The Cell

Overview of Cell Biology

 Cells are the fundamental unit of life; the structural and func tional unit of all living orga nism s.

 The cell th eory: All organisms are co m posed of cell s and all cells co me from pre-existing cells.

 In single-celled organism s such as bacteria and protoz oa, each cell is independent.

 In m u lti-celled organism s, function is distributed am ong different specialized cells.

 Cells to tissues to organs to organism s…

 Biological system s use cells to build high er levels of organization, but the cell rem a ins the fundam e ntal unit. Radiation e ffects at the cellular level can affect the higher-level organization.

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[ L od ish , 20 00 ]

What can cells do?

Cells can:

 use DNA as hereditary material ,

 use proteins as catalysts

 reproduce

 transform matter and energy

 respond to their environm ent

Sizes and shapes vary

0.5 µm bacteria to a se veral cm hen egg (yolk)

shapes can be spherical, fl attened colum n ar, cuboidal.

The cell memb rane

 The interior of cells is an aqueous environment.

 Most of the intracellular m o lecules are water-soluble.

 Most of the ch emi cal reactions carried out inside cells require an aqueous environm ent.

 The environment around cells is also basically an aqueous one.

 Bodily flui ds and blood are aqueous soluti ons of prot eins and small mol ecules.

 Chem ically (pH, ioni c strength) blood is si m i lar to sea water.

For cells to maintain integrity they m u st be surrounded by a mem b rane through which water cannot flow. A mem b rane co m posed of fatty acid m o lecules served this purpose.

All cells have a cell memb rane, a two-layered shell of phospholipi d s. All biological mem b ranes have the sam e ba sic phospholi p id bilayer structure.

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Other functions of membranes Mem b ranes serve many functions other than segregating inside from o u tside.

Many proteins are as sociated with mem b ran e s, either em b e dded in the membran e , or attached to the s u rface.

 Transport of ions or m a crom olecules

 Energy storage . Ion concentration gradi e nt s serve as source of potential energy.

 Receptors for signal transducti on

 Adherence. Cell-cel l contacts.

Tw o major divisions of cells.

P r ok ar yote s (before nucleus). Lack a defined nucl e us. Have a sim p lified internal organization. Have only a plasma me mbrane and a nucle oid, no internal membran e s.

All are single celled organism s (e.g., bacteria, blue-green algae)

Eu karyotes (typical or true nucleus). All memb ers of the plant and ani m al kingdom s are eukaryotes. Eukaryotes have a more com p licat ed internal structure including a defined, mem b rane-lim ited nu cleus, an d several d i stinct, membran e - delim ited organelles .

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[Lodish, 2000]

Tw o major divisions w i thin the cell.

Nuclear region (inform a tion storage and processing)

Cytoplasm (diverse functions, organelles)

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[from Lodish, 2000]

Som e definitions:

Plasma membrane : surrounds the outside of the cell, 7-8 nm thick, responsible for transport, receptors (signal trans duction) adherence, immune recognition.

Nuclear envelope : separates the nucleus and the cy toplasm; a double membrane with nuclear pores (channels for conducti ng materials between the nucleus and the cytoplasm)

Nucleus . Contains DNA associated with proteins called histones and non-hi stone chrom o somal proteins.

Histones - small positively charged proteins mainly structural, that pack DNA into chromatin fibers.

Nonhistone proteins - invol ved in DNA replication, transcription regulat ion of gene activity

Chromosome : each individual linear DNA m o lecule with its associated proteins;

23 pairs of chrom o somes in humans (ranges from 1 to 50) the same in all cells of an organism

only vi sibl e during mitosis

in hum ans each chrom o some cont ains about a m e ter of DNA

Chromatin : DNA and its associated proteins; the collection of chromosomes at any phase of the cell cycle.

Gene : the inform ation encoded in the DNA sequence necessary to make a protein

Genome : all of the DNA, or h e reditary material, in both functional and nonfunctional sequences in an organism .

Cytoplasm : the region of the cell lying outsi de the nucleus.

Ribosomes : site of translation (protein sy nthesis); may be free in cytoplasm or attached to endoplasm ic reticulum.

Rough endoplasmic reticulum : ER with attached ribosom es; proteins form ed here go into ER to be further pro cessed and/or dist ributed to vesicles for transport to other parts of the cell.

Smooth ER : no ribosomes attached; functions include breaking down fats and synthe sis of lipi d s.

Golgi apparatus : di rect mem b rane constit uent s to appropriate locations within the cell; function in the form ation of storage vesicles or secretory vesicles.

Lysosomes : organelles in animal cells that containing digestive enzymes.

Mitochondria : carry out the cell’s energy m e tabolism is carried out.

Mitochondria contain DNA. Function?

