STBP STBP 1023 1023

Cell Cell BiologyBiology

Assoc. Prof. Dr. Hasidah Mohd. Assoc. Prof. Dr. Hasidah Mohd. SidekSidek

hasidah@ukm.my

School of Biosciences & Biotechnology

Faculty of Science & Technology(Level 1 Biological Science Building)

Course ObjectivesCourse Objectives

At the end of the course, At the end of the course, students will :students will :

• understand understand the biochemical the biochemical basisbasis of the living system of the living system

• understand the concept of the understand the concept of the cell as the basic unit of lifecell as the basic unit of life and and appreciate the structure & appreciate the structure & function of cell componentsfunction of cell components

Course Objectives (continued)Course Objectives (continued)

• understand the understand the principles of principles of bioenergeticsbioenergetics in order to in order to appreciate how intermediary appreciate how intermediary metabolism though complex is metabolism though complex is very systematic and orderlyvery systematic and orderly

• understand the concept of understand the concept of energy utilisation and energy utilisation and productionproduction of energy (ATP) in of energy (ATP) in the cellthe cell

Course StructureCourse Structure

•Lectures Lectures

•TutorialsTutorials

EvaluationEvaluation

• Mid-semester Examination (30-Mid-semester Examination (30-40%) 40%)

• Final Examination (60-70%)Final Examination (60-70%)

Course FacilitatorsCourse Facilitators

• Assoc. Prof. Dr. Hasidah Mohd. Assoc. Prof. Dr. Hasidah Mohd. Sidek (HMS)Sidek (HMS)

• Dr. Izyanti Ibrahim (II)Dr. Izyanti Ibrahim (II)

• Dr. Sharom Md. Yusof (SMY) Dr. Sharom Md. Yusof (SMY) **

** Coordinator Coordinator (sharom@ukm.my)(sharom@ukm.my)

Lecture ScheduleLecture Schedule

One SetOne Set

Monday Monday 11:00-12:00 11:00-12:00 DKG129BBDKG129BB Tuesday 08:00-09:00Tuesday 08:00-09:00 DKG129BBDKG129BB ThursdayThursday 08:00-09:0008:00-09:00 DKG130BBDKG130BB

Course OutlineCourse Outline

1. Cell as the basic unit of life (2 1. Cell as the basic unit of life (2 h)h)

- Overview of cell structure & function- Overview of cell structure & function

- Techniques to study cell structure &- Techniques to study cell structure &

function (microscopy, cell function (microscopy, cell

fractionation)fractionation)

2. Structure & function of cells 2. Structure & function of cells (3h)(3h) - Organelles (mitochondria, chloroplasts,- Organelles (mitochondria, chloroplasts,

ribosomes, nucleus, Golgi complex,ribosomes, nucleus, Golgi complex,

endoplasmic reticulum, bacterial &endoplasmic reticulum, bacterial &

plant cell walls, cytoskeleton)plant cell walls, cytoskeleton)

3. Cell buffering system (4 h)3. Cell buffering system (4 h) - Characteristics of water, acids,- Characteristics of water, acids,

bases & properties of buffersbases & properties of buffers

- Physiological buffers- Physiological buffers

4. Macromolecules in cells (5 h)4. Macromolecules in cells (5 h) - Proteins- Proteins

- Polysaccharides- Polysaccharides

- Lipids- Lipids

5. Membrane Biology (4 h)5. Membrane Biology (4 h) - Structure, composition & models of- Structure, composition & models of

membranesmembranes

- Transport across membranes- Transport across membranes

6. Enzymatic reactions in cells 6. Enzymatic reactions in cells (5h)(5h)

- - Types & functions of enzymesTypes & functions of enzymes

- Catalytic characteristics- Catalytic characteristics

- Factors influencing enzyme reactions- Factors influencing enzyme reactions

- Isoenzymes & coenzymes- Isoenzymes & coenzymes

7. Bioenergetics (2 h)7. Bioenergetics (2 h) - Principles of thermodynamics- Principles of thermodynamics

- Involvement of ATP in coupled reactions- Involvement of ATP in coupled reactions

