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Programma del corso di

Ciclo Cellulare e ApoptosiCiclo Cellulare e Apoptosi

(aa 2005/2006)

Titolari: Prof.ssa Carla CarusoDott.ssa Maria Saveria Gilardini Montani

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Il Ciclo Cellulare: Fasi, Controllo e Regolazione. Strategia generale e fasi del ciclo cellulare. Sistemi sperimentali per lo studio del ciclo cellulare: uova

di anfibi e lieviti. Studio sui mutanti cdc e wee in S. pombe. Regolazione di

MPF mediante fosforilazione e defosforilazione. Studio sui mutanti cdc in S. cerevisiae. Cicline G1 e SPF. Cdks e cicline nel ciclo cellulare dei mammiferi. Punti di controllo del ciclo e ruolo del punto di restrizione. Ruolo di Rb nella regolazione del ciclo cellulare. DNA danneggiato da UV e ruolo di p53. Oncogeni e oncoproteine.

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Le basi genetiche del cancro. Cancerogeni, mutageni, virus tumorali.

Tipi di morte cellulare: apoptosi e necrosi. Caratteristiche e significato biologico

I recettori di morte e i ligandi La via delle caspasi Il ruolo dei mitocondri nei processi apoptotici Il sistema Fas/FasL La regolazione dell’apoptosi: le proteine della

famiglia di Bcl-2; IAP e FLIP L’apoptosi caspasi-indipendente Tecniche di laboratorio per lo studio dell’apoptosi

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LIBRI DI TESTO CONSIGLIATI (X Ciclo Cellulare)

Murray A & Hunt T, The cell cycle, an introduction, Oxford University Press, New York.

Alberts B, Johnson A, Lewis J, Raff M, Roberts K & Walter P, Biologia Molecolare della Cellula, Zanichelli, 2004 (IV Edizione)

Lodish H., Berk A., Zipursky S.L., Matsudaira P., Baltimore D., Darnell J., Biologia Molecolare della Cellula, Zanichelli, 2002 (II edizione)

Lewin B, Il gene VI, Zanichelli, Bologna, 1999.

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Prokaryotic cellProkaryotic cell

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ComponentsComponents• Cytoplasm• Ribosomes• Nuclear Zone• DNA• Plasmid• Cell Membrane• Cell Wall• Capsule (or slime layer)• Flagellum

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Eukaryotic cellEukaryotic cell

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ComponentsComponents

• Cytoplasm• Nucleus• Mitochondria• Chloroplast• Ribosomes• RER• Golgi body• Vacuoles

• Lysosomes• Cytoskeleton• Centriole• Cilium and Flagellum• Microvilli• Cell membrane• Cell Wall

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Summary of differences!Summary of differences!

reproduction is asexual or sexualreproduction is always asexual

cell division is by mitosis or meiosiscell division is by binary fission

always has a cytoskeletonno cytoskeleton

ribosomes are large (80S)ribosomes are small (70S)

DNA is linear and associated with proteins to form chromatin

DNA is circular, without proteins

always have nucleus and other membrane-bound organelles

no nucleus or any membrane-bound organelles

often multicellularalways unicellularlarger cells (> 10 mm)small cells (< 5 mm)

Eukaryotic cellsProkaryotic Cells

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Characteristic Features of Bacteria and Characteristic Features of Bacteria and Archaea: Comparison to EukaryotesArchaea: Comparison to Eukaryotes

• Bacteria and Archaea lack membrane-bound nuclei and organelles, and have a single circular chromosome.

• Archaea and Eukaryotes have multiple complex RNA polymerases and begin translation with methionine; bacteria begin translation with formyl-methionine.

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Characteristic Features of Bacteria and Characteristic Features of Bacteria and Archaea: Comparison to EukaryotesArchaea: Comparison to Eukaryotes

• Unique Features of Archaea:– Their cell walls are vary in structure, but

always lack the peptidoglycan of bacterial cell walls.

– The lipids in their membranes are branched and have an ether linkage to glycerol.