Peroxisomes : fatty acids and am i no acids are deg r aded

fibers:

Cytoskeleton : supportive network in the cytoso l com posed of three types of

 Microtubles : fibers built of polymers of tubulin; responsible for cell m ovement,

 Microfilaments , built of the protein actin

 intermediate filaments .

The cytoskeleton gives the cell strength and rigi dity control m ovem e nt of structures within the cell, provi de tracks along which organelles m ove.

[Lodish, 2000]

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Biomol ecules :

Cells carry out a mult itude of chemical transform a tions, which provi de energy, and the m o lecules needed to form its stru cture and coordinate its activities.

Small molecules : Water, inorganic ions, and a small array of small organic mol ecules (e.g., sugars, vita m i ns, fatty acids), com p rise 75-80% of living matter by weight. These ar e i m ported into cells . Cells can also ma ke or transform many small mol ecules as p a rt of the cells m e tabolic activity.

Macromo lecules : proteins, polysaccharid es, phosph olipids and DNA. These ar e

made wit h in cells .

 Carbohydrates : carbon hydrogen and oxygen (ratio 1:2:1); sugars (m onosaccharides) usually 3-7 carbons, straight chains or rings, found in starches, cellulose, glycoprotei ns

 Lipids : di verse group of com p ounds that dissolve m o re readily in nonpolar solvents than in water

Fatty acids

Saturated Unsaturated Polyunsaturated

Neutral lipids Phospholi p ids

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 Proteins: the w o rkhorses of the cell.

Proteins are the m o st abundant and f unctionally versatile of the cellular macromol ecules.

functions include structure, enzymes, m o bility.

Proteins are formed from 20 different m onomers, the amino acids . Linkages are through pe ptide bonds.

Amino acids -subuni t s of protei ns

 20 differe nt am ino acids

 all but one have the same basic stru cture (central carbon, to which is attached a carboxyl group and an am ino group)

 the exception is proli n e (ring st ructure)

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[Lehninger, 1975]

Structure defines function .

Primary s t ructure: the linear sequence of am ino acids -pepti de bond- condensation

Secondary structure : the peptide bond is relatively rigid, relatively little rotation so lim ited num ber of stable arrangements.

Tertiary structure - 3-dim e nsional arrangem e nt; represents minim a l energy state; usually sensitive to tem p erature, pH, salt concentration, disulfide bonds help to maintain conform a tion.

Quaternary structure - associations of multiple proteins. E.g., hem oglobin or antibodies are made of 4 separate protein subunits.

Proteins can co m b ine with other types of m o lecules (glycoproteins, lipoproteins).

En zym e s are proteins that carr y out chemi cal reactions invol ving sm all m o lecules by acting as catalysts . Tertiary structure brings reactants together, lower s free energy barrier, and accelerat e s reac tions by many orders of magnitude.

Inorganic ions may be required as cofactors for enzymatic act ivity (e.g., zinc, copper, iron). “All (alm ost) enzymes ar e proteins, but not all proteins are enzy mes . ”

DNA: th e cell’s mast er molecule.

Deoxyribonucleic acid or DNA is the macro m olecule that garners the m o st public attention.

As we explore the interaction of radia tion with biological material, DNA will assume central impo rtance.

The structure of DNA was proposed in 1 953 by Ja mes Watson and Francis Crick. DNA consists of two long helical st rands that are wound together in a double helix .

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[Lodish, 2000]

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DNA st ructure

Nuclei c acids (DNA and RNA) are linear polymer s co mposed of mono mers called

nucleotides .

All nucleotides have a common structure: a phosphate group is linked by a phosphodiester bond to a pentose sugar (ribose in RNA and deoxyri b ose in DNA)

The base com ponents of the nucleic acids are heterocyclic ring structure s called

purines or pyrimidines .

Purines: adenine, guanine Pyrim i dines: cytosine, thy m ine, uracil

The charge on the phosphate b ackbone gives nucleic acids a net negative charge . Most nucleic acids i n cells ar e associated w i th proteins .

Native DNA is a double helix of co mplementary antiparallel ch ains.

The two sugar-phosphate backbones are on the out side. Th e bases are on the inside connected by hydrogen bonds. Ba se-pair complementarity defines the base bondi ng:

A-T

C-G

Also known as Watson-Crick base pairs .

Each strand of DNA is com posed of 4 di fferent m onomers called nucleotides.

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Note that adenosine and guanosin e are both purines, and thym idine and cy tidin e are pyrim i dines. A pairs with T, and G pairs with C.

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DNA is t h e storage form of genetic information .

DNA contains information about which pr oteins are to be made and when.

Proteins re ad the information in the DNA for use by the cell an d translate it into a transportable unit called messenger RNA ( m RNA).

Other prot eins read the mRNA sequen ce and assemb le the ami no acids into proteins.

