MOLECULAR CELL BIOLOGYSIXTH EDITION

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MOLECULAR CELL BIOLOGY

SIXTH EDITION

CHAPTER 9 Visualizing, Fractionating, and Culturing Cells

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira

Organelles in Degradation

• Lysosomes/endosomes/phagosomes

• (Vacuoles: plants)

• Peroxisomes

• (glyoximes: plants)

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Lysosomes

• Acidic environment (pH 4.8) –Maintained by:

• Hydrogen ion pump: H+ into lumen

• Cl- channel protein: Cl- into lumen

–Helps denature protein = expose for action of hydrolases

Contain acid hydrolases (degradative enzymes) –Nucleases

–Proteases –Phosphatases

Cellular structure that participate in delivering materials to lysosome 1. Endocytic pathway: soluble macromolecules into the cell 2. Phagocytosis:Whole cell and other large insoluble particles 3. Autophagy: organelles and cytoplasmic substance.

Lysosomes are acidic organelles that contain a battery of degradative enzyme

Lysosome:

Lumen: aqueous interior of the compartment Degrade RNA, DNA → mononucleotides Protein → amino acid

Other polymer → monomer

Membrane has H+ transport → more acidic →denature protein Notice: nucleus and cytosolic protein did not degrade in lysosome; It

proteasome pathway

Primary lysosome did has particles

Tay-Sachs disease:

A defect enzyme in lysosome; did not breakbown gangliosides → glycolipid accumulation →nerve devasting →death

A mammalian cell,

EE: early endosome AV: autophagosomes LE: late endosome

Gold-ovalbumin

A scondary lysomes containing fragments of a mitochondrion (M) and a peroxisome (P) Has organelle

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Location of lysosomes and mitochondria in a cultured living bovine pulmonary arter endothelial cell

N: nucleus Green: mitochondria Red: lysosome

Peroxisomes

Responsible for degrading

 fatty acids

 toxic compounds Key Features

– single membrane – contain oxidases and

catalase

Oxidize toxic molecules, oxidase Catalase, degrade hydogen peroxide.

Oxidation of fatty acid, generate acetyl groups.

The oxidation of fatty acids in Mitochondria: produceds CO2 and ATP Peroxisomes: no ATP

Tansport to cytosol and the synthesis of cholesterol

H₂O₂→ catalase → H₂O + O₂

The endoplasmic reticulum (ER) Responsible for

 most lipid synthesis

 most membrane protein synthesis

 Ca⁺⁺ion storage

 detoxification Key Features

– network of interconnected closed membrane tubules and vesicles

– composed of smooth and rough regions

Smooth ER: synthesis of fatty acids and phospholipids; no ribosome; detoxify and modify chemistry

Rough ER: ribosome bound, synthesis of secreted and membrane proteins Cisternae: an extensive network of closed, flatted

membrane-bounded sacs

1. Transport vesicles to golgi complex from RER 2. Concentrated and packaged into immature secretory 3. Vesicles accumulate

4. exocytosis

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Model of the Golgi complex based on three-dimensional reconstruction of electron microscopy. White: transport vesicle; orange and red: trans-Golgi membrane; blue:

vesicles have budded off the RER fuse with cis-membrane.

Modifies and sorts most ER products Key Features

– series of flattened compartments & vesicles – composed of 3 regions:

cis (entry), medial, trans (exit)

– each region contains different set of modifying enzymes

The golgi complex processes and sorts secreted and membrane protein

Site of photosynthesis in plants and green algae

Key Features

– outer membrane – intermembrane space – inner membrane – stroma

– thylakoid membrane – thylakoid lumen

Site of ATP production via aerobic metabolism Occupy up to 25% of the

volume of cytoplasm The two membrane has

different composition and function; out membrane composed of about half lipid and half protein and has porins. Inner membrane less permeable, about 20% lipid and 80% protein, has cristae to increase the area.

