(医学课件)自噬-检测指南ppt.pptx

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1、细胞自噬研究方法概述1 Autophagic Compartments2 Autophagic CompartmentsPhagophore (pre-autophagosomal)： previously called the isolation or sequestration membrane吞噬泡吞噬泡 ：参与自噬体形成早期事件的膜池。也指“隔离膜isolation membrane”或“杯状结构cup-shaped structure”Autophagosome :自噬体自噬体 ：双层膜包裹胞质形成的囊泡Amphisome ：generated by the fusion of au

2、tophagosomes with endosomes, also referred to as an acidic late autophagosome自噬内涵体自噬内涵体：溶酶体和内涵体融合的中间囊泡Autolysosome ：generated by fusion of autophagosomes or amphisomes with a lysosome自噬溶酶体自噬溶酶体：自噬小体和溶酶体融合形成的终末结构3 Induce/ promoteInhibitionAutophagic molecular mechanisms 4 Initiation： Induction，Cargo

3、recognition and selectivityElongation，Closure： Autophagosome formationMaturation，Degradation: Vesicle fusion and autophagosome breakdownAutophagic molecular mechanisms 5 Induction Normal conditionsBasal-level autophagy is very low;Autophagy inhibitor: serine/threonine protein kinase TOR(target of ra

4、pamycin)input information from multiple upstream signal transduction pathways (discussed below) and negatively regulates another serine/threonine kinase, Atg1, in nutrient-rich conditionsStarvation conditons or RapamycinTOR inhibited;Atg1 activated;Atg1 binding affinity to Atg13 and Atg17 ;Promotes

5、the formation of an Atg1-Atg13-Atg17 scaffold ;Atg1-Atg13-Atg17 recruitment of multiple Atg proteins to the PAS to initiate autophagosome formation. 6 Cargo recognition and selectivityP62/sequestosome 1 (SQSTM1) .P62 directly binds both poly- or mono-ubiquitin via its ubiquitin-associated (UBA) doma

6、in and LC3 links the ubiquitinated cargos to the autophagy machinery for autophagic degradation. 7 Class III phosphatidylinositol 3-kinase (PtdIns3K) complex:PtdIns3K Vps34 (vacuolar protein sorting 34),a myristoylated serine/threonine kinase Vps15, Atg14 ; Beclin 1 ; Autophagosome formationVps34Atg

7、14Vps15Beclin1The PtdIns3K complex produces PtdIns3P (phosphatidylinositol 3-phosphate) and is involved in PAS targeting of a number of yeast Atg proteins that bind PtdIns3P, such as Atg18, Atg20, Atg21, and Atg24 .In yeast, Atg20 and Atg24 interact with the Atg1-Atg13-Atg17 complex, and the latter

8、mediates autophagy induction ;The PtdIns3K complex, recruits two interrelated ubiquitin-like (Ubl) conjugation systems, Atg12Atg5-Atg16 and Atg8PE (phosphatidylethanolamine), to the phagophore which play an essential role in regulating the membrane elongation and expansion of the forming autophagoso

9、me.8 The function of Beclin 1 in autophagy is regulated by Bcl-2;Bcl-2 inhibits autophagy by binding and sequestering Beclin 1 ;Dissociation of Beclin 1 from Bcl-2 is required for autophagy induction. Vps34Atg14Vps15Beclin1Bcl-2Atg12 is activated by Atg7 , transferred to Atg10 (E2 conjugating enzyme

10、) and attached to an internal lysine of the substrate protein Atg5 covalently. The Atg12Atg5 conjugate further interacts with a coiled-coil protein Atg16, which links the Atg12Atg5-Atg16 complex into a tetramer by self-oligomerization and attaches it to the phagophore .Autophagosome formation9 Atg8

11、is first processed by a cysteine protease, Atg4, exposing a C-terminal glycine residue. The same E1 enzyme Atg7 activates Atg8 and transfers it Atg8 is finally conjugated to the target lipid PE via an amide bond. In nutrient-rich conditions, the majority of Atg8 is cytosolic（16Kd）; upon autophagy in

12、duction, Atg8 largely exists as the lipid-conjugated form (14Kd)and is localized to both sides of the phagophore .Atg8 controls the size of the autophagosome ,which may result from its abilityto determine membrane curvature. The lipidation of Atg8 and its mammalian homolog LC3 are widely used to mon

