Membrane
• Structure and Function in Detail
• 探討細胞膜表面一些訊息（signals）的機制
（mechanisms）-如何偵測與傳送至細胞內
– Electrical Signals in Chapter 13
– Chemical Signals in Chapter 14
– Cell-Cell recognition and adhesion in Chapter 17
Membranes: Their Structure,
Function, and Chemistry

• The functions of membrane
• Models of membrane structure: An
experimental perspective
• Membrane lipids: The fluid part of the
model
• Membrane protein: The mosaic part of
the model
The Functions of Membranes
1.
Membranes define boundaries and serve as permeability
barrier
– to define the boundaries of cell and its compartments
– to serve as permeability barrier
– plasma membrane and intracellular membranes
2.
Membrane are sites of specific proteins and therefore of
specific functions
– Transport proteins, receptors, enzymes
3.
Membrane proteins regulate the transport of solutes
– endocytosis and exocytosis
4.
Membrane proteins detect and transmit electrical and
chemical signals
– Signal transduction
5.
Membrane proteins mediate cell-to-cell communication
– gap junctions
Membranes: Their Structure,
Function, and Chemistry

• The functions of membrane
• Models of membrane structure: An
experimental perspective
• Membrane lipids: The fluid part of the
model
• Membrane protein: The mosaic part of
the model
• Models of membranes were developed long
before membranes were first seen with electron
microscopes in the 1950s.
– In 1895, Charles Overton hypothesized that
membranes are made of lipids because substances that
dissolve in lipid enter cells faster than those that are
insoluble.
– Twenty years later, chemical analysis confirmed that
membranes isolated from red blood cells are
composed of lipids and proteins.
• Early images from electron microscopes seemed
to support the Davson-Danielli model and until
the 1960s, it was considered the dominant model.
• Further investigation revealed two problems.
– First, not all membranes were alike, but differed in
thickness, appearance when stained, and percentage
of proteins to lipids.
– Second, measurements showed that membrane
proteins are actually not very soluble in water.
• Membrane proteins are amphipathic, with hydrophobic and
hydrophilic regions.
• If at the surface, the hydrophobic regions would be in
contact with water.
Membranes: Their Structure,
Function, and Chemistry

• The functions of membrane
• Models of membrane structure: An
experimental perspective
• Membrane lipids: The fluid part of the
model
• Membrane protein: The mosaic part of
the model
Membrane lipids:
The fluid part of the model
• Membranes contain several major classes of lipids
• Thin-layer chromatography is an important technique
for lipid analysis (TLC)
• Fatty acid are essential to membrane structure and
function
• Membrane asymmetry: Most lipids are distributed
unequally between the two monolayers
• The lipid bilayer is fluid state
• Membrane function properly only in the fluid state
• Most organisms can regulate membrane fluidity
• Lipid rafts are localized regions of membrane lipids
that are involved in cell signaling
• The main macromolecules in membranes are lipids and
proteins, but include some carbohydrates.
• The most abundant lipids are phospholipids.
• Phospholipids and most other membrane constituents
are amphipathic molecules.
– Amphipathic molecules have both hydrophobic
regions and hydrophilic regions.
• The phospholipids and proteins in membranes create a
unique physical environment, described by the fluid
mosaic model.
– A membrane is a fluid structure with proteins
embedded or attached to a double layer of
phospholipids.
系統組成
• 層析系統的兩個主要組成為 固定相
(stationary phase) 及 流動相 (mobile
phase)，二者各有不同的極性或非極性強
度；樣本分子因其自身極性的強弱，與
此二相之親和力不同。 與固定相親和力
大者，易留滯原地； 與流動相親和力大
者，易隨流動相移動，因而達成分離的
目的。
Membrane Function Properly
Only in the Fluid State
• The effects of fatty acid composition on
membrane fluidity
• The effects of sterol on membrane fluidity
Most Organisms Can Regulate
Membrane Fluidity
• Change the lipid composition
– Important for poikilotherms (變溫) :
bacteria, fungi, protozoa, algae , plants,
invertebrates and cold-blooded animals
– Homeoviscous adaptation
• To work properly with active enzymes and
appropriate permeability, membrane must
be fluid, about as fluid as salad oil.
• Cells can alter the lipid composition of
membranes to compensate for changes in
fluidity caused by changing temperatures.
– This allows these organisms to prevent their
membranes from solidifying during winter.
– For example, cold-adapted organisms,
• Micrococcus (bacteria) : increase in the
proportion of 16-carbon versus 18-carbon fatty
acids in plasma membrane
• Winter wheat（小麥）: increase the percentage
of unsaturated phospholipids in the autumn.
• Amphibians and reptiles: increase the proportion
of unsaturated fatty acid in their membranes.
Lipid Rafts(脂筏)
除了含有豐富的膽固醇及神經鞘脂類
(sphingolipid) ，還包含許多細胞受體蛋白，
受體蛋白可以經由與傳遞訊息的蛋白結合
而活化，將細胞外的訊息傳遞到細胞內。
所以脂筏被認為與傳導細胞信號通路的激
活有關。
THP-1單核球細胞lipid rafts的蛋白質體學研究徐園堤(吳烘老師)
Lipid rafts 是細胞膜上的特化結構，其特性為富含cholesterol，sphingolipids與
glycophosphatidylinositol-anchored proteins。在之前的研究指出，lipid rafts在物質運輸與
訊息傳遞中扮演重要的角色。Lipid rafts蛋白質體上的研究大部分是利用非膠體的方法，
例如使用液相層析與質譜儀。相形之下，二維電泳的方法則較少被利用來分析lipid rafts
蛋白質體，儘管它能夠在ㄧ片膠上分離上千個蛋白質。本論文的研究目標主要是爲了
發展一個基於二維電泳的高解析度的分離策略及結合串聯式質譜儀來建立lipid rafts的蛋
白質圖譜。爲了利用二維電泳來分離lipid rafts的成分，我們以THP-1細胞為實驗模型，
首先利用density gradient將lipid rafts分離出來，並使用多種以CHAPS為基礎的detergent
組合來試驗二維電泳分離的效果。經蛋白質圖譜解析度與蛋白質點的數目分析結果顯
示發現使用結合ASB-14與CHAPS來分離lipid rafts的效果較好。在二維電泳膠片上已利
用串聯式質譜儀鑑定出17個蛋白質點，包括actin-gamma 1、alpha-tubulin、G-beta1、
RhoA、Rab、heat shock cognate protein、P58、GRP78以及intracellular chloride channels。
由於lipid rafts與訊息傳遞有關，所以我們利用已建立的二維電泳方法來探討fractalkine
處理的THP-1細胞在lipid rafts成分的變化。在lipid rafts二維電泳圖譜的影像分析顯示細
胞經fractalkine刺激後導致12個蛋白質的表現出現差異。其中有6個蛋白質點的表現增加
與6個蛋白質點的表現被抑制。利用二維電泳與質譜儀技術，本論文已成功地建立一個
高解析度的lipid rafts蛋白質體分析平台。以蛋白質體學為基礎的策略不僅可以探索許多
未知的lipid rafts蛋白質身份並且也進一步提供與lipid rafts相關的訊息傳遞路徑的了解。
Membranes: Their Structure, Function,
and Chemistry

