D103 Apoptosis (ALS 26, Video 39)

apoptosis

regulated form of cell death that occurs during development in homeostasis of organs

• mass turnover in healthy human body

• cotnrolled cell death for homeostasis

characteristics of apoptotic cell death

1. mild convolution; chromatin compaction and margination; condensation of cytoplasm

2. breakup of nuclear envelope; nuclear fragmentation; blebbing; cell fragmentation

3. phagocytosis

blebs

balls formed from PM of cell undergoing apoptosis

• grapes

morphological characteristics of apoptotic cell death

light video-microscopy of HeLa cells undergoing apoptosis in a cell culture dish in response to an anti-cancer drug

DNA fragmentation

1. dna cleaves when in condensed structure via active CAD

2. formation of DNA ladder due to random cleavage of DNA between nucleosomes during apoptosis in mouse lymphocytes (agarose gel electrophoresis)

3. identification of high incidence of DNA cleavage associated with apoptosis in developing chicken limb

movement of phosohatidylserine to the exterior surface of plasma membrane

• in healthy cells, PS is localized to the inner leaflet of the PM by a flippase

• scramblase is off

• apoptosis - PS flippase is inhibited, while scramblase is activated, so PS becomes exposed on cell surface

• PS on the cell surface functions as a cellular “Eat Me” signal

action of a flippase

entire phospholipid is switched, not just the head group

chromatin condensation

nuclear morphology of live (arrowhead) and apoptotic (arrow) cells stained with Hoechst, visualized under fluorescence microscopy

attributes of necrosis

• cell swells

• no chromatin condensation

• no DNA laddering

• cellular rupture

• inflammatory response

• typically affects groups of cells

attributes of apoptosis

• chromatin condensation

• dna laddering

• cellular fragmentation without cellular rupturing

• no inflammatory response

• engulfment of dead cells

• typically affects single cells

necrosis vs apoptosis

necrosis: swelling, rupture, inflammation

apoptosis: condensation, laddering, no cell rupture engulfment of dead cells

examples of function of apoptosis during animal development

sculpting

adjust cell number

deleting unwanted structures

eliminate injured or dangerous cells

disease associated with excessive apoptosis

• aids

• alzheimers

• parkinsons

• als

• aplastic anemia

• ischemia

disease associated with insufficient apoptosis

• follicular lymphoma

• breast cancer

• prostate cancer

• ovarian cancer

• lupus

• viral infections

caspases

apoptosis is effected by an intracellular family of enzymes

• proteases with cysteine in the active site

• cut at asp residues (c asp ases)

how are caspases synthesized

synthesized as a pro-enzyme that has very low proteolytic activity

caspase cascade

• when brought into proximity, initiator pro-capses cleave each other, resulting in full activation

• initiator caspases activate executioner procaspases → executioner caspses disassemble the cell by cleaving essential cellular proteins

• provides robust activation of cell-death processes

• once fully activated = impossible for the cell to prevent cell death

result of capase-mediated activation of DNA endonuclease during apoptosis

DNA fragmentation and DNA ladder

• initiator caspase → executioner caspase activation → iCAD cleaved from the inactive CAD → active CAD → cleaves DNA

what causes accumulation of phospholipids (PS) on the surface of apoptotic cells

caspase-mediated inactivation of flippase and activation of scramblase

• healthy: PS is almost exclusively localized to the inner leaflet of the PM by a flippase

• apoptosis: executioner caspases inactivate flippase and activate scramblase and PS becomes exposed on cell surface

• PS on the cell surface = Eat Me signal

what regulates caspase activation

fas or BCL2 pathways

• both pathways involve activation of proteases (caspases) that disassemble essential cellular proteins

purpsoe of BCL2 and FAS protein families

control activation of caspases required for a cell to undergo apoptosis

BCL2 pathway

• regulates cell death by monitoring inside the cell (cell intrinsic pathway of cell death)

• involves interaction with mitochondria and release of cytochrome c from mitochondria

FAS pathway

regulates cell death by monitoring signals outside the cell (extrinsic pathway of cell death)

