COOH
NH2
4.细胞膜中的脂筏及
膜穴系统
很早以前，人们就发现许多真核生物的细胞中
都可以分离得到抗去垢剂的膜微畴结构，英文简称
为 DRMs（ detergent-resistant membrane domains），
但直到近年来 DRMs才引起人们的广泛关注。这是
因为 DRMs在细胞内的分选和细胞表面信号传导过
程中都表现出其特有的重要性。
这些 在 4℃ 去垢剂不溶的膜区域被认为是由鞘脂
类和胆固醇的动态聚集而形成，它们组成了相对稳
定的具有一定功能的畴结构漂浮于二维流动的细胞
膜中，人们形象地称之为, 脂筏, （ Lipid rafts） 。
Functional rafts in cell
membranes
Nature387(1997)571
A 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 (1997) 569-572.
在胞吞、脂类运输和信号传导过程中，质
膜的表面会出现一种无笼形蛋白覆盖的穴样凹
陷，这些穴样凹陷呈现 4℃ 去垢剂不溶性，人
们把这种 DRMs称作, 膜穴, （ caveolae）。
目前发现膜穴与功能筏在分子水平上有着
类似的组成和结构，因此可以说膜穴是功能筏
的一种特殊表现形式。
Caveolae,lipid rafts in cell surface
invaginations containing caveolin
Nature387(1997)571
Caveolae in 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 signalling proteins are anchored,
such as hetrotrimeric G proteins,Src-family
kinases,H-Ras,
Be involved in endocytosis and transcytosis.
Lipid composition of
rafts
Sphingolipid (glycosphingolipids,
sphingomyelin) and cholsterol
?sphingomyelin- and cholsterol-rich DIGs can
also be isolated from cells.
?glycosphingolipids are not absolutely required!
?DIGs in trans-Golgi-network,rich in
cholesterol and anchored by GPI protein!
以甘油为骨架的磷脂
即甘油分子中三个羟基有两个与高级脂肪酸形
成酯, 另一个与磷酸衍生物形成酯：
其中 R1,R2为脂肪酸碳氢链 。 根据 X的成分不
同, 可以形成不同的磷脂 。
以神经鞘氨醇为骨架的鞘脂类 (sphingolipid)
神经鞘氨醇 (sphinogsine)的 C－ 2上的氨基 (-NH2)与脂肪酸 (R)
缩合生成神经鞘脂类 (sphingolipid),C－ l上的羟基与磷酸衍
生物 (X)缩合即生成磷酸神经鞘脂类 (phosphasphingolipid):
若, X＝磷脂胆碱 (PC)，
则生成 神经鞘磷脂 (sphingomyelin,SM ).
若, X＝－ H,
则生成 神经酸胺 （ ceramide） 。
Sphingolipids:
?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 DPPC 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
?phase separation of two liquid phases
?liquid-cryst,and liquid-ordered phases
?protein induces microdomain formation
Does phase separation occur in biological membrane?
Proteins in DIGs
GPI-anchored proteins ---- the first proteins to
be identified in DIGs.
By acyl tails the proteins bind to the
cytoplasmic leaflet ---- such as the Src-family
kinases.
Proteins associating through their
transmembrane domains ---- such as influenza
virus haemagglutinin(HA).
The function of lipid
rafts
Membrane sorting and
trafficking
Signal transduction
The intracellular transport of
sphingolipid-cholesterol rafts
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 proteins
PNAS 95 (1998) 6460
PNAS 95 (1998) 3966
Functional rafts in neural polarity
EMBO J,16 (1997) 4932
EMBO J,15 (1996) 5218
Signalling occurs in a raft
Following dimerization (or oligomerization) the protein
becomes phosphorylated (blue circle) in rafts.
Signalling occurs by altering
protein partition in a raft
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 mixture of
molecules,such as crosslinkers and enzymes,Clustering could occur either
extracellularly,within the membrane,or in the cytosol (a–c),Raft clustering could also
occur through GPIanchored proteins.
