03. Cell Injury II

Early stage: cell shrinkage •The cell becomes smaller in size •The cytoplasm becomes condensed •The normal-looking organelles are more tightly packed. By contrast, the early stage of necrosis is cell ____, not shrinkage.

Early stage: cell shrinkage •The cell becomes smaller in size •The cytoplasm becomes condensed •The normal-looking organelles are more tightly packed. By contrast, the early stage of necrosis is cell swelling, not shrinkage. The arrow on the far left shows the condensation of the cytoplasm The middle and right-most arrows show how the cell is becoming detached from the basement membrane

Extracellular signals cause apoptosis using the extrinsic pathway: these include ____, ____, ____ and ____. Extrinsic pathway is initiated by engagement of the extracellular signal with plasma membrane death receptors, which are present on the surface of a variety of cells. Death receptors are members of the ____ receptor family that contain a cytoplasmic domain involved in protein-protein interactions that is called the death domain because it is essential for delivering apoptotic signals.

Extracellular signals cause apoptosis using the extrinsic pathway: these include toxins, hormones, growth factors and cytokines. Extrinsic pathway is initiated by engagement of the extracellular signal with plasma membrane death receptors, which are present on the surface of a variety of cells. Death receptors are members of the TNF receptor family that contain a cytoplasmic domain involved in protein-protein interactions that is called the death domain because it is essential for delivering apoptotic signals.

Extrinsic pathway Fas receptor (FasR), a member of the TNF receptor family, has strong death-inducing capability. Fas receptor is located on the _____ Structure of Fas receptor: -Extracellular domain, -Transmembrane domain -Cytoplasmic domain Which part of the Fas receptor contains the death domain? Binding of FAS ligand to FAS receptor causes ____ of Fas receptor --> Associated with this is the clustering of the _____

Extrinsic pathway Fas receptor (FasR), a member of the TNF receptor family, has strong death-inducing capability. Fas receptor is located on the cell surface Structure of Fas receptor: - Extracellular domain, - Transmembrane domain - Cytoplasmic domain (contains death domain) Binding of FAS ligand to FAS receptor causes trimerization of Fas receptor --> Associated with this is the clustering of the death domains

Extrinsic pathway Caspase-8 and caspase-10 trigger the cascade of caspase activation and activate _____ caspases.

Extrinsic pathway Caspase-8 and caspase-10 trigger the cascade of caspase activation and activate executioner caspases.

Intrinsic Pathway Anti-apoptotic and pro-apoptotic members are dimerized --> this allows for the suppression of the pro-apoptotic members

Intrinsic Pathway

Intrinsic Pathway Anti-apoptotic and pro-apoptotic members are ____ --> this allows for the ____ of the pro-apoptotic members

Intrinsic Pathway Anti-apoptotic and pro-apoptotic members are dimerized --> this allows for the suppression of the pro-apoptotic members

Intrinsic Pathway Apaf-1-cytochrome c heterodimers provide a platform for activation of the _____

Intrinsic Pathway Apaf-1-cytochrome c heterodimers provide a platform for activation of the procaspase-9, the critical initiator of caspase activation cascade

Lack of ability to stimulate an inflammatory response is another significant feature of apoptosis. Inflammatory cells (neutrophils and lymphocytes) are not generally seen in the vicinity of apoptotic cells. Only mononuclear phagocytes (____) can be seen containing cellular debris from apoptotic cells. The lack of inflammation ensuing apoptotic cell death is clearly beneficial to the body because avoids collateral tissue damage.

Lack of ability to stimulate an inflammatory response is another significant feature of apoptosis. Inflammatory cells (neutrophils and lymphocytes) are not generally seen in the vicinity of apoptotic cells. Only mononuclear phagocytes (macrophages) can be seen containing cellular debris from apoptotic cells. The lack of inflammation ensuing apoptotic cell death is clearly beneficial to the body because avoids collateral tissue damage. Picture on the left is of necrosis --> the dots are nucleus' of the leukocytes (PMNs) infiltrating the necrotic tissue Picture on the right is of apoptotic --> the arrows are denoting the apoptotic bodies --> you can also note there are not leukocytes (PMN's) present

Electron Microscopic Appearance of Apoptosis

N = normal | A = apoptotic Note: the evenly distributed chromatin in cell "N" --> the chromatin is aggregated in the other two cells The black areas are the condensed chromatin

Necroptosis is a hybrid that shares aspects of both necrosis and apoptosis. •Necroptosis is triggered when ____ or ____ binds to its respective receptors. •Receptor-bound complexes incorporate the receptor-interacting proteins (___ and ____). •There is no ____ activation.