8. Energy metabolism & ATP 8. Energy metabolism & ATP

synthesis (7 h)synthesis (7 h) - Glycolysis- Glycolysis

- TCA cycle- TCA cycle

- Respiratory chain & synthesis of ATP- Respiratory chain & synthesis of ATP

- - -oxidation of fatty acids-oxidation of fatty acids

- Shuttle systems- Shuttle systems

Campbell N.A. & Reece J.B. 2005. Campbell N.A. & Reece J.B. 2005. Biology. 7Biology. 7thth Edition. San Francisco: Edition. San Francisco: Pearson Benjamin Cummings.Pearson Benjamin Cummings.

Campbell M.K. & Farrell S.O. 2006. Campbell M.K. & Farrell S.O. 2006. Biochemistry. 5 Biochemistry. 5thth Edition. Edition. Belmont: Thomson Brooks / Cole.Belmont: Thomson Brooks / Cole.

Nelson, D.L. & Cox, M.M. 2008 Nelson, D.L. & Cox, M.M. 2008 or or 20062006. . Lehninger Principles of Lehninger Principles of Biochemistry. 5Biochemistry. 5thth or 4or 4thth EditionEdition. . New York: W.H. Freeman.New York: W.H. Freeman.

Recommended TextRecommended Text

First 2 lectures…

Cell as the basic unit of life (2 h)Cell as the basic unit of life (2 h)

- Overview of cell structure & function- Overview of cell structure & function

- Techniques to study cell structure &- Techniques to study cell structure &

function (microscopy, cell fractionation)function (microscopy, cell fractionation)

All Living Things are Composed All Living Things are Composed of Cellsof Cells

CellCell• Mass of protoplasmMass of protoplasm• Bound by Bound by

membranemembrane• Smallest Smallest livingliving unit unit

In other words, the cell isIn other words, the cell is

the basic unit of lifethe basic unit of life Anything lower than the cellAnything lower than the cell

is considered non-living is considered non-living

• Cells comprise of Cells comprise of :: - molecules- molecules - atoms- atoms (organised into(organised into macromoleculesmacromolecules))

• Individually, Individually, molecules and molecules and atomsatoms are non-livingare non-living componentscomponents

• molecules and molecules and atomsatoms are not able to are not able to carrycarry out life out life processesprocesses

We will review later on whatWe will review later on what

properties make the cell properties make the cell

‘ ‘living’ as compared to its living’ as compared to its

‘ ‘non-living’ constituentsnon-living’ constituents ((molecules and atoms)molecules and atoms)

http://www.sinauer.com/cooper/4e/chapter01.html July 5th 2009

All present-day cells (both prokaryotes & eukaryotes) All present-day cells (both prokaryotes & eukaryotes)

are descended from a single ancestorare descended from a single ancestor

The first cell arose at least 3.8 billion years ago as a The first cell arose at least 3.8 billion years ago as a

result of enclosure of self-replicating RNA in a result of enclosure of self-replicating RNA in a

phospholipid membrane phospholipid membrane

Present-day prokaryotes are divided Present-day prokaryotes are divided into two groups : into two groups : the archaebacteriathe archaebacteria the eubacteria the eubacteria

Eukaryotic cells:Eukaryotic cells: are larger and more complex than prokaryotic cellsare larger and more complex than prokaryotic cells contain a nucleuscontain a nucleus consist of cytoplasmic organelles consist of cytoplasmic organelles

and a cytoskeletonand a cytoskeleton

The simplest eukaryotes are The simplest eukaryotes are unicellular organismsunicellular organisms (e.g. yeasts & (e.g. yeasts & amoebas)amoebas)

Multicellular organisms Multicellular organisms evolved from evolved from associations between such unicellular associations between such unicellular eukaryoteseukaryotes

Division of labor led to the Division of labor led to the development of the many kinds of development of the many kinds of specialised cells specialised cells that make up that make up present-day plants & animals present-day plants & animals