Rafts 的确定方法
Techniques to identify rafts
The Existence of rafts in
cell membranes
Biochemical crosslinking of GPI-
anchored proteins when they are in
proximity in rafts,
Visualization of rafts and clustered
rafts in IgE signalling by electron
microscopy
Antibody crosslinking of raft proteins
into patches segregating from non-raft,
----The first demonstration 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 domains 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 be
separated.
The Existence of rafts in
cell membranes
Biochemical crosslinking of GPI-
anchored proteins when they are in
proximity in rafts.
Visualization of rafts and clustered
rafts in IgE signalling by electron
microscopy
Clear visualization of raft clustering by
immuno-electron microscopy
Lyn 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 micrographs,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
cell membranes (Rafts in 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 the size of rafts in
living cells.
CFP,EX436; EM476,YFP,EX516; EM529
The Existence of rafts in
cell membranes (Rafts in 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 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 raft 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 300–500 nm (Sako and Kusumi,
1995),The cytoskeletal elements drawn here are 250 nm apart.
Individual raft size is measured by
photonic force microscopy
Lipid Rafts Reconstituted in
Model Membranes
(1) Planar supported lipid layers constitute one model
system that has the important advantage that bilayers
may be constructed from two monolayers of different
composition; thus,the inherent asymmetry of
biological membranes may be mimicked,
(2) Giant unilamellar vesicles (GUVs)
(GUV composed of DOPC /cholesterol/
sphingomyelin (1:1:1) with 1 mol % GM1 ) are a
second model system that provides a free standing
bilayer,without potential substrate effects,
(a) Non-raft mixture (POPC/cholesterol 2:1); (b) Raft-
mixture (POPC/cholesterol/sphingomyelin 2:1:1),Both
contain 0.5 mol % FL-DPPE.
登顶成功，可惜神仙都被一脸幸
福地俺们吓跑了。
膜分子生物学
游击队
NH2
4.细胞膜中的脂筏及
膜穴系统
很早以前，人们就发现许多真核生物的细胞中
都可以分离得到抗去垢剂的膜微畴结构，英文简称
为 DRMs（ detergent-resistant membrane domains），
但直到近年来 DRMs才引起人们的广泛关注。这是
因为 DRMs在细胞内的分选和细胞表面信号传导过
程中都表现出其特有的重要性。
这些 在 4℃ 去垢剂不溶的膜区域被认为是由鞘脂
类和胆固醇的动态聚集而形成，它们组成了相对稳
定的具有一定功能的畴结构漂浮于二维流动的细胞
膜中，人们形象地称之为, 脂筏, （ Lipid rafts） 。
Functional rafts in cell
membranes
Nature387(1997)571
A 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 (1997) 569-572.
在胞吞、脂类运输和信号传导过程中，质
膜的表面会出现一种无笼形蛋白覆盖的穴样凹
陷，这些穴样凹陷呈现 4℃ 去垢剂不溶性，人
们把这种 DRMs称作, 膜穴, （ caveolae）。
目前发现膜穴与功能筏在分子水平上有着
类似的组成和结构，因此可以说膜穴是功能筏
的一种特殊表现形式。
Caveolae,lipid rafts in cell surface
invaginations containing caveolin
Nature387(1997)571
Caveolae in 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 signalling proteins are anchored,
such as hetrotrimeric G proteins,Src-family
kinases,H-Ras,
Be involved in endocytosis and transcytosis.
Lipid composition of
rafts
Sphingolipid (glycosphingolipids,
sphingomyelin) and cholsterol
?sphingomyelin- and cholsterol-rich DIGs can
also be isolated from cells.
?glycosphingolipids are not absolutely required!
?DIGs in trans-Golgi-network,rich in
cholesterol and anchored by GPI protein!
