8. cell cycle (apoptosis)

7/23/2019 8. Cell Cycle (Apoptosis)

http://slidepdf.com/reader/full/8-cell-cycle-apoptosis 1/11

BIO3153 – Cell Biology – Caroline Petit-Turcotte

Cell Cycle (Apoptosis) – Lecture 9Shivan Desai

Apoptosis

- “Programmed cell death”- Number of cells is, in part, controlled by regulation

of cell deatho 1) f the cell isn!t intact one does not "ant

the cell to divide and #eep these mutations

and #eep the process goingo $) %o avoid an in&ammatory response caused

by the cell that e'ploded and their content spread ha(ardly tothe neighbouring cells

o ) *voids +ess

- Diers from necrosis trauma or cytoto'icity leading to ↓*%P and

↓Na./0 .-*%Pase activity, follo"ed by lysis

- Necrosis is triggered by a trauma, something unplanned during the

cells life spano %he sodium potassium pump does not "or# properly, no ion

e'change, membrane becomes permeable to everything, the cell

s"ells and lysis, active en(ymes then are in the vicinity of the

healthy cells- t is a very clean process

o t gets rid of the cells "ithout impacting the neighbouring cells

o o"ever, some neighbouring cells participate in ta#ing up some

of the debris, gain more space and can

divide faster- mportance in development- 2alance of cell division "ith death in adult tissues- Some cells are selectively programmed to die at a

particular moment during its life time to let the

organism gro" and developo %here is a timing and a trigger component

o 3or an organisms development cells are programmed to die and

be born at a certain time- *poptosis is important to regulate ho" many cells "ill live and ho"

many "ill die and "hen they "ill die or live



- *poptosis can also occur because of a situation not related to

development such as a mutation- n order for apoptosis to occur, a cell must undergo signi4cant

biochemical and morphological change- nitiation by intracellular or e'tracellular signal

o Stress, Damage, ormones, %o'ins, ypo'ia, 5'cessiveintracellular 6a$., insu7cient nutrients, etc8

- *ctivation of a series of proteins involved in promoting apoptosis 9or

inhibition of those that prevent it)o During both path"ays there is a series of biochemical events

that leads to the cell dyingo %he cell al"ays begins "ith these biochemical path"ays as a

disposable cell "hich has no impact on the neighbouring tissues

and/or cells- mportant intracellular proteins necessary for survival are cleaved

during apoptosis- :rderly disposal of dead cell

!tructural C"anges #uring Apoptosis

- 2egin by having condensation of chromatin in the nucleus and the

cytoplasm "ill shrin#- Nucleus becomes fragmented and DN* is cut into smaller portions

9called “DN* ;addering”)o %his is a very important hallmar# of apoptosis

o 2ecause as DN* becomes fragmented the ends of these

fragments can be used to determine ho" DN* is fragmented and

can be used to detect it and to con4rm that it is a apoptosis

process- %hen the cells under goes this process and encapsulating "ith the

portions of the membrane 9portions of the cell), called blebbingo 2lebbing consists of little vesicles that contain part of DN* and

organelles "rapped in membraneo %hese blebs are called *poptotic bodies and go through

phagocytosiso Neighbouring cells absorbs these blebs and get rid of them

o %herefore, there is no free debris &ying around such as proteins

or en(ymes



P"agocytosis

- *symmetric distribution of plasma membrane is lost- Negatively charged phosphatidylserine then becomes e'posed on the

outside of cell- %he cell is then mar#ed for phagocytosis by a macrophage- %he membrane of the bleb 9apoptotic body) is made up of t"o bilipid

layers, ho"ever, they are not organi(ed the same- %he negatively charged polar heads are directed

to"ards the inside of the cell membrane 9cytosol)o <hen the apoptotic body is formed these

s"itch and the negatively charged polar

heads are phosphatidylserine molecules and

become e'posed to the e'tracellular

environment/ layer <hich triggers the reaction and the

little vesicles are detected and get rid

of by phagocytosis through other cells

Cell $eat" %utants

- 6aenorhabditis elegans is a nematode

- =>= somatic cells- 11 apoptose, al"ays the same- Discovery of ced- gene that encodes proteins

similar to mammalian protease “caspases”- ced- encodes 65/6aspase 1? ced-@ encodes

*paf-1? ced-= encodes 2cl-$- During the 1ABBs, a group started to study

eleganso 5asy accessible genetic material

o Cuic# to get a ne" generation 9ta#es days

to get a ne" nematode "ith a dierent

function after)o Nematodes have =>= somatic cells

o <hen the nematode is developing and dierentiating 11 cells

go through apoptosis and die8 *l"ays the sameo +echanism "hich decides "hat cell "ill die