Necroptosis is a hybrid that shares aspects of both necrosis and apoptosis. •Necroptosis is triggered when Fas ligand (FasL) or TNF-α binds to its respective receptors. •Receptor-bound complexes incorporate the receptor-interacting proteins (RIP1 and RIP3). •There is no caspase activation.

Necroptosis is being recognized as an important death pathway in pathologic conditions: Examples of necroptosis = cell death in: •Steatohepatitis •Acute pancreatitis •Reperfusion injury •Neurodegenerative diseases (Parkinson disease) •Cytomegalovirus infection

Necroptosis is being recognized as an important death pathway in pathologic conditions: Examples of necroptosis = cell death in: •Steatohepatitis •Acute pancreatitis •Reperfusion injury •Neurodegenerative diseases (Parkinson disease) •Cytomegalovirus infection

Necroptosis pt2. •Activation of RIP1 and RIP3 leads to increased cytosolic ____ •____ and other degradative enzymes are activated •____ membranes are damaged: release of ____ hydrolases into the cytosol. •____ are damaged: impaired ____ generation •Free ____ pool in cytoplasm is increased: increase in ____ and damage to proteins, lipids and DNA. •Loss of ____ integrity: cell swelling, plasma membrane fragmentation and nuclear pyknosis •The cellular morphology is similar to ____ . Release of cellular contents is followed by an ____

Necroptosis pt2. •Activation of RIP1 and RIP3 leads to increased cytosolic Ca2+ •Calpain and other degradative enzymes are activated •Lysosome membranes are damaged: release of lysosomal hydrolases into the cytosol. •Mitochondria are damaged: impaired ATP generation •Free iron pool in cytoplasm is increased: increase in free radicals and damage to proteins, lipids and DNA. •Loss of plasma membrane integrity: cell swelling, plasma membrane fragmentation and nuclear pyknosis •The cellular morphology is similar to necrosis. Release of cellular contents is followed by an inflammatory response

Note: this is a macrophage --> it shows the macrophage phagocytizing the apoptotic cell (you know that it's the apoptotic cell by the condensed chromatin that's black on the periphery) --> directly to the right of the apoptotic cell is the nucleus of the macrophage btw

Note: this is a macrophage --> it shows the macrophage phagocytizing the apoptotic cell (you know that it's the apoptotic cell by the condensed chromatin that's black on the periphery) --> directly to the right of the apoptotic cell is the nucleus of the macrophage btw

p53 is a _____ p53 is activated in response to DNA damage --> describe the two types of responses that can occur.

p53 is a transcription factor p53 is activated in response to DNA damage If DNA damage is limited, p53 activates DNA repair proteins. If DNA damage is so severe that it cannot be repaired, p53 activates the cascade of events leading to apoptosis, and the cell dies.

Describe the difference between necrosis and apoptosis

Apoptosis is a programmed, enzyme-mediated cell death. A form of cell suicide Necrosis will induce an anti-inflammatory event Apoptosis will NOT induce an A-I event

Apoptosis is important in ____ development.

Apoptosis is important in embryological development. During embryonic development the body will produce more cells/structures that we don't need thus will undergo apoptosis Ex. pronephros and mesonephros have functions for early embryonic development of the kidneys however will undergo apoptosis because they are not needed any further --> metanephros does not undergo apoptosis

Activated initiator caspases directly leads to the activation of downstream, executioner caspases-3, -6 and -7. Executioner caspases initiate the death cascade. They coordinate the execution phase of apoptosis by cleaving multiple structural proteins. Executioner caspases target several hundred proteins for restricted proteolysis in a controlled manner that minimizes damage and disruption to neighboring cells and avoids the release of immunostimulatory molecules. Executioner caspases cleave key structural components of the nucleus and cytoskeleton, as well as numerous proteins involved in signaling pathways. Caspase-___ is the primary executioner caspase, which is necessary for cleavage of nuclear lamins, DNA fragmentation, chromatin margination and nuclear collapse. When all kinds of caspase substrates are demolished, the cell will undergo a series of dramatic perturbations to the cellular architecture