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EUBACTERIA-LIKESmallCell wallNo nucleusNo internal membranes or organellesNo eukaryotic cytoskeletal elementsCell division by splittingMany transporters for ions and small molecules

EUKARYOTE-LIKEMachinery for DNA replication, RNA transcription and protein translationRibosomal proteinsFive histone genes are like those in eukaryotic chromatin

Features of archaea (=archaebacteria) based on Features of archaea (=archaebacteria) based on complete genome sequencescomplete genome sequences

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Archaea and Eukarya Archaea and Eukarya share a more recent share a more recent common ancestorcommon ancestor

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The Origin of Mitochondria The Origin of Mitochondria and Chloroplastsand Chloroplasts

• The endosymbiotic theoryThe endosymbiotic theory

– Evidence that supports the theory of endosymbiosis:• Physical similarities exist between

mitochondria, chloroplasts and prokaryotes• Molecular data indicates mitochondria and

chloroplasts are of prokaryotic origin

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Same size and shape as bacteria Double membrane 70 S Ribosomes Circular chromosomes Replicate on their own

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The Origin of Mitochondria The Origin of Mitochondria and Chloroplastsand Chloroplasts

The endosymbiotic theoryThe endosymbiotic theoryLarger anaerobic eukaryotes engulfed aerobic prokaryotes, which became endosymbionts that enabled the host cell to become aerobic

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THE ENDOSYMBIOTIC THEORYTHE ENDOSYMBIOTIC THEORY

Reduced carboncompounds + O2

High ATPyield

Electron transport

Reduced carboncompounds

Low ATPyield

Fermentation

Aerobic bacterium

Anaerobic eukaryote

Pyruvateand O2

ATP

chain

1. Eukaryotic cellsurrounds andengulfs bacterium.

2. Bacterium liveswithin eukaryotecell.

3. Eukaryote suppliesbacterium withreduced carboncompounds;bacterium supplieseukaryote with ATP.

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Evidence for endosymbiotic origin of Evidence for endosymbiotic origin of mitochondria and chloroplasts is very strong mitochondria and chloroplasts is very strong

• Organelles and bacteria have similar size and structure.• Mitochondria and chloroplasts replicate by binary fission. • Mitochondria and chloroplasts have their own DNA

(circular like bacteria).• They have their own transfer RNA and ribosomes and

produce some of their own enzymes. • The ribosomes are structurally like bacteria • Analysis of rRNA gene sequence identifies the gene as in

the bacterial clade.

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Where did the Features of Eukaryote cell come from?Where did the Features of Eukaryote cell come from?

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Origin of Nucleus and Endoplasmic ReticulumOrigin of Nucleus and Endoplasmic Reticulum

The nucleus may have originated as a specialization of a portion of the internal membrane. Note this would have generated a double membraneNote this would have generated a double membrane

As the cell evolved toward larger size, the endoplasmic reticulum likely evolved as an adaptation to increase surface area.

24Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Endosymbiosis likely happened while the oxygen was “poisoning” anaerobic archaea-eukarya ancestor. Aerobic respiration and photosynthesis evolved once in bacteria and were then imported into the eukarya lineage

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The three domains seem to have genomes that are chimeric mixes of DNA that was transferred across the boundaries of the domains

This has lead some researchers to suggest replacing the classical tree with a web-like phylogeny

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La divisione cellulareLa divisione cellulare

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STEPS IN BACTERIAL CELL DIVISIONSTEPS IN BACTERIAL CELL DIVISION

1. chromosome attaches to one point on plasma membrane

2. chromosome is replicated: replicated chromosome attached to plasma membrane at a different nearby point

3. cell elongates – new plasma membrane is added between chromosomes, pushing them towards opposite ends of cell

4. plasma membrane grows inward at middle of cell

5. parent cell is divided into two identical daughter cells

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Bacterial Cell DivisionBacterial Cell Division DNA replication produces two copies of the DNA replication produces two copies of the

genomegenome The cell grows to approximately double in sizeThe cell grows to approximately double in size The two chromosomes separate as the cell The two chromosomes separate as the cell

growsgrows A new cell wall is formed between the two A new cell wall is formed between the two

chromosomes is a process called binary fissionchromosomes is a process called binary fission Under optimal conditions, the entire process Under optimal conditions, the entire process

can occur in 20 minutes can occur in 20 minutes

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Why does a cell divide?Why does a cell divide?

-As a cell absorbs nutrients and gets larger, the volume of the cell increases faster than the surface area.

-Therefore, the demands of the cell (the volume) exceed the ability of the cell to bring in nutrients and export wastes.

Solution?Solution? Divide into two smaller cells Divide into two smaller cells

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When is cell division occurring?When is cell division occurring?

Different kinds of cells divide at different rates:Yeast cell – 2 hoursAmoeba – a few daysHuman embryo cell – 15-20 minutesHuman adult cell – 8 hours to 100 days

GROWTH -increase number of cellsREPAIR -replace lost cells due to injury, diseaseCANCER – Abnormally high rates of cell division due to mutation

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AgingAging

All cells die after a certain number of divisions (programmed cell death). At any given time some cells are dividing and some cells are dying.