Discovered the ced--gene that encodes proteins similar to

mammalian protease



%he group mutated the gene and none of the 11 cells died

in the nematode %here are more of these genes such as ced- and ced @

etc

Caspases

- denti4cation of ced- gene led to the discovery of a homologous

family of proteins, called “caspases”, in mammals- %hey are proteases that cleave essential proteins- nvolved in most changes observed during cell death- %his en(yme has a cysteine residue at its catalytic site and cleaves

other proteins at an aspartate site- 6aspases may also cleave each other, leading to their activation- $ main classes of caspases nitiator 96*SP$,A,=,1B) and 5'ecutioner

96*SP,,E) caspases

- %he group #ept studying the genes and discovered that they are afamily of proteins called caspases

o %hey are en(ymes, and they have a cysteine residue at the

catalytic site and they cleave other proteins at their aspartatic /

ate siteo %here are t"o main classes of caspases

nitiator caspases

5'ecutioner caspases

- %hey are en(ymes that cleave by proteolysis and they themselves are

activated by being cleaved

- Some caspases have the Fob of activating other caspases- %he machinery of the cell them puts together everything to ma#e sure

the cells go through the apoptosis process- 6aspases cleave essential proteins

o 1) 6aspases being proteiosis can cleave of other caspases and

proteins in the cell %hey can cleave certain #inases, for e'ample 3*0 9focal

adhesion #inase), these cleaved and non-active #inases

cannot function and perform their Fob 3a# allo"s the cell to stay adhered/ attached

o $) 6aspases can also cleave ;amins, "hich can no longermaintain the cell structure of the nucleus and the nuclear lamina

brea#s do"n and nucleus shrin#s %his occurs "hen the DN* is being fragmented and

chopped into smaller pieces



6aspases undergo 6*D 9caspase activated DN*ase)

cleavage "hich is the only 6*D that grams the caspases

added activity and activates 6*Do ) 6aspases change cell shape, structure and si(e, cytos#eleton

%hey need to brea# the strong net"or# made up of

4laments, membrane to ma#e vesicles

Procaspase Acti&ation

- 6aspases are 4rst produced as procaspases

and are inactive- %hese are activated by proteolytic interactions

9cleavage) "ith other active caspases- %hey have a protein "hich #eeps them inactive

and that portion needs to be cleaved o by a

proteolytic cleavage by another active caspase- :nce they are active they can perform their

Fob in the cascade depending on "hichcaspase is activated

T"e Caspase Casca#e

- *n active caspase activates another caspase- rreversible, “all-or-none”- *mpli4cation- Gesult is intracellular proteins are cleaved- %his is a domino eect because one caspase

activates another and the process continues "hich

is irreversible once it has begun- *mpli4ed process is "here one signal can activate

many caspases and begin to "or# to"ards apoptosis

for the cell- 6aspases are responsible for the events that govern

apoptosis and "hat ampli4es the signals in the cells



' %aor Apoptotic Pat"ays

- 1) 5'trinsic path"ayo Procaspase activation triggered from outside the cell

o “6ell death” receptors

- $) ntrinsic path"ay

o Procaspase activation triggered from inside the cello e8g8 intracellular damage

o e8g8 via 2cl-$ family of proteins 9note this may involve an

e'tracellular survival factor)o Something on the inside of the cell triggers apoptosis and may or

may not involve survival factorso Survival factors are outside the cell and bind the receptors

o"ever, in the absence of survival factors it is the inside

of the cell that responses to the damage/ trigger

*+trinsic Pat"ay- 5'tracellular signal 93as ligand) activates death receptor 93as protein)- Gecruitment of adaptor proteins and procaspase activation- 6aspase cascade- %he signal is called the 3as ;igand

o 3as is a protein "hich is e'pressed on the surface of the cell and

this is mostly used in immunologyo 3as binds to death receptor on the cell surface

%he binding triggers a conformation change "ithin the cell

o Sub units of another protein called 3*DD "hich recruits the

procaspase and adaptor proteins "hich produces a DS6 ;eads to the cleavage of initaitor caspase "hich renders it

activeo %hese caspases activate the e'trinsic caspases

o 6ell adherence decreases, and the e'ecutioner caspases are

going to cleave the tyrosine #inases that are responsible for cell

adhesion, lamins, 6*D and "ill easily access the nucleus because

cytos#eleton is "ea#- 5'ecutioner caspases cleave everything



Intrinsic Pat"ay

- *n intracellular death signal initiates caspase

cascade and cell death- nitiated by e8g8 DN* damage or loss of survival

factor- 2cl-$ protein family

o 2cl-$ inhibits apoptosis

o 2a' and 2a# act on mitochondria and release

cytochrome c- 2alance of these determines fate 9i8e8 life or death) of cell

- %riggered from signal inside cell because of a damage to DN*, to'ins

etc8- *n intracellular death signal initiates caspase cascade and cell death- Need a family of proteins called 2cl-$ "hich is responsible to inhibiting

apoptosis and they are responsible for the ba' and ba# protein "hich

interact "ith the mitochondria and allo" it to release cytochrome co 6alcium is in mitochondria and the cytochrome c resides

bet"een the inner and outer membrane of mitochondriao 6ytochrome is released from mitochondria by punching a hole in

the outer layer of the mitochondria and the pore is triggered

from the mitochondria because the internal concentration of

calcium increases and so mitochondria punches a hole to #ic#

the cytochrome c- *poptotic signal triggers dephosphorylation of 2ad, "hich activates