In research, cells have been In research, cells have been used as experimental modelsused as experimental models

Examples of cells as Examples of cells as experimental modelsexperimental models

The The bacteria, bacteria, E. coliE. coli is useful is useful for investigating fundamental for investigating fundamental aspects of biochemistry & aspects of biochemistry & molecular biology (related to molecular biology (related to the genetic simplicity of the the genetic simplicity of the bacteria thus easily studied)bacteria thus easily studied)

science.howstuffworks.com 12 Sept 2011 en.wikipedia.org 12 Sept 2011

• some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls

• the harmless strains are part of the normal flora of the gut

• E. coli is the most widely studied prokaryotic model organism

• an important species in the fields of biotechnology and microbiology (it has served as the host organism for the majority of work with recombinant DNA)

senescence.info 12 Sept 2011

More examples of cells More examples of cells (and organisms) as(and organisms) as

experimental modelsexperimental models

YeastsYeasts, as the simplest , as the simplest eukaryotic cells, are important eukaryotic cells, are important models for studying various models for studying various aspects of eukaryotic cell aspects of eukaryotic cell biologybiology

The The nematode, nematode, Caenorhabditis Caenorhabditis eleganselegans is a simple is a simple multicellular organism that multicellular organism that serves as an important model serves as an important model in developmental biologyin developmental biology

The The fruitfly, fruitfly, Drosophila Drosophila melanogaster melanogaster has been has been extensively studied in terms of its extensively studied in terms of its genetics and contribute to major genetics and contribute to major advances in understanding advances in understanding animal developmentanimal development

Also the Also the small flowering plant, small flowering plant, Arabidopsis thalianaArabidopsis thaliana is widely used is widely used as a model for studies of plant as a model for studies of plant molecular biology & developmentmolecular biology & development

genotyping.wordpress.com Sept 12 2011

- Its small stature and short generation time facilitates rapid genetic studies

- Arabidopsis was the first plant to have its genome sequenced

In addition, many kinds of In addition, many kinds of vertebrate cells vertebrate cells can be grown in can be grown in culture where they can be culture where they can be studied under controlled studied under controlled laboratory conditionslaboratory conditions

Specialised cell types, such as Specialised cell types, such as neurons & muscle cells, provide neurons & muscle cells, provide useful models for investigating useful models for investigating particular aspects of cell biologyparticular aspects of cell biology

Epithelial cells in culture, stained for keratin (red) and DNA (green)

Growth of Animal Cells in Culture:Growth of Animal Cells in Culture: The propagation of animal cells in The propagation of animal cells in culture has allowed studies of the culture has allowed studies of the mechanisms that control cell mechanisms that control cell growth & differentiationgrowth & differentiation

Culture of Plant Cells:Culture of Plant Cells: Cultured Cultured plant cells can differentiate to form plant cells can differentiate to form specialised cell types &, in some specialised cell types &, in some cases, can regenerate entire plants cases, can regenerate entire plants

Viruses:Viruses: Viruses provide simple Viruses provide simple models for studies of cell functionmodels for studies of cell function

The The frog frog Xenopus laevisXenopus laevis & & zebrafishzebrafish are important models are important models for studies of early vertebrate for studies of early vertebrate development development

The The mousemouse is a mammalian is a mammalian species suitable for genetic species suitable for genetic analysis and also for analysis and also for in vivo in vivo trialstrials

At the level of organisms…

The ease of manipulation in amphibian embryos has given them an important place in modern developmental biology.