Childhood Cell division > cell death

Adulthood Cell division = cell death

Aging Cell division < cell death

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THE CELL CYCLE: THE CELL CYCLE: 3 phases: Interphase-Mitosis-Cytokinesis3 phases: Interphase-Mitosis-Cytokinesis

Interphase- 90% of the time

G1: Little new cell absorbs nutrients and grows larger (protein synthesis).

S phase: Synthesis of new DNA (DNA replication) for daughter cells in preparation for mitosis.

G2: Cell continues to grow …… gets too large, needs to divide.

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Cell cycle movieCell cycle movie

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Cell cycle schemeCell cycle scheme

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How long is one cell cycle? Depends. Eg. Skin cells every 24 hours. Some bacteria every 1-2 hours. Some cells every 3 months. Nerve cells, never. Cancer cells very short.

Programmed cell death: Each cell type will only do so many cell cycles then die. (Apoptosis)

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Mammalian Cell CycleG1:G1: Highly variable,

Absent in rapidly dividing cells, long in slow-growing cells

S:S: 6-8 hours G2:G2: 3-6 hours

M:M: 1-2 hours

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Determinazione della Determinazione della durata delle fasi del ciclo durata delle fasi del ciclo

cellularecellulare

Marcatura per brevi periodi con 3H-timidina

Osservazione delle cellule mitotiche marcate

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Mitotic Cell DivisionMitotic Cell Division2 major processes:

• mitosis – nuclear division => preserves diploid number of chromosomes

• cytokinesis – cytoplasmic division => cell divides into two daughter cells

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MitosisMitosis4 phases:

1st – Prophase (3 major events)

2nd – Metaphase

3rd – Anaphase

4th – Telophase and Cytokinesis

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1. Prophase1. Prophase

i) chromosomes condense

• 3 major events

ii) spindle fibers form

iii) nuclear membrane breaks down

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Mitotic Spindle FormsMitotic Spindle Forms

• spindle fibers are specialized microtubules• spindle fibers radiate out from centrioles,

forming the “aster”• centrioles occur in pairs, and are duplicated

during interphase• one pair of centrioles migrates to one pole of

cell, the other pair migrates to opposite pole of cell

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Spindle Captures ChromosomesSpindle Captures Chromosomes

1. When spindle fibers are fully formed nuclear envelope disintegrates and nucleolus disappears

2. Spindle fibers attach to chromosomes at the kinetochore, a structure located at the centromere

3.Other spindle fibers do NOT attach to chromosomes, but retain free ends that overlap at cell’s equator => “free spindle fibers”

4. Function of spindle fibers is to organize division of sister chromatids into daughter cells

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• Prophase – Inside nucleus

• Chromosomes condense• Nucleoli begin to break down and disappear

– Outside nucleus• Centrosomes move apart and migrate to opposite ends

of the cell• Interphase microtubules disappear and are replaced by

microtubules that grow from the MTOC

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• Prometaphase– Nuclear envelope breaks down– Microtubules invade nuclear area– Chromosomes attach to microtubules through

kinetochore– Mitotic spindle includes other microtubules that

are involved in the process

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•Metaphase – Chromosomes move towards imaginary equator called

metaphase plate– This occurs with opposite forces of the microtubules:

pulling, pushing and sliding– Note, no nuclear membrane, the chemical changes keep

the membrane bits from reforming the nuclear membrane

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• Anaphase– Separation of sister chromatids allows each

chromatid to be pulled towards spindle pole connected to by kinetochore microtubule

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3. Anaphase3. Anaphase

• spindle fibers attached to kinetochores shorten and pull chromatids poleward

• free spindle fibers lengthen and push poles of cell apart

AnaphaseAnaphase AA

AnaphaseAnaphase BB

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• Telophase– Spindle microtubules disassemble– Nuclear envelope forms around group of

chromosomes at each pole– Nucleoli reappear– Chromosomes decondense

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CytokinesisCytokinesis Cytokinesis occurs, enclosing each daughter nucleus

into a separate cell Starts during anaphase and ends in telophase Animal cells: contractile ring pinches cells into two

halves Plant cells: cell plate forms dividing cell into two

halves

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Animal cells: – microfilaments attached to plasma membrane

form a ring around equator of cell – ring contracts, like a drawstring, dividing the

cytoplasm

Plant cells:- stiff cell wall makes pinching impossible- Golgi complex buds off vesicles filled with

carbohydrate- vesicles line up at equator and fuse, producing a

structure called the cell plate- cell plate becomes new cell wall between the two

cells

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Mitosis: an overviewMitosis: an overview

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Mitosis: an overviewMitosis: an overview