBS. Non-specific reactions were blocked with 5% bovine serum albumin and 5% Goat serum in PBS for 30 minutes before the addition of antibodies. Cells were incubated with labelled antibody or first and labelled secondary antibody sequentially and washed with PBS. The antibodies used are li

40、sted in table 2. Nuclei were stained with DAPI (Sigma, D9542). The specificity of the anti-spastin antibody was confirmed by preincubation of the antiserum with purified polyhistidine tagged spastin or polyhistidine tagged spastin-EGFP. The cells were post-fixed with 2% paraformaldehyde in 1x PBS so

41、lution, mounted in VectaShield H1000 (Vector Laboratories) and stored at 4C. Organelle tracker staining was undertaken according to the manufacturers protocol. Cells stained with ER-Tracker Blue-White DPX and MitoTracker Red CMXRos were photographed following treatment with 4% paraformaldehyde in 1x

42、 PBS solution or acetone fixation respectively. Cells stained with LysoTracker Red DND-99 were observed directly after staining in the culture medium with no further manipulation.The Meta 510 Confocal Microscope (Zeiss, Germany) was used for observation and analysis of the cells. Slice thickness was

43、 set at 0.5 or 1.0mm. The slices though the cell equator or those showing the best structures were photographed.Colocalization analysis on 20-25 cells expressing ECFP tagged spartin or spartin mutants was undertaken with colocalization analysing software of Zeiss Meta 510 Confocal Microscope. Accumu

44、lated data was processed with Sigma Plot statistical analysis software (SPSS Inc). P value 0.05 was considered as statistically significant.Fluorescence Resonance Energy Transfer (FRET) Hela cells were double transfected with pEYFP-Tub (BD biosciences 6118-1) and one of the pECFP-Spartin, pECFP- 1-3

45、47Spartin or pECFP- spartin Spartin347-666 plasmids. Hela cells transfected with pEYFP or pECFP only, or double transfected with pEYFP and pECFP, pEYFP-Tub and pECFP-SPG4 (Spastin) were used as controls. Consecutive 20 positive cells expressing both EYFP-alpha tubulin and ECFP-spartin or its mutant

46、fusion proteins were subject to the following FRET examination. From each cell, 1-3 regions of interest (ROI) were set for photobleaching. The laser of Zeiss LSM510Meta confocal microscope (Carl Zeiss. Inc) was tuned to lines at 458, 488 and 514 nm. The filter setting of Karpova (2003) was used to o

47、ptimize the imaging of CFP and YFP and to eliminate cross talk between the channels. PMT settings were established by the preceding procedure consistently yielded no cross talk when CFP-only or YFP-only cells were imaged (Supplementary FRET Figures). FRET was measured using the acceptor photobleachi

48、ng method (Kenworthy, 2001). The FRET energy transfer efficiency was calculated as percentage of (Ef) Ef= (IafterIbefore) x100/Iafter, where Ibefore/after is the CFP intensity before or after the photobleaching. This formula yields the increase in CFP fluorescence following a YFP bleach normalized b

49、y CFP fluorescence after the bleach. Similar calculation in non-bleached regions of the specimen: Cf= (IafterIbefore) x100/Iafter was always performed on the same cell as controls (Karpova 2003). ResultsLocalization of spartin to the cytoplasm To gain insights into spartin subcellular localization,

50、we examined spartin expression in a variety of neuronal and non-neuronal cells including neuronal cells from primary culture, human neuroblastoma SH-SY5Y and murine neuroblastoma neuro-2A cell lines at various stages of differentiation, and non-neuronal rat glioma C6 cells as well as HeLa cells. Spa

51、rtin expression was monitored using fluorescent confocal microscopy to ECFP tagged protein. We observed cytoplasmic expression (figure 1a-d) in all transfected cells examined. We did not observe any evidence of nuclear expression even when ECFP-spartin was expressed at high levels (figure 1d). In pr