- Fully-sequenced genome - Well understood, easily observable and testable developmental behaviors

- relatively easy to maintain and handle- reproduce quickly- share a high degree of homology with humans (mouse genome already sequenced)

Tools which have Tools which have facilitated studies in facilitated studies in cell biology include… cell biology include…

Light Microscopy allows for:Light Microscopy allows for: - - visualisation of cells & subcellularvisualisation of cells & subcellular structuresstructures - determination of intracellular- determination of intracellular localisation of specific moleculeslocalisation of specific molecules

Electron Microscopy (with a Electron Microscopy (with a resolution resolution 100 fold greater 100 fold greater than light microscopy) allows:than light microscopy) allows:

- - detailed analysis of cell structure detailed analysis of cell structure

Subcellular Fractionation:Subcellular Fractionation: allows organelle isolation allows organelle isolation from eukaryotic cells for from eukaryotic cells for further biochemical further biochemical analysisanalysis

(via (via differential centrifugation)differential centrifugation)

thin slices of cork thin slices of cork as observed by as observed by Robert HookeRobert Hooke

internal ultrastructure

of cells-transmission electron microscope

surface details of cilia- scanning electron microscope

Blood components (surface details)

– scanning electron microscope

The early microscope!The early microscope!

SEM

Development of Development of microscopy microscopy

contributed to the contributed to the formulation offormulation of

the cell theorythe cell theory

Cell theory historical timelineCell theory historical timeline

Basic tenets of the Cell Basic tenets of the Cell TheoryTheory

1.1. All organisms consist of All organisms consist of one or more cellsone or more cells

2.2. The cell is the basic unit of The cell is the basic unit of structure for all organismsstructure for all organisms

3.3. All cells arise only from All cells arise only from pre-existing cellspre-existing cells

The combined work of The combined work of Schleiden, Schwann & Schleiden, Schwann & VirchowVirchow led to the Cell Theory led to the Cell Theory

This would not have been This would not have been possible without the possible without the

contribution of contribution of ‘microscopists’‘microscopists’

Robert Hooke

Anton van LeeuwenhoekAnton van Leeuwenhoek

Cells are the basic Cells are the basic structural units of all structural units of all livingliving organisms organisms

Organisms may be Organisms may be made up of singlemade up of single

or multiple cellsor multiple cells

in other words, in other words,

organisms are unicellular organisms are unicellular or multicellularor multicellular

The cell is the lowest level of The cell is the lowest level of organisation that can perform all organisation that can perform all activities required for lifeactivities required for life

25 µmFigure 1.5

Therefore…Therefore…

Any lower level of organisation is non-living

General General characteristicscharacteristics

of cellsof cells

Cell structure is diverse but all cells Cell structure is diverse but all cells share common characteristicsshare common characteristics

The chemical composition of all The chemical composition of all cells are basically the samecells are basically the same

Basic elements in cells – C, H, N, O, P, SBasic elements in cells – C, H, N, O, P, S

‘‘Non-living’ elements form cellular Non-living’ elements form cellular macromoleculesmacromolecules - proteins, nucleic - proteins, nucleic acids, carbohydrates & lipidsacids, carbohydrates & lipids

Activities carried out by cells depend Activities carried out by cells depend on properties & functions of on properties & functions of macromoleculesmacromolecules

All cells are bounded by a All cells are bounded by a plasma membraneplasma membrane

The plasma membrane :The plasma membrane :

• separates activities occurring in separates activities occurring in neighboring cellsneighboring cells

• gives each cell an independent gives each cell an independent entityentity

Lateral movement(~107 times per second)

Flip-flop(~ once per month)

(a) Movement of phospholipids

Lipid bilayer of the plasma membraneLipid bilayer of the plasma membrane

Hydrophillic head exposed to aqueous environmentHydrophillic head exposed to aqueous environmentHydrophobic tail hidden from the environmentHydrophobic tail hidden from the environment

Cells are highly complexedCells are highly complexed & organised& organised

Organisation of Organisation of LifeLife• CellCell

• TissueTissue• OrganOrgan• Organ SystemOrgan System• OrganismOrganism• PopulationPopulation• CommunityCommunity• EcosystemEcosystem• BiosphereBiosphere

Smallest Smallest LevelLevel

Largest LevelLargest Level

Cells possess a Cells possess a genetic program genetic program & the means to & the means to use ituse it

Genetic information is contained in genes

Genes constitute blueprints for cell structure, activities & multiplication

The Cell’s Heritable Information Cells contain chromosomes made partly of DNA Cells contain chromosomes made partly of DNA

(the substance of genes)(the substance of genes)

