生物物理課- ppt課件

1、俺怎樣越俺怎樣越看越象看越象乒乒乓乓，不要扔乒乒乓乓，不要扔西紅柿西紅柿 -_-b-_-b俺們的進(jìn)山俺們的進(jìn)山全家福，全家福，hohohohoCOOHNH24.4.細(xì)胞膜中的脂筏及細(xì)胞膜中的脂筏及膜穴系統(tǒng)膜穴系統(tǒng) 很早以前，人們就發(fā)現(xiàn)許多真核生物的細(xì)胞中很早以前，人們就發(fā)現(xiàn)許多真核生物的細(xì)胞中都可以分別得到抗去垢劑的膜微疇構(gòu)造，英文簡稱都可以分別得到抗去垢劑的膜微疇構(gòu)造，英文簡稱為為DRMsDRMsdetergent-resistant membrane detergent-resistant membrane domainsdomains，但直到近年來，但直到近年來DRMsDRMs才引起人們

2、的廣泛關(guān)才引起人們的廣泛關(guān)注。這是由于注。這是由于DRMsDRMs在細(xì)胞內(nèi)的分選和細(xì)胞外表信號在細(xì)胞內(nèi)的分選和細(xì)胞外表信號傳導(dǎo)過程中都表現(xiàn)出其特有的重要性。傳導(dǎo)過程中都表現(xiàn)出其特有的重要性。 這些在這些在44去垢劑不溶的膜區(qū)域被以為是由鞘脂去垢劑不溶的膜區(qū)域被以為是由鞘脂類和膽固醇的動態(tài)聚集而構(gòu)成，它們組成了相對穩(wěn)類和膽固醇的動態(tài)聚集而構(gòu)成，它們組成了相對穩(wěn)定的具有一定功能的疇構(gòu)造漂浮于二維流動的細(xì)胞定的具有一定功能的疇構(gòu)造漂浮于二維流動的細(xì)胞膜中，人們籠統(tǒng)地稱之為膜中，人們籠統(tǒng)地稱之為“脂筏脂筏Lipid raftsLipid rafts。 Functional rafts in cell

3、membranesNature387(2019)571A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. - Simons & Ikonen, Nature 387 (2019) 569-572. 在胞吞、脂類運輸和信號傳導(dǎo)過程中，質(zhì)在胞吞、脂類運輸和信號傳導(dǎo)過程中，質(zhì)膜的外表會出現(xiàn)一種無籠形蛋白覆蓋的穴

4、樣凹膜的外表會出現(xiàn)一種無籠形蛋白覆蓋的穴樣凹陷，這些穴樣凹陷呈現(xiàn)陷，這些穴樣凹陷呈現(xiàn)44去垢劑不溶性，人去垢劑不溶性，人們把這種們把這種DRMsDRMs稱作稱作“膜穴膜穴caveolaecaveolae。 目前發(fā)現(xiàn)膜穴與功能筏在分子程度上有著目前發(fā)現(xiàn)膜穴與功能筏在分子程度上有著類似的組成和構(gòu)造，因此可以說膜穴是功能筏類似的組成和構(gòu)造，因此可以說膜穴是功能筏的一種特殊表現(xiàn)方式。的一種特殊表現(xiàn)方式。 Caveolae: lipid rafts in cell surface invaginations containing caveolinNature387(2019)571Caveolae in

5、 endocytic traffic Caveolae are non-clathrin coated invaginations (50-100 nm) in the plasma membrane in many cell types. Caveolae are formed by self-associating caveolin molecules (making a hairpin loop) in the membrane interacting with raft lipids. To form small signalling compartments: a number of

6、 signalling proteins are anchored, such as hetrotrimeric G proteins, Src-family kinases, H-Ras. Be involved in endocytosis and transcytosis.Lipid composition of raftsSphingolipid (glycosphingolipids, sphingomyelin) and cholsterol sphingomyelin- and cholsterol-rich DIGs can also be isolated from cell

7、s. glycosphingolipids are not absolutely required! DIGs in trans-Golgi-network: rich in cholesterol and anchored by GPI protein!以甘油為骨架的磷脂以甘油為骨架的磷脂即甘油分子中三個羥基有兩個與高即甘油分子中三個羥基有兩個與高級脂肪酸構(gòu)成酯，另一個與磷酸衍級脂肪酸構(gòu)成酯，另一個與磷酸衍生物構(gòu)成酯：生物構(gòu)成酯：其中其中R1、R2為脂肪酸碳?xì)滏?。根為脂肪酸碳?xì)滏?。根?jù)據(jù)X的成分不同，可以構(gòu)成不同的的成分不同，可以構(gòu)成不同的磷脂。磷脂。以神經(jīng)鞘氨醇為骨架的鞘脂類以神經(jīng)鞘氨醇為

8、骨架的鞘脂類(sphingolipid)神經(jīng)鞘氨醇神經(jīng)鞘氨醇(sphinogsine)的的C2上的氨基上的氨基(-NH2)與脂肪酸與脂肪酸(R)縮合生成神經(jīng)鞘脂類縮合生成神經(jīng)鞘脂類(sphingolipid), Cl上的羥基與磷酸衍生物上的羥基與磷酸衍生物( X ) 縮 合 即 生 成 磷 酸 神 經(jīng) 鞘 脂 類縮 合 即 生 成 磷 酸 神 經(jīng) 鞘 脂 類(phosphasphingolipid):假設(shè)假設(shè): X磷脂膽堿磷脂膽堿(PC)，那么生成神經(jīng)鞘磷脂那么生成神經(jīng)鞘磷脂(sphingomyelin, SM ). 假設(shè)假設(shè): XH, 那么生成神經(jīng)酸胺那么生成神經(jīng)酸胺ceramide。Sph