Key Features – outer membrane – intermembrane space – inner membrane – matrix

Mitochondria are the principal sites of ATP production in aerobic cells

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Bone marrow stem cell Nuclear pore: regulates material transport

Nucleolus: synthesize rRNA Heterochromatin: condensed DNA Lamin: fibrous structure Separates

– DNA from cytosol – transcription from

translation Key Features

– outer membrane – inner membrane – nuclear pores – Nucleolus – nucleoplasm

The nucleus contains the DNA genome, RNA synthetic apparatus and a fibrous matrix

nucleus

核仁

Site of photosynthesis in plants and green algae Key Features

– outer membrane – intermembrane space – inner membrane – stroma

Chloroplasts contain internal compartments in which photosynthesis

Thylakoids are flattened to form disks → formed stacks (called grana) → embedded in matrix → stroma; contain green pigments, generated ATP during photosynthesis

The apical part of a detergent extracted intestinal epithelial cell

The cytoskeleton: components and structural functions

About ¼- ½ protein in cytosol Most water soluble protein

bound to filament or localized in specific regions Triton X-100 can not extract the

cytoskeleton and organelles.

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（一）、普通光學顯微鏡普通生物顯微鏡由3部分

構成，即︰

照明系統，包括光源和聚光器；

光學放大系統，由物鏡和目鏡組成，是顯微鏡的主體，為了消除球差和色差，目鏡和物鏡都由複雜的透鏡組構成；

機械裝置，用于固定材料和觀察方便。

顯微鏡物象是否清楚不僅決定于放大倍數，還與顯微鏡的分辨力（resolution）有關，分辨力是指顯微鏡（或人的眼睛距目標25cm處）能分辨物體最小間隔的能力，

分辨力的大小決定于光的波長和鏡口率以及介質的折射率，用公式表示為︰

R=0.61λ /N．A． N．A．＝ｎsinα/2

式中︰n=介質折射率；α=鏡口角（標本對物鏡鏡口的張角），N.A.=鏡口率（numeric aperture）。鏡口角總是要小于180°，所以sina/2的最大值必然小于1。

相差顯微鏡

相差顯微鏡（phasecontrast microscope，圖2-8、9）由 P．Zernike于1932年發明，並因此獲1953年諾貝拉物理獎。這種顯微鏡最大的特點是可以觀察未經染色的標本和活細胞。

相差顯微鏡的基本原理是，把透過標本的可見光的光程差變成振幅差，從而提升了各種架構間的對比度，使各種架構變得清晰可見。

光線透過標本后發生折射，偏離了原來的光路，同時被延遲了1/4λ（波長），如果再增加或減少1/4λ，則光程差變為1/2λ，兩束光合軸后干涉加強，振幅增大或減下，提升反差。

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微分干涉差顯微鏡

1952年，Nomarski在相差顯微鏡原理的基礎上發明了微分干涉差顯微鏡

（differential interference contrast microscope）。

DIC顯微鏡又稱Nomarski相差顯微鏡（Nomarki contrast microscope），其優點是能顯示架構的三維立體投影影像。與相差顯微鏡相比，其標本可略濃一點，折射率差別更大，故影像的立體感更強。

DIC顯微鏡下的矽藻

（偽彩色）

Bright filed

Differential interference contrast (DIC)

Phase contrast

•觀察未經染色的標本和活細胞。

倒置顯微鏡

組成和普通顯微鏡一樣，只不過物鏡與照明系統顛倒，前者在載物台之下，后者在載物台之上（圖 2-11），用于觀察培養的活細胞，具有相差物鏡。

進入20世紀80年代以來，光學顯微鏡的設計和製作又有了很大的發展，其發展趨勢主要表現下，注重實用性和多功能方面的改進。

在裝配設計上趨于採用組合模式，集普通光鏡加相差、熒光、

暗視野、DIC、攝影裝置於一體，從而操作靈活，使用方便。

螢光顯微鏡

細胞中有些物質，如葉綠素等，受紫外線照射后可發螢光；另有一些物質本身雖不能發螢光，

但如果用螢光染料或螢光抗體染色后，經紫外線照射亦可發螢光，螢光顯微鏡就是對這類物質進行定性和定量研究的工具之一。

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Fluorescence microscopy can localize and quantify specific molecules in live cells

Transgenic GFP in hydra

An optical section of a living CHO-K1 cell expression a recombinant lamin A-GFP chimeric protein

Detection of etracysteine-tagged connexin 43, a gap- junction by both light and electro microscopy

Fura-2 staining, Ca2+ sensitive flurochrome

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鐳射共聚焦掃描顯微鏡

鐳射共聚焦掃描顯微鏡（laser confocal scanning microscope）用鐳射作掃描光源，逐點、逐行、逐面快速掃描成像，掃描的鐳射與熒光收集共用一個物鏡，物鏡的焦點即掃描鐳射的聚焦點，也是瞬時成像的物點。