DNA programs the cellular production of proteins DNA programs the cellular production of proteins and transmit information from parents to offspringand transmit information from parents to offspring

Egg cell

Sperm cell

NucleicontainingDNA

Fertilized eggwith DNA fromboth parents

Embyro’s cells with copies of inherited DNA Offspring with traits

inherited fromboth parentsFigure 1.6

Cells are capable Cells are capable of producing more of producing more of themselvesof themselves

Reproduce by Reproduce by division (mitosis)division (mitosis)

Developing and maintaining complexity Developing and maintaining complexity requires requires energy from the sunenergy from the sun

Photosynthesis converts light energy Photosynthesis converts light energy into into chemical energy – sucrose and chemical energy – sucrose and starchstarch

Animal cells – contains Animal cells – contains prepackaged prepackaged energy in the form of glucoseenergy in the form of glucose

Glucose metabolism produces ATPGlucose metabolism produces ATP

Cells acquire and utilise energy

Cells perform a variety of Cells perform a variety of chemical reactionschemical reactions

Cell processes based on Cell processes based on biochemical reactionsbiochemical reactions

Require enzymesRequire enzymes

Biological catalysts increase Biological catalysts increase reaction rate without increase in reaction rate without increase in temperaturetemperature

Cells engage in numerous Cells engage in numerous mechanical activitiesmechanical activities

Transport, assembly and Transport, assembly and degradationdegradation

These activities are based on These activities are based on dynamic changes in protein dynamic changes in protein structurestructure

Cells are able to respond to Cells are able to respond to stimulistimuli

Visible responses – cilliate moves away Visible responses – cilliate moves away from object or moves towards a source of from object or moves towards a source of nutrientsnutrients

Less obvious for multicellular organismLess obvious for multicellular organism Receptors on cell surface interact with Receptors on cell surface interact with

substances – hormones, growth factors substances – hormones, growth factors etcetc

Respond by altering metabolic Respond by altering metabolic activities, preparing for cell division, activities, preparing for cell division, committing suicide(!)committing suicide(!)

Cells are capable of self-regulationCells are capable of self-regulation

To maintain constant ordered stateTo maintain constant ordered state

Failure to correct mistake during DNA Failure to correct mistake during DNA replication may cause mutation leading replication may cause mutation leading to diseases e.g. cancerto diseases e.g. cancer

SummarySummaryThe chemical composition of all cells are basically the same

Cells acquire and utilize energy

All cells are bounded by a plasma membrane

Cells perform a variety of chemical reactions

Cells are highly complexed & organized

Cells engage in numerous mechanical activities

Cells possess a genetic program & the means to use it

Cells are able to respond to stimuli

Cells are capable of producing more of themselves

Cells are capable of self-regulation

Thus far, we have looked at Thus far, we have looked at common characteristics of common characteristics of cellscells

There are differences too…There are differences too…

Cells vary in sizeCells vary in size smallest bacteria - 0.2 smallest bacteria - 0.2 m in m in

diameterdiameter

longest in mammalslongest in mammals (nerve cells, giraffe neck)(nerve cells, giraffe neck)

largest volumelargest volume (yolk of ostrich egg-also the (yolk of ostrich egg-also the

largest single cell in the world)largest single cell in the world)

Animal and plant cells large Animal and plant cells large enough to be seen with a light enough to be seen with a light microscopemicroscope

Smaller molecules only Smaller molecules only observed with an electron observed with an electron microscopemicroscope

Cells vary in shape & Cells vary in shape & functionfunction

Nerve cells are enormously Nerve cells are enormously extended to allow transmission extended to allow transmission of electrical signalsof electrical signals

Human red blood cells are Human red blood cells are flattened to allow transport of flattened to allow transport of OO22

Cell Cell classificationclassification Based on the presence or Based on the presence or

absence of a nucleusabsence of a nucleus

Two basic types of cellsTwo basic types of cells Eukaryotes (from the Greek word Eukaryotes (from the Greek word

eueu meaning ‘truly’ and meaning ‘truly’ and karyonkaryon, a , a ‘nucleus’)‘nucleus’)