13、itor autophagy induction.Autophagosome formation10 Vesicle fusion and autophagosome breakdownIn mammalian cells, the fusion event requires the lysosomal membrane protein LAMP-2 and the small GTPase Rab7.After fusion, degradation of the inner vesicle is dependent on a series of lysosomal/vacuolar aci

14、d hydrolases, including proteinases A and B (encoded by PEP4 and PRB1, respectively) and the lipase Atg15 in yeast and cathepsin B, D (a homolog of proteinase A), and L in mammalian cellsThe resulting small molecules from the degradation, particularly amino acids, are transported back to the cytosol

15、 for protein synthesis and maintenance of cellular functions under starvation conditions.11 1. Transmission electron microscopy2. Atg8/LC3 detection and quantification 3.SQSTM1/p62 and related LC3 binding protein turnover assays4. MTOR, AMPK and Atg1/ULK15. Additional autophagy-related markers6. Tra

16、nscriptional and translational regulation7. Autophagic protein degradation8. Selective types of autophagyMethods for Monitoring Autophagy9. Autophagic sequestration assays10. Turnover of autophagic compartments11. Autophagosome-lysosome colocalization and dequenching assay12. Tissue fractionation13.

17、 Analyses in vivo14. Cell death15. Chaperone-mediated autophagy12 Transmission electron microscopy一、取材一、取材快速、低温细胞株：细胞株：胰酶消化：细胞完整性保存良好，但是对自噬有一定影响细胞刮片：最大程度维持细胞原生理状态，细胞物理损伤大先固定，后刮取：细胞完整性、生理性维持良好，但细胞分散，不易成团组织：组织：优选低温灌注固定时间点：12h，8h，24h13 Transmission electron microscopy二、结构特点二、结构特点Autophagosomes ：12h, 8h

18、double membrane，visible as two parallel membrane bilayers separated by an electron-lucent cleft contain cytosol and/or organelles that look morphologically intactAmphisomescan sometimes be identified by the presence of small internal vesicles inside the autophagosome/autophagic vacuole (AV). These i

19、nternal vesicles are delivered into the lumen by fusion with multivesicular endosomes. Late/degradative autophagic vacuoles and autolysosomes (AVd) ：24husually have only one limiting membrane, and contain cytoplasmic material and/or organelles at various stages of degradation 14 Transmission electro

20、n microscopy15 Cautionary notesFixation of excised tissues requires care to avoid sampling a nonrepresentative or uninformative section of tissue. Quantify autophagosome (and/or autolysosome) profiles per total cytoplasmic or cellular area in sections.At least 20 cell profiles per sample.Each imaged

21、 cell profile is captured and scored at the same magnification.16 Cautionary notesNot all double-membrane structures are autophagosomesApoptotic bodies from neighboring cells are readily phagocytosed by surviving cells of the same tissue.Phagosomes have double limiting membranes, inner one is from t

22、he plasma membrane of the apoptotic body and the outer one is that of the phagocytizing cell. A major difference, is that the surrounding membranes are the thicker than the thinner sequestration membrane type (910 nm, vs. 78 nm, respectively). A good feature to distinguish between autophagosomes and

23、 double plasma membrane-bound structures is the lack of the distended empty space between the two membranes of the phagocytic vacuoles.Engulfed apoptotic bodies usually have a larger average size than autophagosomes.17 Due to the cisternal structure of the ER, double membrane-like structures surroun

24、ding mitochondria or other organelles are often observed after sectioning.Employ tomographic reconstructions of the TEM images to confirm that the autophagic compartments are spherical and are not being confused with endomembrane cisternae or damaged mitochondria .If there are ribosomes associated w

25、ith these membranes they can help distinguish them from the ribosome free double-membrane of the phagophore and autophagosome. Cautionary notes18 a. Western blotting and ubiquitin-like protein conjugation systems b. Turnover of LC3-II/Atg8PE c. GFP-Atg8/LC3 lysosomal delivery and proteolysis d. GFP-

26、Atg8/LC3 fluorescence microscopy e. Tandem mRFP/mCherry-GFP fluorescence microscopy f. Autophagic flux determination using flow and multispectral imaging cytometry g. Immunohistochemistry. Atg8/LC3 detection and quantification 19 Western blottingThe mammalian homologs of Atg8 LC3 (microtubule-associ