以甘油为骨架的磷脂
即甘油分子中三个羟基有两个与高级脂肪酸形
成酯, 另一个与磷酸衍生物形成酯：
其中 R1,R2为脂肪酸碳氢链 。 根据 X的成分不
同, 可以形成不同的磷脂 。
以神经鞘氨醇为骨架的鞘脂类 (sphingolipid)
神经鞘氨醇 (sphinogsine)的 C－ 2上的氨基 (-NH2)与脂肪酸 (R)
缩合生成神经鞘脂类 (sphingolipid),C－ l上的羟基与磷酸衍
生物 (X)缩合即生成磷酸神经鞘脂类 (phosphasphingolipid):
若, X＝磷脂胆碱 (PC)，
则生成 神经鞘磷脂 (sphingomyelin,SM ).
若, X＝－ H,
则生成 神经酸胺 （ ceramide） 。
Sphingolipids:
?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 DPPC 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
?phase separation of two liquid phases
?liquid-cryst,and liquid-ordered phases
?protein induces microdomain formation
Does phase separation occur in biological membrane?
Proteins in DIGs
GPI-anchored proteins ---- the first proteins to
be identified in DIGs.
By acyl tails the proteins bind to the
cytoplasmic leaflet ---- such as the Src-family
kinases.
Proteins associating through their
transmembrane domains ---- such as influenza
virus haemagglutinin(HA).
The function of lipid
rafts
Membrane sorting and
trafficking
Signal transduction
The intracellular transport of
sphingolipid-cholesterol rafts
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 proteins
PNAS 95 (1998) 6460
PNAS 95 (1998) 3966
Functional rafts in neural polarity
EMBO J,16 (1997) 4932
EMBO J,15 (1996) 5218
Signalling occurs in a raft
Following dimerization (or oligomerization) the protein
becomes phosphorylated (blue circle) in rafts.
Signalling occurs by altering
protein partition in a raft
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 mixture of
molecules,such as crosslinkers and enzymes,Clustering could occur either
extracellularly,within the membrane,or in the cytosol (a–c),Raft clustering could also
occur through GPIanchored proteins.
Rafts 的确定方法
Techniques to identify rafts
The Existence of rafts in
cell membranes
Biochemical crosslinking of GPI-
anchored proteins when they are in
proximity in rafts,
Visualization of rafts and clustered
rafts in IgE signalling by electron
microscopy
Antibody crosslinking of raft proteins
into patches segregating from non-raft,
----The first demonstration 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 domains 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 be
separated.
The Existence of rafts in
cell membranes
Biochemical crosslinking of GPI-
anchored proteins when they are in
proximity in rafts.
Visualization of rafts and clustered
rafts in IgE signalling by electron
microscopy
Clear visualization of raft clustering by
immuno-electron microscopy
Lyn 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 micrographs,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
cell membranes (Rafts in 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 the size of rafts in
living cells.
CFP,EX436; EM476,YFP,EX516; EM529
The Existence of rafts in
cell membranes (Rafts in 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 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 raft 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 300–500 nm (Sako and Kusumi,
1995),The cytoskeletal elements drawn here are 250 nm apart.
Individual raft size is measured by
photonic force microscopy
Lipid Rafts Reconstituted in
Model Membranes
(1) Planar supported lipid layers constitute one model
system that has the important advantage that bilayers
may be constructed from two monolayers of different
composition; thus,the inherent asymmetry of
biological membranes may be mimicked,
(2) Giant unilamellar vesicles (GUVs)
(GUV composed of DOPC /cholesterol/
sphingomyelin (1:1:1) with 1 mol % GM1 ) are a
second model system that provides a free standing
bilayer,without potential substrate effects,
(a) Non-raft mixture (POPC/cholesterol 2:1); (b) Raft-
mixture (POPC/cholesterol/sphingomyelin 2:1:1),Both
contain 0.5 mol % FL-DPPE.
登顶成功，可惜神仙都被一脸幸
福地俺们吓跑了。
膜分子生物学
游击队
