1、細(xì)胞自噬研究方法概述,研究生：王穎 導(dǎo)師：劉乃豐 教授,Autophagic Compartments,Autophagic Compartments Phagophore (pre-autophagosomal)： previously called the isolation or sequestration membrane 吞噬泡 ：參與自噬體形成早期事件的膜池。也指“隔離膜isolation membrane”或“杯狀結(jié)構(gòu)cup-shaped structure” Autophagosome : 自噬體 ：雙層膜包裹胞質(zhì)形成的囊泡 Amphisome ：generated by th

2、e fusion of autophagosomes with endosomes, also referred to as an acidic late autophagosome 自噬內(nèi)涵體：溶酶體和內(nèi)涵體融合的中間囊泡 Autolysosome ：generated by fusion of autophagosomes or amphisomes with a lysosome 自噬溶酶體：自噬小體和溶酶體融合形成的終末結(jié)構(gòu),Autophagic molecular mechanisms,Initiation： Induction，Cargo recognition and selec

3、tivity Elongation，Closure： Autophagosome formation Maturation，Degradation: Vesicle fusion and autophagosome breakdown,Autophagic molecular mechanisms,Induction,Normal conditions Basal-level autophagy is very low; Autophagy inhibitor: serine/threonine protein kinase TOR(target of rapamycin) input inf

4、ormation from multiple upstream signal transduction pathways (discussed below) and negatively regulates another serine/threonine kinase, Atg1, in nutrient-rich conditions,Starvation conditons or Rapamycin TOR inhibited; Atg1 activated; Atg1 binding affinity to Atg13 and Atg17 ; Promotes the formatio

5、n of an Atg1-Atg13-Atg17 scaffold ; Atg1-Atg13-Atg17 recruitment of multiple Atg proteins to the PAS to initiate autophagosome formation.,Cargo recognition and selectivity,P62/sequestosome 1 (SQSTM1) . P62 directly binds both poly- or mono-ubiquitin via its ubiquitin-associated (UBA) domain and LC3

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

7、Beclin1,The 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 media

8、tes 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 autophagosome.,

9、Atg12 is activated by Atg7 , transferred to Atg10 (E2 conjugating enzyme) 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-oligomeri

10、zation and attaches it to the phagophore .,Autophagosome formation,Atg8 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-ric

11、h conditions, the majority of Atg8 is cytosolic（16Kd）; upon autophagy induction, 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. Th

12、e lipidation of Atg8 and its mammalian homolog LC3 are widely used to monitor autophagy induction. .,Autophagosome formation,Vesicle fusion and autophagosome breakdown,In mammalian cells, the fusion event requires the lysosomal membrane protein LAMP-2 and the small GTPase Rab7.,After fusion, degrada

13、tion of the inner vesicle is dependent on a series of lysosomal/vacuolar acid 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 cells,The resulting small molecules from t

14、he degradation, particularly amino acids, are transported back to the cytosol for protein synthesis and maintenance of cellular functions under starvation conditions.,1. Transmission electron microscopy2. Atg8/LC3 detection and quantification 3.SQSTM1/p62 and related LC3 binding protein turnover ass

15、ays 4. MTOR, AMPK and Atg1/ULK15. Additional autophagy-related markers 6. Transcriptional and translational regulation7. Autophagic protein degradation 8. Selective types of autophagy,Methods for Monitoring Autophagy,9. Autophagic sequestration assays10. Turnover of autophagic compartments11. Autoph

16、agosome-lysosome colocalization and dequenching assay 12. Tissue fractionation 13. Analyses in vivo 14. Cell death15. Chaperone-mediated autophagy,Transmission electron microscopy,一、取材 快速、低溫 細(xì)胞株： 胰酶消化：細(xì)胞完整性保存良好，但是對自噬有一定影響 細(xì)胞刮片：最大程度維持細(xì)胞原生理狀態(tài)，細(xì)胞物理損傷大 先固定，后刮?。杭?xì)胞完整性、生理性維持良好，但細(xì)胞分散，不易成團(tuán) 組織： 優(yōu)選低溫灌注固定 時間點(diǎn)：1

17、2h，8h，24h,Transmission electron microscopy,二、結(jié)構(gòu)特點(diǎn) Autophagosomes ：12h, 8h double membrane，visible as two parallel membrane bilayers separated by an electron-lucent cleft contain cytosol and/or organelles that look morphologically intact Amphisomes can sometimes be identified by the presence of small

18、 internal vesicles inside the autophagosome/autophagic vacuole (AV). These internal vesicles are delivered into the lumen by fusion with multivesicular endosomes. Late/degradative autophagic vacuoles and autolysosomes (AVd) ：24h usually have only one limiting membrane, and contain cytoplasmic materi