27、ated protein 1 light chain 3)LC3A, B, B2 and C GABARAP: GABAA receptor-associated protein GABARAPL1/GEC1: GABAA receptorassociated protein like 1/Glandular Epithelial Cell 1 GABARAPL2/GATE-16/GEF2: GABAA receptor-associated protein like 2/Golgi-associated ATPase enhancer of 16 kDa/gangliosideexpress

28、ion factor 2 GABARAPL3: GABAA receptor associated protein like 320 There is not always a clear precursor/product relationship between LC3-I and LC3-II, changes in LC3-II amounts are tissue- and cell context-dependentMoreover, LC3-I is more labile than LC3-II, being more sensitive to freezingthawing

29、and to degradation in SDS sample buffer, fresh samples should be heated and assessed as soon as possible and should not be subjected to repeated freeze-thaw cycles.PVDF membranes may result in a stronger LC3-II retention than nitrocellulose membranesTriton X-100 may not efficiently solubilize LC3-II

30、 in some systemsHeating in the presence of 1% SDS, or analysis of membrane fractions, may assist in the detection of this protein. In some cases beta-actin levels decrease when autophagy is inducedCautionary notes:21 Western blotting22 23 Turnover of LC3-II/Atg8PE.Prevent Lysosomal Degradation Autop

31、hagic flux can be measured by inferring LC3-II/Atg8PE turnover by western blot in the presence and absence of lysosomal degradation;The relevant parameter in this assay is the difference in the amount of LC3-II in the presence and absence of saturating levels of inhibitors;If flux is occurring, the

32、amount of LC3-II will be higher in the presence of the inhibitor.24 Protease inhibitors pepstatin A and E-64d:Neutralize the lysosomal PHbafilomycin A1（洛霉素A1）chloroquine ：氯喹NH4Cl ：氯化铵Block fusion of autophagosomes with lysosomes bafilomycin A1 Knocking down or knocking out lysosomal-associated membr

33、ane protein 2 (LAMP2) Prevent Lysosomal Degradation 注：抑制剂作用时间：12h； 设阳性对照组 时间点：4h/24h bafilomycin A1, NH4Cl or chloroquine, also directly inhibit the endocytosis /uncoating of viruses and other endocytic events requiring low pH monitor both turnover of LC3-II and an autophagosome substrate in paralle

34、l.25 1 h of pre-incubation with 10 mg/ml E-64d is sufficient in most cases, since this inhibitor is membrane permeable and rapidly accumulates within lysosomes. pepstatin A is membrane impermeable (ethanol or preferably DMSO must be employed as a vehicle) and requires a prolonged incubation (. 8 h)

35、and a relatively high concentration (. 50 mg/ml) to fully inhibit lysosomal cathepsin D 26 GFP-Atg8/LC3 lysosomal delivery and proteolysisWerstern blotGFP-LC3在自噬溶酶体的酸性环境中被降解GFP单体释放到胞质中，蛋白印迹检测GFP单体条带Cautionary notes:A reduction in the intensity of the free GFP band may indicate reduced flux, but it m

36、ay also be due to efficient turnover. Using a range of concentrations and treatment times of compounds that inhibit autophagy can be useful in distinguishing between these possibilities.27 GFP-Atg8/LC3 fluorescence microscopyFluorescence microscopyA constant increase in the number of cells accumulat

37、ing GFP-LC3 puncta is suggestive of defective fusion of autophagosomes with lysosomes;Conversely, a decline implies that GFP-LC3 is consumed within newly formed autolysosomes. Fluorescence microscopy + lysosomal protease or fusion inhibitors Monitoring changes in the number of puncta. The presence o

38、f lysosomal inhibitors should increase the number of GFP-LC3-positive structures, and the absence of an effect on the total number of GFP-LC3 puncta or on the percentage of cells displaying numerous puncta is indicative of a defect(s) in autophagic flux.28 Tandem mRFP-GFP fluorescence microscopyThe

39、GFP signal is sensitive to the acidic and/or proteolytic conditions of the lysosome lumen, whereas mRFP is more stable. Therefore, colocalization of both GFP and mRFP fluorescence indicates a compartment that has not fused with a lysosome, such as the phagophore or an autophagosome. In contrast, an