• when activated, extrinsic FAS pathway can also activate the BCL2 cell intrinsic pathway

extrinsic activation of caspase cascade Via Fas-signaling

fas = death receptor located on cell surface

• trimeric fas ligand binds to fas receptor and causes the receptor to trimerize

• conf change in receptor recruit FADD adaptor proteins and procaspase-8, bringing the procaspases into proximity

• procaspases have low intrinsic proteolytic activity

• aggregation allows cleavage of procaspase-8 by each other → caspase cascade

intrinsic activation of caspase cascade

• cell damage or stress can trigger apoptosis

• cyto-c (plus other molecules) released from mitochondria and binds to an adaptor protein (Apaf1)

• Apaf1 undergoes a conformational change, which allows it to multimerize → exposes binding sites for procaspase-9 to form the apoptosome (proteolytic buzz saw of death)

• cleavage and activation of procaspase-9 → caspase cascade

function of caspase recruitment domain and death effector domain

recruitment of procaspases into proximity thereby facilitating their activation

what do BCL2 family proteins control

release co cytochrome c required for activation of procaspases

• regulate apoptosis by controlling release of cytochrome c and other inner membrane space proteins from mito

pro-apoptotic bcl2 proteins

promote release of cytochrome c and other proteins from the mitochondria

anti-apoptotic bcl2 proteins

try to counteract the effects of their pro-apoptotic family members by preventing release of cytochrome c and other proteins

pro and anti apoptotic bcl2 members

can bind each other directly to inhibit each other function

• balance between these competing actions regulates whether a cell lives or dies

BH3 only BCL2 family proteins

pro-apoptotic and drive the release of cytochrome c during the intrinsic pathway of apoptosis

• healthy cells: anti-apoptotic BCL2 family members prevent effector BCL2 family proteins from aggregating to form a channel in outer mito membrane

• cell receives death signal → BH3 only domain pro-apoptotic BCL2 family members can bind to, and inhibit anti-apoptotic BCL2 family members, thereby allowing effector BCL2 family porteins to aggregate and form a channel

• channels permit release of cytochrome c and other proteins from mito inter-membrane space

what inhibits apoptosis

extracellular survival factors alter the conc and activity of BCL2 family members

survival factors suppress apoptosis

stimlating transcription of genes encoding anti-apoptotic BCL2 family members

• EpoR → STAT → BCLX

survival pathways for activation of S/T protein kinases

AKT, PKB

• phosphorylate and cause temporary inactivation of BH3-inly pro-apoptotic BCL2 family members (BAD)

supply of extracellular survival factors

suppress apoptosis but limited in supply

• animal cells require constant signals to promote cell survival

• absence of signals = cells activate apoptosis program

• ensures cells survive only when and where they are needed (ex - nerve cells using nerve growth factor)

nerve growth factors

limited and cells compete for it

• ultimately only those cells that receive enough factor will survive

• musical chairs

what provides extracellular survival signals

cell adhesion

• cells require anchorage to survive, growth, and proliferate

• FAKs

focal adhesion kinase

associates in the cytoplasm at points of cellular contact with the substratum

• signals generated at these sites stimulate cell growth, division, and survival

• involve actin stress fibers and phospho-tyrosine

integrins for cell adhesion

when integrins are attached to ECM, the integrins send a signal into the cell, instructing FAK to phosphorylate tyrosines on target proteins

• phosphorylation of these target proteins results in transduction of survival stimuli cell, which prevents induction of apoptosis

Which of the following is not a feature of cells undergoing apoptosis ?

A. Condensation of chromatin.

B. Ligation of DNA fragments.

C. Typically affects single cells.

D. No inflammatory response.

E. Shrinkage of cell.

B. Ligation of DNA fragments.

Which of the following genetically engineered proteins could protect cells from activating the cell-extrinsic pathway of programmed cell death when expressed in mammalian cells ?

A. APAF1 that cannot bind cytochrome c.

B. BAX that cannot oligomerize.

C. BH3-only protein that cannot interact with any BCL2 pro-survival member.

D. Pro-caspase 9 that cannot be cleaved.

E. A Fas death receptor lacking the cytoplasmic region of the protein.

E. A Fas death receptor lacking the cytoplasmic region of the protein.