Prokaryotes (from Prokaryotes (from propro, meaning , meaning ‘before’)‘before’)

Prokaryotes evolved Prokaryotes evolved earlier than eukaryotesearlier than eukaryotes

A Brief History of Life on EarthA Brief History of Life on Earth

Earth formedEarth formed4.5 billion 4.5 billion yearsyears ago ago

3.5 billion years ago3.5 billion years ago

1.5 billion years ago1.5 billion years ago

0.5 billion years ago0.5 billion years ago

First life - prokaryoticFirst life - prokaryoticbacteria dominatebacteria dominate

Nucleated cells arise - eukaryoticNucleated cells arise - eukaryotic

Cambrian explosionCambrian explosionmulticellular eukaryotes arisemulticellular eukaryotes arise

Two basic types of cells

Diagrams:

Prokaryotic & Eukaryotic Cell, Mariana Ruiz

__________________________________________

http://cellbiologypowerpoints.googlepages.com/home July 5th. 2009

Prokaryote Eukaryote

Generally,…Generally,… Prokaryotes are almost always single-Prokaryotes are almost always single- celledcelled (except for prokaryote colonies) (except for prokaryote colonies)

Prokaryotes do not contain any cell Prokaryotes do not contain any cell nucleus or any other membrane-nucleus or any other membrane-

boundbound organellesorganelles ((DNA travels openly around the cell)DNA travels openly around the cell)

Prokaryotes reproduce by Prokaryotes reproduce by binary fissionbinary fission

(generation of another copy by dividing)(generation of another copy by dividing)

BinarBinaryyFissiFissionon

All bacteria All bacteria (Kingdom=Monera)(Kingdom=Monera)

are prokaryotesare prokaryotes

Eukaryotic vs prokaryotic Eukaryotic vs prokaryotic cellscells

Also to put things in perspective in terms of size

Thiomargarita (Sulfur Pearl of Namibia)

Atypically-sized prokaryote

>100 X bacterial size (typical size 1-5

m)

large nitrate-storing vacuole contributes

to the size

When compared to prokaryoticWhen compared to prokaryotic cells, eukaryotic cells are morecells, eukaryotic cells are more complex complex

containing membrane-enclosedcontaining membrane-enclosed organelles absent in prokaryotes organelles absent in prokaryotes

Eukaryotes are 10-20 X Eukaryotes are 10-20 X largerlarger

than prokaryotes (~10-than prokaryotes (~10-100 100 m)m)

Multicellular eukaryotes Multicellular eukaryotes (human, animal, plant, fungus, (human, animal, plant, fungus,

protist)protist) Unicellular eukaryotes Unicellular eukaryotes (yeast, Paramecium)(yeast, Paramecium)

YeastYeast

ParameciParameciumum

Candida albicans

Acetabularia Acetabularia (Mermaid's (Mermaid's wineglass algae)wineglass algae)

Atypically-sized eukaryotic Atypically-sized eukaryotic cellcell

A single giant cell ~5-7 cm in A single giant cell ~5-7 cm in length length

The nucleus is in holdfast The nucleus is in holdfast (root)(root)

Red blood cell is a Red blood cell is a eukaryoticeukaryoticcell without nucleuscell without nucleus Unable to undergo mitosisUnable to undergo mitosis

Some eukaryotic cells Some eukaryotic cells have more than one have more than one nucleinuclei

Fungi - fused cells,Fungi - fused cells,

multinucleatemultinucleate

Human skeletal muscle cells -Human skeletal muscle cells -

multinucleatemultinucleate

Cross section of Cross section of skeletal muscle :skeletal muscle : with with peripheral nuclei and large amounts of peripheral nuclei and large amounts of cytoplasm / small extracelluarcytoplasm / small extracelluar spacespace

                                                                                                