Activated initiator caspases directly leads to the activation of downstream, executioner caspases-3, -6 and -7. Executioner caspases initiate the death cascade. They coordinate the execution phase of apoptosis by cleaving multiple structural proteins. Executioner caspases target several hundred proteins for restricted proteolysis in a controlled manner that minimizes damage and disruption to neighboring cells and avoids the release of immunostimulatory molecules. Executioner caspases cleave key structural components of the nucleus and cytoskeleton, as well as numerous proteins involved in signaling pathways. Caspase-3 is the primary executioner caspase, which is necessary for cleavage of nuclear lamins, DNA fragmentation, chromatin margination and nuclear collapse. When all kinds of caspase substrates are demolished, the cell will undergo a series of dramatic perturbations to the cellular architecture

Apoptosis is important in embryological development. •Excess ____ are pruned from the developing brain. •Redundant epithelium is removed following fusion of the palatine processes during development of the roof of the mouth. •Redundant tissue is removed during the closure of the ____

Apoptosis is important in embryological development. •Excess neurons are pruned from the developing brain. •Redundant epithelium is removed following fusion of the palatine processes during development of the roof of the mouth. •Redundant tissue is removed during the closure of the neural tube.

Apoptosis is important in the following situations: 1. Apoptosis eliminates ____ cells. •A normal turnover of cells in many organs is essential to maintain the size and function of that cellular compartment. •In the mucosa of the small intestine, cells in the depths of the crypts proliferate, young cells migrate from the depths of the crypts to the tips of the villi, where they undergo apoptosis and are sloughed into the lumen. •As new leukocytes are continuously supplied to the circulating blood, the old ones must be eliminated to maintain the normal complement of the white blood cells.

Apoptosis is important in the following situations: 1. Apoptosis eliminates obsolescent (unneeded) cells. •A normal turnover of cells in many organs is essential to maintain the size and function of that cellular compartment. •In the mucosa of the small intestine, cells in the depths of the crypts proliferate, young cells migrate from the depths of the crypts to the tips of the villi, where they undergo apoptosis and are sloughed into the lumen. •As new leukocytes are continuously supplied to the circulating blood, the old ones must be eliminated to maintain the normal complement of the white blood cells.

Apoptosis is important in the following situations: 2. Involution of ____-dependent tissues upon ____ withdrawal.

Apoptosis is important in the following situations: 2. Involution of hormone-dependent tissues upon hormone withdrawal. Hormone-sensitive cells cannot survive if deprived of the relevant hormone or growth factor. Growth factor deprivation triggers apoptosis. Ex. in the beginning of the months estrogen is being produced and influences the growth of the endometrium --> progresterone begins to be produced that influences the maturation of the endometrium --> the last few days of the cycle estrogen production is halted thus the endometrium tissue undergoes apoptosis

Apoptosis often affects a relatively (small or large) number of cells and causes no lasting tissue damage. •The cell detaches from ____ •The cytoplasm becomes deeply ____. Nucleus becomes ____ or fragmented. By contrast, necrosis involves (small or large) numbers of cells causing massive tissue destruction (e.g. myocardial infarction).

Apoptosis often affects a relatively small number of cells and causes no lasting tissue damage. •The cell detaches from neighboring cells. •The cytoplasm becomes deeply eosinophilic. Nucleus becomes pyknotic or fragmented. By contrast, necrosis involves (small or large) numbers of cells causing massive tissue destruction (e.g. myocardial infarction). The picture on the right shows an area of necrosis (lots of cells) The picture on the left shows a singular apoptotic cell in the epidermis --> difference here is necrosis covers a region (lots of cells) whereas apoptosis is only a singular/a few cells

Cells destined to die activate enzymes that degrade the cells' own nuclear DNA and nuclear cytoplasmic proteins. Apoptotic cells break up into fragments, called ____, which contain portions of the ____ and ____ and ____ The ____ of the apoptotic cell and bodies remains intact, but its structure is altered in such a way that these become "tasty" targets for phagocytes. The dead cell and its fragments are rapidly devoured, before the contents have leaked out, and therefore cell death by this pathway does not elicit an inflammatory reaction in the host.