This is the central dogma of biology: the coded ge netic information hard-wired into DNA is transcribed into transporta ble cassettes , co mposed of mRNA, each mRNA cassette contains the inform ation for synthesis of a particular protein.

DNA → mRNA → protein

Gene exp ression is the process of translating the inform ation in the DNA into functional proteins.

Higher order stru cture: organi z ing DNA into chromosomes.

The total length of the DNA in a cell can be up to 100,000 tim es the cells diam eter. Packaging the DNA is crucial to the cell architecture and function. The longest DNA m o lecules in h u man chrom o somes (2-3 x 10 8 base pairs) are alm o st 10 cm long!!

 Eukaryotic nuclear DNA associates with histone proteins to form chromatin .

Chromatin consists of an approximately equal m a ss of DNA and protein in a highl y com p act structure known as chromatin . The general structure of chromatin is sim ilar in all eukaryotic cells.

The m o st abundant of the DNA-a ssociated proteins are called histones . Histones are a family of basic (posi tively charged) proteins present in all eukaryoti c cell nuclei. H1, H2A, H2B, H3 an d H4

Histone sequences are highly conserved am ong species.

 Chromatin exists in exte nded and condensed forms .

Chromatin appearance depends on the cond itions (primarily the salt concentration or ionic strength) use d in the solutions to isolate it from th e cell.

At low salt concentration chromatin ap p ears like “ b eads on a string” or in an

extended form .

The beadlike structures ar e call ed nucleosomes. Nu cleoso mes are co mp osed of DNA and histones, are about 10 nm in diameter an d are the basic structural unit of chrom a tin.

At high salt concentration, isol ated chro matin assu mes a mor e condensed fiberlike form that is 30 nm in diameter .

Structure of nucleosomes;

 A protein core with DNA wou nd on its surface like thread on a spool.

 Nucleosomes contain146 base pairs wra pped slightl y less than two turns around the protein core.

 The DNA of nucleosomes is more pr otected from en zy mes ( a nd radiation?) than the linker DNA between n u cleosomes.

Structure of condensed chromatin:

 When extract ed from cells in is otonic buffers, most chromatin appears as fibers, 30 nm in diameter.

 Nucleosomes are thought to be packed into a spiral or solenoid arrangem ent , with six nucleosomes per turn.

Modification of the histones (acetylation) can affect the binding and the assembly into conde nsed chromatin.

Condensed chromatin structure m a y be dyna m i c, with sections partially unfol ding and then refolding.

Chromatin in regions not being transcri bed exists in the condensed form , and possibly higher order structures built from it.

Chromatin in regions actively being transc ribed is thought to exist in the extended “beads on a string” form .

Eukaryoti c chrom o somes cont ain one linear DNA mol ecule.

It is diffic u lt to isola t e high m o lecular weight DNA without breaking it.

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[Lodish, 2000]

Chromatin is further organ ized into chromosomes

Chromatin is further organized into larg e units hundreds of kil obases in length called chromosomes .

Chromosome nu mber, size and shape at m e taphase (the k a ryotype ) are s p ecies specific.

In non-dividing cells the ch romosomes are not visible .

During mitosis (and meiosis) the chrom o somes condense and becom e visible. Alm o st all cytogenetic work has been done on metaphase cells.

The condensation results from several orders of foldi ng and coiling of the 30 nm chromatin fibers.

At m itosis, cells have progressed thr ough S phase and duplicated the DNA. Metaphas e chromoso mes are com posed of two sister chromatids .

Non-hist one proteins provi de a sc affold for long chrom a tin loops.

Megabase-long l oops of 30 nm DNA fibers are thought to associate with the flexible chomosome scaffold , yielding an extended form characteristic of chrom o somes during interpha se.

Coiling of the scaffold into a helix and fu rther packing of thi s helical structure produces the highly condensed structure characteristic of metaphase chromosomes .

Genes are located primarily wit h in chromatin loops . Scaffold associated regions

serve as the att achment points of the loop t o the protein scaffold.

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The Cell Cycl e

 New cells arise when one cell divides or t w o cells (like sperm and egg) fuse.

 These events initiate a cell-replication program that is encoded in the DNA and executed by prot eins.

 A period of cell growth, and replication of DNA is then followed by cell divisi on.

 Cell growth and divi sion is hi ghly regulat ed in the body.

 Cancer occurs when a cell escapes fro m this regulat ion and growth is unchecked .

Most eukaryotic cells live acco rding to an internal cl ock, they proceed through a series of phases called the cell cycle .

 G 1 : the g ap between the end of m itosis and beginning of S phase

 S phase : DNA is duplicated: cells have a DNA content that progressivel y increases from 2n to 4n.

 G 2 : tim e between the end of S and the beginning of m itosis

 M - m itosis: Cell division: t h e two daught er cells receive all of the genetic inform ation of the parent cell.

 G 0 : quiescent cells, not actively cycling.