9、ingolipids:long and saturated fatty acyl chians higher Tm: Glycosphingolipid 60-70 oC. Sphingomyelin 37-41 oC. Detergent-insoluble glycolipid-enriched complexes (DIGs) arise from lipid-lipid interactions detergent insolubility was observed even in the absence of protein. saturated-chain, high-Tm DPP

10、C is Triton insoluble in DIGs-containing liposomes, while the low Tm, unsaturated-chain DOPC is much Triton soluble. correlation of acyl chain structure, Tm, and detergent insolubility not from lipid head-group interactions.Phase separation exists in model membranes co-existing gel and fluid phases

11、phase separation of two liquid phases liquid-cryst. and liquid-ordered phases protein induces microdomain formationDoes phase separation occur in biological membrane?Proteins in DIGsGPI-anchored proteins - the first proteins to be identified in DIGs.By acyl tails the proteins bind to the cytoplasmic

12、 leaflet - such as the Src-family kinases.Proteins associating through their transmembrane domains - such as influenza virus haemagglutinin(HA).The function of lipid raftsMembrane sorting and traffickingSignal transductionThe intracellular transport of The intracellular transport of sphingolipid-cho

13、lesterol rafts sphingolipid-cholesterol rafts shows a apical route.shows a apical route.Apical sorting signal GPI anchors specific membrane-spanning regions N-glycans basolateral sorting signals tyrosine or dileucine containing motifs of the cytoplasmic domains of basolaterally targeted proteinsPNAS

14、 95 (2019) 6460PNAS 95 (2019) 3966Functional rafts in neural polarity EMBO J. 16 (2019) 4932EMBO J. 15 (2019) 5218Signalling occurs in a raftFollowing dimerization (or oligomerization) the protein becomes phosphorylated (blue circle) in rafts.Signalling occurs by altering protein partition in a raft

15、 Following dimerization (or oligomerization) the protein becomes phosphorylated (blue circle) in rafts.Clustering of rafts triggers signalling There are several rafts in the membrane, which differ in protein composition. Clustering would coalesce rafts (red), so that they would now contain a new mix

16、ture of molecules, such as crosslinkers and enzymes. Clustering could occur either extracellularly, within the membrane, or in the cytosol (ac). Raft clustering could also occur through GPIanchored proteins.Rafts 確實定方法Techniques to identify raftsThe Existence of rafts in The Existence of rafts in ce

17、ll membranescell membranesBiochemical crosslinking of GPI-anchored proteins when they are in proximity in rafts. Visualization of rafts and clustered rafts in IgE signalling by electron microscopyAntibody crosslinking of raft proteins into patches segregating from non-raft .-The first demonstration

18、that clusters of rafts segregate away from non-raft proteins. Bulk separation of membrane phases caused by clustering of membrane components. (A) Microdomains with membrane proteins in these domains are dispersed in the plasma membrane.(B) Cross-linking generates large and stabilized membrane domain

19、s that coalesce to form patches. If two membrane components share a preference for a lipid environment such as raft microdomains the markers will copatch into tightly associated domains. If two markers partition into different membrane environments such as raft and non-raft markers the patches will

20、be separated.The Existence of rafts in The Existence of rafts in cell membranescell membranesBiochemical crosslinking of GPI-anchored proteins when they are in proximity in rafts. Visualization of rafts and clustered rafts in IgE signalling by electron microscopyClear visualization of raft clusterin

21、g by immuno-electron microscopyLyn associates with FceRI in resting mast cells. Membrane sheets were prepared from untreated RBL-2H3 cells and labeled from the inside with 5-nm gold particles specific for Lyn and with either 3- (A) or 10-nm (B) gold particles specific for FceRI b. In both micrograph

22、s, a substantial portion of 5-nm gold particles marking Lyn are colocalized with FceRI b (circles). (C) Demonstrates the absence of background binding when both sizes of gold particles are incubated with membrane sheets in the absence of specific antibodies. The Existence of rafts in The Existence o

23、f rafts in cell membranes (Rafts in cell membranes (Rafts in living cells)living cells) Fluorescence resonance energy transfer measurements using fluorescent folate to show interactions of folate receptors when they are in proximity in rafts in living cells. Photonic force microscopy measurements of

24、 the size of rafts in living cells.CFP: EX436; EM476. YFP: EX516; EM529The Existence of rafts in The Existence of rafts in cell membranes (Rafts in cell membranes (Rafts in living cells)living cells) Fluorescence resonance energy transfer measurements using fluorescent folate to show interactions of

25、 folate receptors when they are in proximity in rafts in living cells. Photonic force microscopy measurements of the size of rafts in living cells.Figure 1. Scaled model of the experimental situation: a sphere (r 5 108 nm) bound via an adsorbed antibody to a GPI-anchored protein that is part of a ra

26、ft domain. The lipid bilayer is symbolized by the double row of gray dots with black sections symbolizing raft domains. The extent of the thermal position fluctuations observed in the experiments (6 60 nm) is marked. It is much smaller than the smallest estimates of the spacing of immobile cytoskeleton-anchored obstacles to free diffusion of 300500 nm (Sako and Ku