Cells destined to die activate enzymes that degrade the cells' own nuclear DNA and nuclear cytoplasmic proteins. Apoptotic cells break up into fragments, called apoptotic bodies, which contain portions of the cytoplasm and nucleus and tightly packed organelles The plasma membrane of the apoptotic cell and bodies remains intact, but its structure is altered in such a way that these become "tasty" targets for phagocytes. The dead cell and its fragments are rapidly devoured, before the contents have leaked out, and therefore cell death by this pathway does not elicit an inflammatory reaction in the host. Note: The picture on the left is right before the fragmentation into membrane-bound apoptotic bodies à once the protuberances bud off they become membrane-bound apoptotic bodies

Chromatin condensation •This is the most characteristic feature of apoptosis. •The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. •The central area of nucleus remains clear.

Chromatin condensation •This is the most characteristic feature of apoptosis. •The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. •The central area of nucleus remains clear.

Extrinsic pathway Clustering of the death domains forms a binding site for an adaptor protein called ____ When this protein is attached to the trimerized death domain, in turn, binds an inactive form of caspase-__ or caspase-__ in order to generate the active forms of the caspases

Extrinsic pathway Clustering of the death domains forms a binding site for an adaptor protein called FADD (Fas associated death domain) When this protein is attached to the trimerized death domain, in turn, binds an inactive form of caspase-8 or caspase-10 in order to generate active caspase-8 or -10.

Failure of apoptosis is a factor in the development of syndactyly (webbed fingers). Exaggeration of apoptosis is a factor in the development of cleft palate and spina bifida.

Failure of apoptosis is a factor in the development of syndactyly (webbed fingers). Exaggeration of apoptosis is a factor in the development of cleft palate and spina bifida.

Ferroptosis is triggered when excessive intracellular levels of ____ produce overwhelming amounts of ____ causing unchecked membrane lipid peroxidation

Ferroptosis is triggered when excessive intracellular levels of iron produce overwhelming amounts of ROS causing unchecked membrane lipid peroxidation

Intrinsic Pathway Bcl-2 family has 20 proteins divided into three groups: antiapoptotic, proapoptotic and sensors •Anti-apoptotic (such as _____, _____ and _____) [guardians] •Pro-apoptotic (such as _____, _____) •Sensors of cell damage (_____, _____, _____, _____, _____).

Intrinsic Pathway Bcl-2 family has 20 proteins divided into three groups: antiapoptotic, proapoptotic and sensors •Anti-apoptotic (such as Bcl-2, Bcl-XL and Mcl-1) [guardians] •Pro-apoptotic (such as Bax, Bak) •Sensors of cell damage (Bad, Bim, Bid, Puma, Noxa). Sensors respond to cellular stress and initiate the mitochondrial pathway of apoptosis following transcriptional up-regulation. Sensors promote apoptosis by both directly activating pro-apoptotic proteins -Bax and Bak and by suppressing the anti-apoptotic proteins

Intrinsic Pathway Oligomerized Bak and Bax form a high conductance channel termed "_____" in the (inner or outer) mitochondrial membrane. Bak or Bax oligomers are an integral part of MAC.

Intrinsic Pathway Oligomerized Bak and Bax form a high conductance channel termed "mitochondrial apoptosis-induced channel (MAC)" in the outer mitochondrial membrane. Bak or Bax oligomers are an integral part of MAC.

Intrinsic Pathway Once formed MAC mediates the release of ____ from the intermembrane space to the cytosol

Intrinsic Pathway Once formed MAC mediates the release of cytochrome c from the intermembrane space to the cytosol

Intrinsic Pathway Once released in cytosol, cytochrome c binds to a protein called _____ This stimulates the formation of a multiunitprotease activation complex called the ____

Intrinsic Pathway Once released in cytosol, cytochrome c binds to a protein called Apaf-1 (apoptosis activating factor-1) This stimulates the formation of a multiunitprotease activation complex called the apoptosome --> Apoptosome is a wheel-like structure consisting of 7 molecules of Apaf-1 and 7 molecules of cytochrome c