• The functions of membrane
• Models of membrane structure: An
experimental perspective
• Membrane lipids: The fluid part of the
model
• Membrane protein: The mosaic part of
the model
Membrane Protein:
The “Mosaic” Part of the Model
• The membrane consists of mosaic of proteins: Evidence from
freeze-fracture microscopy
• Membranes contain integral, peripheral and lipid-anchored
proteins
• Proteins can be separated by SDS-polyacrylamide gel
electrophoresis
• Molecular biology has contributed greatly to our understanding
of membrane proteins
• Membrane protein have a variety of functions
• Membrane proteins are oriented asymmetrically across the
lipid bilayer
• Many membrane proteins are glycosylated
• Membrane proteins vary in their mobility
Membranes contain integral, peripheral
and lipid-anchored proteins
• Integral membrane proteins (transmembrane protein)
– Hydrophobic regions are embedded within the
membrane interior.
• Peripheral membrane proteins
– Too hydrophilic to penetrate into the membrane but
are attached to the membrane by electrostatic and
hydrogen bonds to adjacent membrane.
• Lipid-anchored proteins
– are hydrophilic and do not penetrate into the membrane ; they
are covalently bound to lipid molecules (saturated fatty acid)
Peripheral Membrane Proteins
• Bind with hydrophilic portions of integral proteins
– through weak electrostatic forces and hydrogen
bonds with the hydrophilic portions of integral
protein or with the polar head groups of membrane
lipids
• More readily removed from membranes than integral
proteins
– Extracted by changing the pH and ionic strength.
• Bound to the inner surface of the plasma membrane
– Form a skeleton meshwork to support the plasma
membrane and maintain the shape of the erythrocyte
Lipid-Anchored Proteins
• Fatty Acid-anchored Membrane Proteins
（Prenylated Membrane Protein）– Bound to inner surface
– Synthesized in the cytosol
• GPI-anchored Membranes (glycosylphosphatidylinositol)
– synthesized in ER, transmembrane protein
– Released from Phospholipase C
– Attached to external surface of plasma
membrane
Membrane carbohydrates are
important for cell-cell recognition
• The membrane plays the key role in cell-cell
recognition.
– Cell-cell recognition is the ability of a cell to
distinguish one type of neighboring cell from another.
– This attribute is important in cell sorting and
organization as tissues and organs in development.
– It is also the basis for rejection of foreign cells by the
immune system.
– Cells recognize other cells by keying on surface
molecules, often carbohydrates, on the plasma
membrane.
• Membrane carbohydrates are usually branched
oligosaccharides with fewer than 15 sugar units.
• They may be covalently bonded either to lipids,
forming glycolipids, or, more commonly, to
proteins, forming glycoproteins.
• The oligosaccharides on the external side of the
plasma membrane vary from species to species,
individual to individual, and even from cell
type to cell type within the same individual.
– This variation marks each cell type as distinct.
– The four human blood groups (A, B, AB, and O)
differ in the external carbohydrates on red blood
cells.
Membrane Proteins Vary in Their Mobility
Membrane proteins are much more variable
than lipids in their mobility. Some proteins
appear to move freely within the lipid bilayer
whereas others are constrained, often because
they are anchored to protein complex located
adjacent to one side of the membrane or the
other.
動物細胞融合技術
• 病毒誘導融合
– 利用仙台病毒(Sendai virus)誘導細胞融合
• 化學誘導融合
– 利用聚乙二醇(polyethylene glycol: PEG)誘導
細胞融合
– PEG分子量1000左右；濃度30%40%
• 電擊誘導融合