40、mRFP signal without GFP corresponds to an amphisome or autolysosome. One of the major advantages of the tandem mRFP/mCherry-GFP reporter method is that it enables simultaneous estimation of both the induction of autophagy and flux through autophagic compartments in essentially native conditions, wit

41、hout requiring any drug treatment. 29 自噬体和自噬溶酶体分别成黄色和红色标记，如果自噬潮增加，两种颜色的点状聚集均增加。如果自噬体向自噬溶酶体成熟受阻，黄色点状聚集物增加，红色不增加。30 Flow and multispectral imaging cytometry在自噬诱导一开始，GFP-LC3 点状聚集显著增多，随后信号可能会出现下降，代表着自噬性降解的发生高通量检测31 ImmunohistochemistryWhen autophagosomes are absent, the localization pattern of LC3 in th

42、e cells of various tissues is diffuse and cytosolic.One problem with immunohistochemistry for LC3 is that in some tissues this protein can be localized in structures other than autophagosomes. For example, in murine hepatocytes and cardiomyocytes under starved conditions, endogenous LC3 is detected

43、not only in autophagosomes but also on lipid droplets.In neurons in ATG7-deficient mice, LC3 is accumulated in ubiquitin- and SQSTM1-positive aggregates.32 p62 and related LC3 binding protein turnover assays The SQSTM1 protein serves as a link between LC3 and ubiquitinated substrates.decreased SQSTM

44、1 levels are associated with autophagy activationThe phosphorylation of SQSTM1 at Ser403 appears to regulate its role in the autophagic clearance of ubiquitinated proteins, and anti-phospho-SQSTM1/p62 antibodies can be used to detect the modified form of the protein.33 Western blot analysis using NP

45、40 or Triton X-100 lysis in autophagic conditions typically shows a reduction in SQSTM1 levels. However, this does not necessarily indicate that SQSTM1 is degraded, because SQSTM1 aggregates are insoluble in these detergent lysis conditions.Whereas LC3 changes may be rapid, clearance of autophagy su

46、bstrates may require a longer time. Therefore, if LC3 changes are assessed at 6 h or 24 h after a drug treatment, SQSTM1 levels can be tested not only at the same time points, but also at later time points (24 h or 48 h) for determining the maximal impact on substrate clearance. Cautionary notes 34

47、TOR, AMPK and Atg1/ULK1 TORC1 is an autophagy-suppressive regulator that integrates growth factor, nutrient and energy signals. inhibition of mTOR leads to induction of autophagyThe enzyme activity of AMPK is absolutely dependent on phosphorylation of the a-subunit on Thr172, and can monitored by we

48、stern blotting with a phosphospecific antibody against this site. Activation/assembly ULK1 complex in mammals (ULK1-RB1CC1-ATG13-C12orf44/ATG101) is one of the first steps of autophagy induction.，activation of this complex can be assessed to monitor autophagy induction.the phosphorylation status of

49、ULK1 at the activating sites (Ser317, 467, 555, 637, 777, or Thr574) or dephosphorylation at inactivating sites (Ser638, 757) can be determined using phospho-specific antibodies, or by western blotting.35 根据自噬机制主要负责降解长寿命蛋白的特性，先让细胞在含有同位素标记氨基酸(如 14C- 或 3H- 缬氨酸或亮氨酸)的培养基中生长一段时间(数小时至数天)细胞在此期间合成的蛋白质都将被同位素标记，然后换成不含同位素的培养基，让一些被标记的短寿命蛋白通过蛋白酶体途径降解在自噬诱导后，通过检测培养上清中释放的自噬性降解产物的放射性活度即可反映细胞自噬性降解的能力同时加入自噬抑制剂作为对比，更能特异性地反映自噬引起的蛋白质降解.长寿命蛋白降解检测长寿命蛋白降解检测36 自噬的实验性调控自噬的实验性调控二、基因干预二、基因干预通过 RNAi 干扰 Atg3、Atg5、Atg7 及 Beclin1等自噬相关基因的表达后，细胞表现为自噬功能缺失与工具药相比，通过基因沉默或敲除技术来抑制自噬具有相对强的特异性一、药物干预一、药物干预37 谢谢聆听！38