Week 15

Cell death is triggered by an absence of

Survival signals and grow/divide signals

And a presence of pro-death signals

programmed cell death is carefully

regulated and maintains a balance between cell proliferation and constant cell numbers (e.g. tissue growth).

programmed cell death eliminates

potentially dangerous cells (e.g. infected or damaged cells)

Abnormalities of cell death are associated with

cancers, autoimmune diseases, and neurodegenerative diseases

Programmed Cell Death In Nervous system development:

up to 50% of neurons are eliminated by programmed cell death (surviving neurons have established proper connections with target cells, which secrete growth factors that block cell death programs)

Mutation of the myostatin gene leads to a

dramatic increase in muscle mass

Programmed Cell Death in Developing Plant Vasculature

Starts out as: protoxylem and protophloem – fully intact, isolated functioning cell, with all organelles present

Matures to: xylem and phloem with full and partial organelle loss, respectively, but fully connected to top and bottom and neighboring cells

Programmed cell death

Apoptosis-like process

• Necrosis (Necroptosis)

• Autophagy

• as a localized process and is often quite rapid

Senescence

death as a recycling process

• In Plants - slower, systemic process that includes nutrient remobilization, and usually culminates in the death of cells

• In Animals – happens in fibroblast cell lines, and is induced in cells with an inflammatory response

Programmed Cell Death (PCD) Pathways:

Apoptosis, Autophagic, Necrosis

Apoptosis

“Eat me” signal: phosphatidylserine on the outer leaflet of the plasma membrane.

• Phosphatidylserine is typically restricted to the inner leaflet of the plasma membrane.

• Highly regulated process that involves the breakdown of organelles, plasma membrane, nuclear envelope, and cellular fragmentation.

A synonym for “ pro-survival” is

Anti-apoptotic

Membrane Polarity and Composition

Phagocytosis of apoptotic cells

In normal cells, phosphatidylserine is restricted to the inner leaflet of the plasma membrane

After receiving ”death” signals, lipids are swapped to the outside of the cell. Don by Scramblase

Is Necrosis reversible?

Yes

Is apoptosis reversible?

No. Breakdown of organelles, pm, nuc env, and cellular fragmentation

What results in a potential for recovery with necrosis?

ER and Mito swelling, mem blebbing

When is Necrosis committed?

Bd of pm, organelles and leakage of cellular contents

What is an alternative pathway of cell death if apoptosis doesn’t occur?

Regulated necrosis - nercroptosis.

What signal can induce apoptosis or necrosis?

“death” signal TNF (Tumor necrosis factor) which can activate RIPK3 (receptor interacting protein kinase-3) is stimulated and phosphorylates MLKL monomers (effector protein) → forms oligomers of MLKL that disrupsts PM → neroptosis

Why is it important that cell proliferation and cell death are balanced during development?

What are some examples of developmental processes in which regulated cell death is important?

What are some examples of damage to cells that can result in cell death?

What would be the result of inhibiting apoptosis during development of the mouse paw?

What is the result of inhibiting or knocking out the myostatin gene?

What are some key distinctions between apoptosis, necrosis, and autophagic cell death?

What types of morphologies would you use to distinguish apoptotic vs necrotic vs autophagic cell death by transmission electron microscopy?

What is the cellular “eat me” signal?

Is Apoptosis Reversible?

If death signals (extrinsic - from the outside) or intrinsic (autophagy) win out, then the scale is tipped to pro-apoptotic (pro-death) fate.

Autophagy:

turnover of the cell’s own components. Important in embryonic development and programmed cell death.

Phagocytosis:

specialized cells such as macrophages take up and degrade large particles— bacteria, cell debris, aged cells, cells with eat me signals

Apoptosis is dependent on what?

Caspase

What happens to the cell in Apoptosis?

DNA fragmentation, chromatin condensation, fragmentation of the nucleus and cell. Apoptotic cells are fragmented and fragments are phagocytosed by neighboring cells or macrophages

What is autophagy dependent upon?

Cathepsin.

What can autophagy result in?

may not result in cell death, targeted degradation of damaged organelles. In autophagic cell death, dying cells are characterized by an accumulation of lysosomes.

Cathepsins are

proteases found in lysosomes.

Autophagy eliminates

proteins, organelles and pathogens from the cytoplasm

Autophagy in plants vs. humans

Plants: implicated in defense, starvation response, and senescence

Humans: implicated in Alzheimer’s disease

What regulates autophagy?

ATG genes. Highly conserved in yeast, plants, and animals.

Autophagosome

What does the autophagosome fuse with?

Lysosome

What can autophagosomes engulf?

entire cells!

Damaged mitochondria are degraded by…

autophagy - important for cellular homeostasis

Autophagosomes are

double- membrane enclosed organelles that selectively recruit poly-ubiquitylated substrates and deliver them to lysosomes for terminal degradation

Polyubiquitylated substrates (proteins, organelles, vesicles) are recognized by selective autophagy receptors and recruited into the autophagosome through interaction with ___

LC3 (ATG receptor proteins). backup to the ubiquitn proteasome pathway

atg mutants…

Cannot tolerate starvation (days recovery after darkness.

Starvation-sensitive and shows runaway cell death.

Autophagy allows for recovery from starvation damage.

Autophagy is considered both a _____ and a _______ process

regulated and constitutive

Constitutively active =

(always on) in cells

always some low level of autophagy occurring to manage misfolded and long-lived proteins, for regular turnover of organelles, and for degradation of damaged organelles.

Programmed Cell Death – C. elegans

Proper C. elegans development includes/relies the death of 131 specific cells. Clumps of dying cells are easily visible.

• Clumps of dying cells are not visibly present – proper development cannot occur

In C. elegans, the genes _____ and _____ are required for developmental cell death

ced-3 (caspases) and ced-4 (pro-caspases, activate ced-3). Considered pro-apoptotic

ced-9`

(BCL-2) is a negative regulator. Considered anti-apoptotic

Ced genes are

central regulators that are highly conserved.

How do caspases trigger apoptosis?

Ced-3 is the prototypical caspase protease

• Caspases are cys proteases

• cys residues at their active sides and cleave after asd residues in their substrate prot.

ultimate executioners of pcd by cleaving over 100 different target proteins

Activation of an initiator caspase starts a

Activation of an initiator caspase (e.g. Caspase 8, 9, or 10) starts a chain reaction of caspase activation leading to cell death

Caspases are synthesized as

precursors (procaspases) that convert to the active form upon proteolytic cleavage

Initiator caspases are activated by

post- translational modification (dimerization, phosphorylation, and ultimately by cleavage)

Apoptotic signal trigger the assembly of

adaptor proteins that dimerize the initator procaspase monomers

inactive monomers of initiator procaspases are activated by

cleavage in thier protease domains that leads to a conformational change.

Regulation of Caspases

Executioner procaspases are also synthesized, but

Inactive as oligomers

What cleaves executioner procaspases?

Active initiator caspases.

What happens when executioner procaspases are cleaved again?

Induces a conformational change, activating the executioner caspase.

Regulation of Caspases

How were C. elegans used to discover critical apoptotic genes?

What is the role of Ced-9 genes in regulating apoptosis? What is the role of Ced-4 genes in regulating apoptosis? What is the role of Ced-3 genes in regulating apoptosis?

Describe the general structure of caspases. What are some ways of classifying caspases?

Caspases are classified as cysteine proteases. What does this mean?

How is the caspase cascade activated? What are the roles of initiator and executor caspases in this cascade? Name the initiator caspases and the executor caspases.