由於鐳射束的波長較短，光束很細，所以共焦鐳射掃描顯微鏡有較高的分辨力，大約是普通光學顯微鏡的3倍。系統經一次調焦，掃描限制在樣品的一個平面內。

調焦深度不一樣時，就可以獲得樣品不同深度層次的圖像，這些圖像訊息都儲于計算機內，透過計算機分析和類比，就能顯示細胞樣品的立體架構

鐳射共聚焦掃描顯微鏡既可以用于觀察細胞形態，也可以用于細胞內生化成分的定量分析、光密度統計以及細胞形態的測量。

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Confocal Microscope (共軛焦顯微鏡)

C-CCD camera

Confocal Cell

圖2-6 LCSM照片，

藍色為細胞核，綠色為微管

Deconvolution fluorescence microscopy yields high resolution optical sections that can be reconstructed into one 3-D image

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免疫細胞化學（immunocytochemistry）是根據免疫學原理，利用抗體同特定抗原專一結合，對抗原進行定位測定的技術。抗原主要為大分子或與大分子相結合的小分子；抗體則是由漿細胞針對特定的抗原分泌的γ球蛋白。

如果將抗體結合上標記物，再與組織中的抗原發生回應，即可在光鏡或電鏡下顯示出該抗原存在于組織中的部位。

常用的標記物有熒光素和。熒光素標記的稱為免疫熒光法（immunofluorescent technique）

常用的螢光素有異硫氰酸熒光素（fluorescein

isothiocyanate）、羅丹明（rhodamine）等。標記的稱為標免疫法（enzyme-labeled antibody method），

常用的有辣根過氧化物（horseradish peroxidase），與底物發生回應后形成不透明的沈積物，從而顯示出抗原存在的部位。

Immunocytochemistry

Immunohistochemistry

Immunofluorescent

Immunochemistry

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電子顯微鏡

透射電子顯微鏡

1、基本原理

在光學顯微鏡下無法看清小于0.2μm的細微架構，這些架構稱為亞顯微架構

（submicroscopic structures）或超微架構（ultramicroscopic structures；

ultrastructures）。

要想看清這些架構，就必須選擇波長更短的光源，以提升顯微鏡的分辨率。

1932年Ruska發明了以電子束為光源的透射電子顯微鏡（transmission electron microscope，TEM），電子束的波長要比可見光和紫外光短得多，