2a' or 2a# to form aggregates in outer mitochondrial membrane, *ND

inactivates 2cl$- nduces cytochrome c release from intermembrane space- <hen bad becomes phosphorylated it becomes active and activates

2a' and 2a#o *lso inactivates 2cl-$ and #ic#s it out by activating ba' and ba#

o 6ytochrome c is then released out of the mitochondria to get the

cascade started



- 3ormation of “apoptosome”- 6aspase cascade

- 6ytochrome c Foins *paf1 9apoptotic protease activating factor) "hichis triggered by P> and *paf1 is readily available to greet cytochrome c

o %his forms a dierent compound called 6*GD H caspase

recruitment domaino 6*GD "ill be able to interact "ith the caspases and recruits them

o %herefore, cytochrome . *paf1 I 6*GD comple' recruits

caspaseso %his is because they are inactive as procaspases and they are

ready to become protiolytically cleaved and form a structure

called “*poptosome”o 5ach card domain interacts "ith an initiator caspase and bind

together to form this “apoptosome” structureo %he apoptosome then activates the e'ecutioner caspases

- Some mechanisms of the intrinsic path"ayo ;eft side

Survival factor receptor binds "ith activated P02 9 Protein#inase 2) "hich then activates 2cl-$ and put the bra#es on

apoptosiso +iddle

*nother "ay to trigger this event is by having damaged

DN* P> then becomes stable because it is phosphorylated



t then triggers 2a' and *paf1

%he middle and right side path"ay leads to forming a

apoptosome8 *nd our cascade of e'ecutioner caspase is

being activated and your intercellular proteins such as

DN*, lamins, cytos#eleton and #inases lead to the cell

being s"allo"ed up by phagocytosiso Gight side

f no survival factor, you do not activate P02 and 2cl-$ is

not active but you are able to dephosphorylate bad 2ad then allo"s activation of 2a' and *paf1

*+ecutioner Caspase

- Gegardless of path ta#en, once e'ecutioner caspase cascade is

activated it undergoes the process- 2oth path"ays have it that "hen you activate the caspases cascade

your intracellular structures are bro#en do"n by the e'ecutioner

caspaseso 6ell becomes disorgani(ed and proteins are cleaved

o 2oth "ays are clean and orderly disposal of the cell

- Gegardless of the trigger the result is the same



Apoptosis in t"e $e&eloping ,er&ous !yste

- * gro"th factor, but also a survival factor- Geleased by “target” cells- 2a' is an important mediatorJ- *dvantages of apoptosis are that the nervous system as it develops

produced a large amount of cells "hich all see# targets8- *n organism produces huge amount of neurons and cells

becauseo t is more e7cient to have more of something then to

have less and not be able to functiono Neurons are special because only a fe" of them are able

to divide in special conditionso %o ensure that all of your reKuired targets are set you produce

"ay too many neuronso Some neurons are told from e'tracellular factors that their

service is doneo %hese factors include survival factors and gro"th factors

o n the absence of receiving gro"th factors from the target for the

cell, the cell "ill no longer gro"/ reach the target and go through

apoptosiso %arget cells release the gro"th factors to reach the target

o %his is an intrinsic path"ays because the gro"th factor "ithin the

cell doesn!t allo" the target to communicate "ith the neuron- +utations in genes encoding important proteins in the apoptotic

path"ay have drastic conseKuenceso +utations in genes hinders the encoding of proteins in the

apoptotic path"ays and causes the cell to face conseKuenceso Such as cells "ill not be removed

o 6ell cycle "ill have a hard time functioning and cell "ill #eep

dividing- n developing mouse embryo, forebrain protrusion and e'cessive

mitosis and dierentiation result



Apoptosis

- 6ell death is characteri(ed by the blebbing of the plasma membrane

and the fragmentation of the nuclei- Suddenly, cells "ea#en attachment to the substrate that they had

been gro"ing on and shrivel up leading to lysis- %he neighbouring cells have more space and their cytos#eleton is

going to gro" and they "ill e'perience elongation of microtubules and

micro4laments and it "ill reorgani(e its intermediate 4laments- %he mechanism of apoptosis involves very tight steps

o 1) Sudden release of cytochrome c from the mitochondria into

the cytosolo $) n normal cell, phosphotidelserine is found only on the face of

the cytosolic membrane 2ut during apoptosis, it becomes e'posed to the

e'tracellular spaceo ) %his helps neighbouring cells to detect the dying cell and be

consumes from the process of phagocytiseso @) %he membrane of the dying cell becomes permeable to small

molecules

8. cell cycle (apoptosis)

Documents