Figure 4 - Skeletal muscle transverse section (Bright field illumination). Bar is 30 microns

www.bris.ac.uk/.../m1_index/histprac/page3.htm 7 July 2007

Multinucleatedeukaryotic cells

Different methods forDifferent methods for

enhancing visualisationenhancing visualisation of of

cellular structures in cellular structures in microscopymicroscopy

EUnstained versus stained human cheekUnstained versus stained human cheekepithelial cellsepithelial cells Brightfield microscopy : light Brightfield microscopy : light passes directly through specimenpasses directly through specimen

Image has little contrast unless Image has little contrast unless cell is naturally pigmented orcell is naturally pigmented orartificially stainedartificially stained

(a)

Staining with various dyesStaining with various dyes enhances contrastenhances contrast

Most staining proceduresMost staining proceduresrequire that cells be fixedrequire that cells be fixed(preserved)(preserved)

(b)

Phase-contrast. Enhances contrast in unstained cells by amplifying variations in density within specimen; especially useful for examining living, unpigmented cells.

50 µm

Brightfield (stained specimen). Staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved).

(b)

In phase-contrast microscopy, In phase-contrast microscopy, contrast of unstained cellscontrast of unstained cellsmay be enhanced by may be enhanced by optical modificationoptical modification

For differential-interference-contrast For differential-interference-contrast (Nomarski) microscopy, (Nomarski) microscopy, image appearsimage appears 3 D through optical modification3 D through optical modification

In fluorescence microscopy,In fluorescence microscopy, the locations the locations of specific molecules in the cell of specific molecules in the cell are shown by tagging the moleculesare shown by tagging the molecules with fluorescent dyes or antibodieswith fluorescent dyes or antibodies

These fluorescent substances absorbThese fluorescent substances absorb ultraviolet radiation and emit visible light, ultraviolet radiation and emit visible light, as shown here in a cell from an arteryas shown here in a cell from an artery

Confocal. Uses lasers and special optics for “optical sectioning” of fluorescently-stained specimens. Only a single plane of focus is illuminated; out-of-focus fluorescence above and below the plane is subtracted by a computer. A sharp image results, as seen in stained nervous tissue (top), where nerve cells are green, support cells are red, and regions of overlap are yellow. A standard fluorescence micrograph (bottom) of this relatively thick tissue is blurry.

50 µm

In electron microscopy,In electron microscopy,

a beam of electrons is a beam of electrons is

focusedfocused

throughthrough a specimen (TEM) or a specimen (TEM) or

ontoonto its surface (SEM) its surface (SEM)

Transmission electron micrograph allows for detailed study of the

internal ultrastructure of cells

Transmission electron micro-scopy (TEM). A transmission electron microscope profiles a thin section of a specimen. Here we see a section through a tracheal cell, revealing its ultrastructure. In preparing the TEM, some cilia were cut along their lengths, creating longitudinal sections, while other cilia were cut straight across, creating cross sections.

Figure 6.4 (b)

In contrast, scanning electronIn contrast, scanning electron

micrograph…. micrograph…. allows for detailed study of the allows for detailed study of the

surface of a specimensurface of a specimenTECHNIQUE

Scanning electron micro-scopy (SEM). Micrographs takenwith a scanning electron micro-scope show a 3D image of the surface of a specimen. This SEM shows the surface of a cell from a rabbit trachea (windpipe) covered with motile organelles called cilia. Beating of the cilia helps moveinhaled debris upward toward the throat.

Figure 6.4 (a)

RESULTS

Importance of cellular Importance of cellular fractionationfractionation

for the study of for the study of cell structure & cell structure &

functionfunction

Cell FractionationCell Fractionation• A combination of A combination of

various methods used to various methods used to separate cell organelles & separate cell organelles & componentscomponents

• Consists of two phases :Consists of two phases :― homogenisationhomogenisation― centrifugation centrifugation

http://www.freewebs.com/ltaing/ july 9 2008

HomogenisationHomogenisation• The process of breaking open cellsThe process of breaking open cells• Accomplished with the use of : Accomplished with the use of :

― chemicals― chemicals ― ― enzymesenzymes

― ― sound wavessound waves• Forcing cells through small spaces Forcing cells through small spaces

at high pressure may also break at high pressure may also break cells apartcells apart

CentrifugationCentrifugation• Isolation of cell organellesIsolation of cell organelles• Results in the isolation of Results in the isolation of mitochondria, nucleus, mitochondria, nucleus, chloroplast etc.chloroplast etc.

