Ciclo Cellulare e Apoptosi

Programma del corso di
Ciclo Cellulare e Apoptosi
(aa 2005/2006)
Titolari: Prof.ssa Carla Caruso
Dott.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 cell
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Components
•
•
•
•
•
•
•
•
•
Cytoplasm
Ribosomes
Nuclear Zone
DNA
Plasmid
Cell Membrane
Cell Wall
Capsule (or slime layer)
Flagellum
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Eukaryotic cell
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Components
•
•
•
•
•
•
•
•
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!
Prokaryotic Cells
Eukaryotic cells
small cells (< 5 mm)
larger cells (> 10 mm)
always unicellular
no nucleus or any membranebound organelles
often multicellular
always have nucleus and other
membrane-bound organelles
DNA is circular, without proteins DNA is linear and associated with
proteins to form chromatin
ribosomes are small (70S)
ribosomes are large (80S)
no cytoskeleton
always has a cytoskeleton
cell division is by binary fission cell division is by mitosis or meiosis
reproduction is always asexual
reproduction is asexual or sexual
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Characteristic Features of Bacteria and
Archaea: 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
Archaea: 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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Features of archaea (=archaebacteria) based on
complete genome sequences
EUBACTERIA-LIKE
Small
Cell wall
No nucleus
No internal membranes or organelles
No eukaryotic cytoskeletal elements
Cell division by splitting
Many transporters for ions and small molecules
EUKARYOTE-LIKE
Machinery for DNA replication, RNA transcription and protein
translation
Ribosomal proteins
Five histone genes are like those in eukaryotic chromatin
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Archaea and Eukarya
share a more recent
common ancestor
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The Origin of Mitochondria
and Chloroplasts
• The 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
and Chloroplasts
The endosymbiotic theory
Larger anaerobic eukaryotes engulfed
aerobic
prokaryotes,
which
became
endosymbionts that enabled the host cell to
become aerobic
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THE ENDOSYMBIOTIC THEORY
Reduced carbon
compounds
Reduced carbon
compounds
+ O2
Electron transport
chain
Fermentation
High ATP
yield
Low ATP
yield
Aerobic bacterium
Anaerobic eukaryote
1. Eukaryotic cell
surrounds and
engulfs bacterium.
2. Bacterium lives
within eukaryote
cell.
Pyruvate
and O2
ATP
3. Eukaryote supplies
bacterium with
reduced carbon
compounds;
bacterium supplies
eukaryote with ATP.
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Evidence for endosymbiotic origin of
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?
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Origin of Nucleus and Endoplasmic Reticulum
As the cell evolved toward larger size, the endoplasmic reticulum likely
evolved as an adaptation to increase surface area.
The nucleus may have originated as a specialization of a portion of the
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internal membrane. Note this would have generated a double membrane
Endosymbiosis likely
happened while the
oxygen was “poisoning”
anaerobic archaeaeukarya ancestor.
Aerobic respiration
and photosynthesis
evolved once in
bacteria and were
then imported into the
eukarya lineage
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Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
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 cellulare
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STEPS 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 Division
 DNA replication produces two copies of the
genome
 The cell grows to approximately double in size
 The two chromosomes separate as the cell
grows
 A new cell wall is formed between the two
chromosomes is a process called binary fission
 Under optimal conditions, the entire process
can occur in 20 minutes
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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?
Divide into two smaller cells
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When is cell division occurring?
GROWTH -increase number of cells
REPAIR -replace lost cells due to injury, disease
CANCER – Abnormally high rates of cell division
due to mutation
Different kinds of cells divide at different rates:
Yeast cell – 2 hours
Amoeba – a few days
Human embryo cell – 15-20 minutes
Human adult cell – 8 hours to 100 days
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Aging
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:
3 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 movie
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Cell 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 Cycle
G1: Highly variable,
Absent in rapidly
dividing cells, long in
slow-growing cells
S: 6-8 hours
G2: 3-6 hours
M: 1-2 hours
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Determinazione della
durata delle fasi del ciclo
cellulare
Marcatura per brevi
periodi con 3H-timidina
Osservazione delle cellule
mitotiche marcate
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Mitotic Cell Division
2 major processes:
• mitosis – nuclear division
=> preserves diploid number of
chromosomes
• cytokinesis – cytoplasmic division
=> cell divides into two daughter cells
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Mitosis
4 phases:
1st – Prophase (3 major events)
2nd – Metaphase
3rd – Anaphase
4th – Telophase and Cytokinesis
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1. Prophase
• 3 major events
i) chromosomes condense
ii) spindle fibers form
iii) nuclear membrane breaks down
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Mitotic 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 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. Anaphase
• spindle fibers attached to kinetochores shorten
and pull chromatids poleward
• free spindle fibers lengthen and push
poles of cell apart
Anaphase
A
Anaphase
B
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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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 Cytokinesis
 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 overview
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Mitosis: an overview
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