G1 and G2 are periods of apparent inactiv ity between the maj o r discernable ev ents in the cell cycle.

 Bacteria can replicat e their single chrom o some and divide in about 20 m i nutes.

 Most m a mmalian cells have a cell cycle time on the order of 10-12 hours.

 Som e cells (nerve cells, striated m u scle cells) do not divide at all. They have tem porarily exited from the cell cy cle and ent e red a quiescent state called G 0 .

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Checkpoints

“Proofreading before division”

Progress through the cell cy cl e is regulated at key check p oints along the way that m onitor the status of the cell.

Before entering m ito sis, the integr ity of the DNA is checked. Exposure t o radiation causes a block in G 2.

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Mitosis is the process for partitioning the genome equally duri n g cell division.

The mitotic apparatus captures the chrom o somes and pulls them to the opposite sides of the dividing cell.

 Prophase : the replicated chrom o somes (each containing two identical chrom a tids) are condensed and releas ed to the cytoplasm when the nuclear m e m b rane breaks down.

 Metaphase and anaphase: the chromosomes are so rted and moved to opposit e ends of the cell.

 Telophase : marks the end of mitosis as a mem b rane is reform ed around each set of chromoso mes.

 Cytok i nesis : division of the cytoplasm and separation of t h e two daughter cells.

[Lodish, 2000]

What’s cancer?

Cancer -uncontrol l ed cell growth

 Tumor - mass formed by growing cancer cells

 Malignant - destructive, invasive, can met a stasize

 Benign - slowly growing, does not invade surroundi ng tissues or m e tastasize, usually encapsulated .

 Metastasis - spread of the tum o r cells to ot her sites, where they establish secondary areas of growth.

 Angiogenesis - both prim ary and secondary tum o rs require new blood vessels to sustain growth.

E.g. ,

 Carcinom a - solid tum o r arising from epithelia l tissue such as skin, colon, lung, breast.

 Sarcoma - solid tum o r arising from connective tissue such as bone.

Cancer cel ls can mul tiply in t h e absence of norm a lly required growth factors and are resistant to signa ls that normally program cell d eath.

Malignant cells show m a ny differe nces from normal counterparts.

 Less well differentiated

 More rapid growth

 Loss of normal attachments to ne ighbors, loss of contact inhibition

 Growth factor independent cell growth

 Genetically unstable (phenot ype and genotype can change with time)

 Disruptions to cytoskeleton

 Higher m e tabolic rate

 Changes i n cytoplasmi c ion co ncentrations

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G e ne s inv o l ve d in c a nc er

Seven types of proteins participat e in the control of cell growth.

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Oncogenes - cancer causing genes

Many oncogenes are altered fo rm s of normal cellular ge nes called proto-oncogenes Gain-of-function m u tations convert proto-oncogenes into oncogenes.

Tumor su ppressor genes - genes which code for cell cycle control proteins

 Loss-of-function m u tations in t u m o r suppressor genes are oncogenic.

 Usually act recessively, both copies m u st be deleted or m u tated to lose the norm a l cell growth suppressi on effect.

Multi-step progression of cancer

Develop m ent of cancer re quires several mutations

Consistent with the o b servation of in creas ed incidence as a function of age

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 Cancer: a clone from a single cell?

 Colorectal car cinoma well studied with biopsy m a terial and genetic analysis at all stages of developm ent.

o Visible on endoscopy

o Biopsy material avail a ble

o Analyze genetic m u tations

Carcin o gens - agents that can caus e cancer

Viruses - can introduce oncogenes, or suppre ss genes that inhi bit cell growth

 Retroviruses - RNA viruses, integrate into the genome of the i n fected cel l as a DNA copy and can carry oncogene s derived from cellular proto- oncogenes.

 DNA tumor viruses - contain oncogenes of purely viral origi n

Chemi c als - usually thought to act as carcinogens by causing DNA damage.

 Som e of the m o st potent are alkylating agents that are capabl e of adding organic groups to D NA.

 Others, (polycyclic hydrocarbons) b ecome carcinogenic when they are biochem i cally m odified in cells, ofte n as the cell attem p ts deto xification

 Phorbol esters- do not cause DNA damage, but prom ote growth (may only work when DNA damage also occurs from another agent)

Radiation - also thought to act by causing DNA danage

 UV - is absorbed directly by DNA, causi ng base ch anges, in s unlight can lead to skin cancer.

 Ioniz i ng radiation i s actually a relatively weak carcinogen.

Apoptosis - now recognized that cancer is due not only to uncontrol led cell growth, but also to loss of control i n regulated cell growth.

In an adult, normal ti ssues are i n homeost asis- no net increase i n cell nu mbers because of a bal a nce between cell divisi on and cell death due to apoptosis.

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Apoptosi s vs. Necrosis

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