19、al and/or organelles at various stages of degradation,Transmission electron microscopy,Cautionary notes,Fixation 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 c

20、ellular area in sections. At least 20 cell profiles per sample. Each imaged cell profile is captured and scored at the same magnification.,Cautionary notes,Not all double-membrane structures are autophagosomes Apoptotic bodies from neighboring cells are readily phagocytosed by surviving cells of the

21、 same tissue. Phagosomes have double limiting membranes, inner one is from the 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

22、 nm, respectively). A good feature to distinguish between autophagosomes and 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.,Due to

23、 the cisternal structure of the ER, double membrane-like structures surrounding 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 endomem

24、brane cisternae or damaged mitochondria . If there are ribosomes associated with these membranes they can help distinguish them from the ribosome free double-membrane of the phagophore and autophagosome.,Cautionary notes,a. Western blotting and ubiquitin-like protein conjugation systems b. Turnover

25、of LC3-II/Atg8PE c. GFP-Atg8/LC3 lysosomal delivery and proteolysis d. GFP-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 quantificatio

26、n,Western blotting,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-dependent Moreover, LC3-I is more labile than LC3-II, being more sensitive to freezingthawing and to degradation in SDS sample buffer, fresh

27、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 membranes Triton X-100 may not efficiently solubilize LC3-II in some systems Heating in the presence of 1

28、% SDS, or analysis of membrane fractions, may assist in the detection of this protein. In some cases beta-actin levels decrease when autophagy is induced,Cautionary notes:,Western blotting,Turnover of LC3-II/Atg8PE.,Prevent Lysosomal Degradation Autophagic flux can be measured by inferring LC3-II/At

29、g8PE 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 amount of LC3-II will be higher in the presence

30、 of the inhibitor.,Protease inhibitors pepstatin A and E-64d: Neutralize the lysosomal PH bafilomycin A1（洛霉素A1） chloroquine ：氯喹 NH4Cl ：氯化銨 Block fusion of autophagosomes with lysosomes bafilomycin A1 Knocking down or knocking out lysosomal-associated membrane protein 2 (LAMP2),Prevent Lysosomal Degr

31、adation,注：抑制劑作用時間：12h； 設(shè)陽性對照組 時間點(diǎn)：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 parallel.,1 h of pre-incubation with 10 mg/ml E-64d

32、 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) and a relatively high concentration (. 50 mg/m

33、l) to fully inhibit lysosomal cathepsin D,GFP-Atg8/LC3 lysosomal delivery and proteolysis,Werstern blot GFP-LC3在自噬溶酶體的酸性環(huán)境中被降解 GFP單體釋放到胞質(zhì)中，蛋白印跡檢測GFP單體條帶 Cautionary notes: A reduction in the intensity of the free GFP band may indicate reduced flux, but it may also be due to efficient turnover. Using

34、a range of concentrations and treatment times of compounds that inhibit autophagy can be useful in distinguishing between these possibilities.,GFP-Atg8/LC3 fluorescence microscopy,Fluorescence microscopy A constant increase in the number of cells accumulating GFP-LC3 puncta is suggestive of defectiv

35、e 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 of lysosomal inhibitors should increase the

36、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.,Tandem mRFP-GFP fluorescence microscopy,The GFP signal is sensitive to the acidic and/or

37、 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 mRFP signal without GFP corresponds to an am

38、phisome 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, without requiring any drug treatment.,自噬體和自噬溶酶體

39、分別成黃色和紅色標(biāo)記，如果自噬潮增加，兩種顏色的點(diǎn)狀聚集均增加。 如果自噬體向自噬溶酶體成熟受阻，黃色點(diǎn)狀聚集物增加，紅色不增加。,Flow and multispectral imaging cytometry,在自噬誘導(dǎo)一開始，GFP-LC3 點(diǎn)狀聚集顯著增多， 隨后信號可能會出現(xiàn)下降，代表著自噬性降解的發(fā)生 高通量檢測,Immunohistochemistry,When autophagosomes are absent, the localization pattern of LC3 in the cells of various tissues is diffuse and cyto

40、solic. 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 not only in autophagosomes but also on lipid

41、droplets. In neurons in ATG7-deficient mice, LC3 is accumulated in ubiquitin- and SQSTM1-positive aggregates.,p62 and related LC3 binding protein turnover assays,The SQSTM1 protein serves as a link between LC3 and ubiquitinated substrates. decreased SQSTM1 levels are associated with autophagy activa

42、tion The 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.,Western blot analysis using NP40 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 ly