Generally…Generally…• Applications for centrifugation Applications for centrifugation are many including :are many including :― sedimentation of cells & virusessedimentation of cells & viruses― isolation of macromolecules (e.g.isolation of macromolecules (e.g.

DNA, RNA, proteins, or lipids)DNA, RNA, proteins, or lipids) ― separation of sub-cellularseparation of sub-cellular

organellesorganelles

In cell biology, cell In cell biology, cell fractionation…fractionation…

• increases knowledge on increases knowledge on organelle functionsorganelle functions― isolate organelles into pure groups isolate organelles into pure groups

i.e.i.e.

specific cell components specific cell components • e.g. by centrifugation, a specific e.g. by centrifugation, a specific

cell fraction was determined to cell fraction was determined to have enzymes that function in have enzymes that function in cellular respirationcellular respiration

• Because this cell fraction is rich inBecause this cell fraction is rich in mitochondria, this is evidence that mitochondria, this is evidence that thethe mitochondria is the site for mitochondria is the site for cellularcellular respirationrespiration

Microscopy is used to identify the Microscopy is used to identify the organelles in each pelletorganelles in each pellet

Biochemical methods are then used Biochemical methods are then used to determine the metabolic functions to determine the metabolic functions associated with each type of associated with each type of organelleorganelle Cell fractionation is now widely used Cell fractionation is now widely used to isolate particular organelles in to isolate particular organelles in order to study further details of their order to study further details of their functionfunction

Subcellular

Fraction

Marker Enzyme

MitochondriMitochondriaa

Succinate Succinate DehydrogenasDehydrogenas

ee

Lysosomes Acid Phosphatase

MicrosomesMicrosomes Glucose-6-Glucose-6-PhosphatasePhosphatase

CytosolLactate

Dehydrogenase

http://www.wpi.edu/Academics/Depts/Chemistry/Courses/General/fractionation.html9 July 2007

Uses of centrifugationUses of centrifugation• separation of blood components ― plasma mostly H2O content ― erythrocytes (red blood cells) ― buffy coat (white blood cells & platelets)

Uses of differential Uses of differential centrifugationcentrifugation• sperm separation (fertility sperm separation (fertility

clinic)clinic)

― sperm with an X chromosome (for sperm with an X chromosome (for girls) weigh a little more than girls) weigh a little more than sperm with a Y chromosome (for sperm with a Y chromosome (for boys)boys)

― sperms can be sorted out & sperms can be sorted out & prepared for insemination prepared for insemination

Course Outline Course Outline (revisited)(revisited) 1. Cell as the basic unit of life (2h)1. Cell as the basic unit of life (2h)

- Overview of cell structure & function- Overview of cell structure & function

- Techniques to study cell structure &- Techniques to study cell structure &

function (microscopy, cell function (microscopy, cell

fractionation)fractionation)

2. Structure and function of cells (2h)2. Structure and function of cells (2h)

- Organelles (mitochondria, - Organelles (mitochondria, chloroplasts,chloroplasts,

ribosomes, nucleus, Golgi complex,ribosomes, nucleus, Golgi complex,

endoplasmic reticulum, bacterial &endoplasmic reticulum, bacterial &

plant cell walls, cytoskeleton)plant cell walls, cytoskeleton)

Course Outline Course Outline (continued)(continued) 3. Cell buffering system (4 h)3. Cell buffering system (4 h)

- Characteristics of water, acids,- Characteristics of water, acids,

bases & properties of buffersbases & properties of buffers

- Physiological buffers- Physiological buffers

4. Macromolecules in cells (6 h)4. Macromolecules in cells (6 h)

- Proteins- Proteins

- Polysaccharides- Polysaccharides

- Lipids- Lipids