並且電子束的波長與發射電子束的電壓平方根成反比，也就是說電壓越高波長越短。目前TEM的分辨力可達0.2nm。

JEM-1011透射電子顯微鏡

電子顯微鏡與光學顯微鏡的成像原理基本一樣，所不同的是前者用電子束作光源，用電磁場作透鏡。

另外，由於電子束的穿透力很弱，因此用于電鏡的標本須製成濃度約50nm左右的超薄切片。這種切片需要用超薄切片機

（ultramicrotome）製作。

電子顯微鏡的放大倍數最高可達近百萬倍、由電子照明系統、電磁透鏡成像系統、真空系統、記錄系統、電源系統等5部分構成。

製樣技術 1）超薄切片

通常以鋨酸和戊二醛固定樣品，以環氧樹脂包埋，以熱膨脹或螺旋推進的模式推進樣品切片，切片濃度 20~50nm，切片採用重金屬鹽染色，以增大反差。

肌動蛋白纖維的負染電鏡照片 2）負染技術

負染就是用重金屬鹽（如磷鎢酸、醋酸雙氧鈾）對鋪展在載網上的樣品進行染色；吸去染料，樣品乾燥后，

樣品凹陷處鋪了一薄層重金屬鹽，而凸的出地方則沒有染料沈積，從而出現負染效果（圖2-15），分辨力可達1.5nm左右。

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3）冰凍蝕刻

冰凍蝕刻（freeze-etching）亦稱冰凍斷裂

（freeze-fracture）。

標本置於-100°C的干冰或-196°C的液氮中，進行冰凍。然後用冷刀驟然將標本斷開，

升溫后，冰在真空條件下迅即昇華，暴露出斷面架構，稱為蝕刻（etching）。

蝕刻后，向斷面以45度角噴涂一層蒸汽鉑，

再以90度角噴涂一層碳，加強反差和強度。

然後用次氯酸鈉溶液消化樣品，把碳和鉑的膜剝下來，此膜即為複膜（replica）。

複膜顯示出了標本蝕刻面的形態，在電鏡下得到的影像即代表標本中細胞斷裂面處的架構（圖2-16）。

掃描電子顯微鏡

掃描電子顯微鏡（scanning electron microscope，SEM）于20世紀60年代問世，用來觀察標本的表面架構。

其工作原理是用一束極細的電子束掃描樣品，在樣品表面激發出次級電子，次級電子的多少與電子束入射角有關，也就是說與樣品的表面架構有關，次級電子由探測體收集，並在那裡被閃爍器轉變為光信號，再經光電倍增管和放大器轉變為電信號來控制熒光屏上電子束的強度，顯示出與電子束同步的掃描圖像。

圖像為立體形象，反映了標本的表面架構。為了使標本表面發射出次級電子，標本在固定、脫水后，要噴涂上一層重金屬微粒，重金屬在電子束的轟擊下發出次級電子信號。

目前掃描電鏡的分辨力為6~10nm，人眼能夠區別熒光屏上兩個相距 0.2mm的光點，則掃描電鏡的最大有效放大倍率為

0.2mm/10nm=20000X。

圖2-17 JEOL掃描電子顯微鏡

圖2-18 光學顯微鏡、

TEM、SEM成像原理比較

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Structure of the nuclear pore complex (NPC) by cryoelectron tomography

Electron microscopy of metal –coated specimens can reveal surface features of cells and their compoents

雲母

Transmission electron microscopy (TEM)

Operates in vacuum

Specimen usually fixed, embedded, sectioned, and stained with an electron-dense material

Special techniques:

Metal shadowing: visualize surface structures, cell components Cryoelectron: visualize unfixed, unstained samples

Freeze fracture, freeze etch: visualize membrane interior Freeze etch: visualize cell interior

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5.1 The transmission electron microscope

Figure 5-16 Figure 5-15

Scanning electron microscopy

Can visualize surfaces of tissues, cells, isolated cell parts Specimen is fixed and coated with thin layer of heavy metal Images secondary electrons, resolution = 10 nm

Figure 5-20

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SEM produces a three dimensional image of the surface of an unsectioned specimen

Centrifugation can separate many types of organelles

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A mixed organelle fraction can be further separated by equilibrium density- gradient centriguation

Small coate vesicles can be purified by binding of antibody specific for a vesicle surface protein and linkage to bacterial cells

Flow cytometry separates different cell types

By cell marker DNA dye 流式細胞儀

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Flow Cytometry and Cell Sorting

‘FACS’ has become a generic term for ALL flow cytometry FACS : Fluorescence Activated Cell Sorter

Is actually a trade name of Becton Dickinson (BD).

FACS is one version of Flow Cytometry, which can sort cells by their surface markers Individual cell is positively or negatively charged based on their fluorescence color When charged cells pass through an electric field, they are deflected and hence separated

0.1 1 10 100 1000

dye 1 intensity (for CD4) dye 2 intensity

(for CD8)

Double Negative (CD4-,CD8-

)

Double positive (CD4+,CD8+) Single

positive (CD4-,CD8+)

Single positive (CD4+,CD8- die ^{ T cell} )

(CD4-,CD8-₎

die

Fluorescence intensity

細胞大小細胞的顆粒度

細胞表面分子︰CD 系列細胞漿內分子︰胞內細胞因子細胞核內分子︰P53

細胞功能檢測︰細胞週期、凋亡

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Differentiation of mouse myoblast cell into muscule cells

Hoechst stain

Myosin heavy chain for red

Primary cell culture can be used to study cell differentiation

Primary cell cultures and cell strains have a finite (有限) life span

transformation

C2C12 cell line (transformed mouse myoblast) differentiate to muscule

STSL1 cell line (preadipocytes) differentiated into adipocyte and expressed adipocyte- specific mRNA

Insulin responsive glucose transporter Adipocyte gene, transcription factor

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How to proof the Ca2+ dependent adhesion

Ca2+ concentration change

有孔 When Ca2+ → generate cell-

cell adhesion (anchoring junctions and tight junctions) Block by add cahderin

antibody Cahderin induced cell

adhesion is Ca2+ dependent

Madin-Darby canine kidney

MDCK in specialized container provide a useful experimental system for studying epithelial cells

NS-1 Hybrid cell called hybridomas produce abundant monoclonal antibodies