52、imary cultured neuron cells, the distribution was mostly dotty or punctuate, suggesting association with cellular organelles. This dotty expression could be seen both in the cell body and in the extending neurites. In cultured neuroblastoma cells expressing ECFP tagged spartin, the dotty punctuate d

53、istribution pattern was also seen together with larger aggregations or clusters of dots, which could be seen accumulated at one side of the nucleus (figure 1d). A weak diffuse cytoplasmic signal was also observed. ECFP-spartin also formed vesicle structure in the cells (figure 1c). The mitochondria

54、is the major site of spartin localization The punctuate cytoplasmic expression pattern observed for spartin is suggestive of localization (packaging and/or functioning) inside cellular vesicles or organelles. To investigate this, we have used a series of organelle specific markers (trackers) and ant

55、ibodies to show the relationship between these organelles/ vesicles and full-length ECFP-spartin. Following transfection of primary cultured mouse neurons, neuronal SH-SY5Y and non-neuronal HeLa cells with pECFP-SPG20, the cells were exposed to either organelle specific trackers for endoplasmic reti

56、culum, mitochondria, or lysosomes, or to an organelle specific antibody; anti-mitochondria ATP synthase subunit beta for mitochondria, anti-calreticulin to visualize endoplasmic reticulum or anti-early endosome antigen 1 (EEA1) antibody to show early endosomes. As can be seen from figure 1e-1l, ECFP

57、-spartin colocalizes with mitochondria in mouse neuron, human neuroblastoma (SH-SY5Y) and HeLa cells. This result was consistently observed using the widely accepted mitochondrial specific marker, MitoTraker. Mitochondrial localization was further confirmed by colocalization examination (Supplementa

58、ry figure 2) and immunocytochemistry with antibody to mitochondrial ATP synthase subunit beta (another mitochondria marker). This is the first reported observation of the localization of spartin in cell organelles. Using confocal microscopy, we were able to optimise both the intensity of signal and

59、the thickness of the sections to obtain better resolution. Taking sections at 0.5mm, we were able to show that in SH-SY5Y and Hela cells expressing ECFP-Spartin within the mitochondria, the tagged protein seemed to associate with the mitochondrial membrane (figure 1j, 1l). These results suggest that

60、 spartin may function as a membrane related protein in the mitochondria. The C-terminal region contains the primary determinants for mitochondrial localization of spartin The region of the spartin protein primarily involved in determining mitochondrial localization was further defined by. To assess

61、the importance of sequences in the C terminal region of spartin, we constructed plasmids expressing prematurely truncated spartin which consists of amino acids 1-398 or 1 347 fused in frame to ECFP (pECFP- spartin1-398, pECFP- spartin1-347). To assess the role of sequences in the N terminal region w

62、e created a plasmid that would express an N terminal deleted spartin, lacking amino acids 1-346 fused in frame to ECFP (pECFP- spartin 347-666. These plasmids were transfected into SH-SY5Y and HeLa cells. The N-terminal deleted ECFP-Spartin347-666 fusion protein had punctuate or clumpy expression pa

63、ttern similar to the pattern observed for full-length spartin, while the prematurely truncated ECFP-Spartin1-398 fusion protein was diffusely distributed in the cytoplasm (figure 2 a-d). ECFP-Spartin1-347 showed the same distribution pattern (data not shown). We then used MitoTracker or anti-mitocho

64、ndrial ATP synthase subunit beta immunocytochemistry on SH-SY5Y and HeLa cells transfected with pECFP- Spartin1-398or pECFP-Spartin347-666 to examine the localization of these mutant spartin fragments in relation to mitochondria. N-terminal deleted ECFP-Spartin347-666 was found to be colocalized wit

65、h mitochondria, while prematurely truncated spartin lacking the C-terminal lost this localization (figure 2e-l, and supplementary figure 2). In some cells, the colocalization of ECFP-Spartin347-666 with mitochondria was not clear-cut, but higher magnification showed that the ECFP-Spartin347-666 was inside the mitochondria forming vesicular structures (figure 2i). These results suggest that ECFP-Spartin347-666 is localised on the inner wall of the mitochondria.ECFP-Spar