Intrinsic Pathway Pairing of anti-apoptotic and pro-apoptotic members is accomplished by interaction between _____

Intrinsic Pathway Pairing of anti-apoptotic and pro-apoptotic members is accomplished by interaction between BH domains

Intrinsic Pathway The intrinsic pathway begins in the _____ The intrinsic pathway is controlled by _____ family of proteins

Intrinsic Pathway The intrinsic pathway begins in the mitochondria The intrinsic pathway is controlled by Bcl-2 family of proteins --> Bcl-2 family of proteins reside within mitochondria or cell cytoplasm

Intrinsic Pathway There are four distinct BH domains based on amino acid homology, named: BH1, BH2, BH3, and BH4. The BH domains are known to be crucial for function. •The anti-apoptotic proteins conserve all four BH domains. •The pro-apoptotic proteins have only 3 BH domains (BH__, BH__, BH__) •While sensors have only one BH domain (BH__).

Intrinsic Pathway There are four distinct BH domains based on amino acid homology, named: BH1, BH2, BH3, and BH4. The BH domains are known to be crucial for function. •The anti-apoptotic proteins conserve all four BH domains. •The pro-apoptotic proteins have only 3 BH domains (BH3, BH2, BH1) •While sensors have only 1 BH domain (BH3).

Intrinsic Pathway Upon activation of caspase-9 within the apoptosome, the death signal is propagated through the stepwise activation of additional downstream caspases, whereby caspases-____ and -___ appear to be simultaneously activated

Intrinsic Pathway Upon activation of caspase-9 within the apoptosome, the death signal is propagated through the stepwise activation of additional downstream caspases, whereby caspases-3 and -7 appear to be simultaneously activated

Intrinsic Pathway p53 induces apoptosis by physically interacting with the pro-apoptotic Bcl-2 family members, ____ and _____ What is the result of this interaction between p53 and these two Bcl-2 members?

Intrinsic Pathway p53 induces apoptosis by physically interacting with the pro-apoptotic Bcl-2 family members, Bak and Bax The interaction of p53 with Bak and Bax liberates these molecules from the interaction with the mitochondrial anti-apoptotic Bcl-2 proteins, Mcl1 and Bcl-XL and activates them --> they will oligomerized after activation

Phagocytosis of apoptotic cells by macrophages. The apoptotic bodies are rapidly taken up by macrophages and degraded within lysosomes. They can be taken up by adjacent cells (such as epithelial cells) and degraded by their lysosomal enzymes. The surrounding cells close ranks and the cell deletion is effected without disruption of overall tissue architecture.

Phagocytosis of apoptotic cells by macrophages. The apoptotic bodies are rapidly taken up by macrophages and degraded within lysosomes. They can be taken up by adjacent cells (such as epithelial cells) and degraded by their lysosomal enzymes. The surrounding cells close ranks and the cell deletion is effected without disruption of overall tissue architecture.

Picture on the left shows normal PMN's and then the arrow denotes the PMN that is undergoing apoptosis Picture on the right with the arrow denotes the liver cell that is undergoing apoptosis compared to the normal liver cells surrounding it

Picture on the left shows normal PMN's and then the arrow denotes the PMN that is undergoing apoptosis Picture on the right with the arrow denotes the liver cell that is undergoing apoptosis compared to the normal liver cells surrounding it

_____ occurs in cells infected by bacteria

Pyroptosis occurs in cells infected by bacteria

Pyroptosis occurs in cells infected by bacteria. •It involves activation of caspase-___ which cleaves the precursor form of IL-___ to generate active IL-___. •Caspase-___ is produced by a structure called an _____ in monocytes, macrophages and neutrophils.

Pyroptosis occurs in cells infected by bacteria. •It involves activation of caspase-1 which cleaves the precursor form of IL-1 to generate active IL-1. •Caspase -1 is produced by a structure called an inflammasome in monocytes, macrophages and neutrophils. Note: •IL-1 is mediator of fever in infections. •IL-1 also induces apoptosis of infected cells.

Describe membrane changes that occur during apoptotic body production.

So in normal cells phosphatidylserine (PS) is present on the inside of the plasma membrane --> when signaled for apoptosis the PS is flipped from the inside to the outside --> since PS is being expressed on the external membrane it is a signal that is recognized by several receptors of macs

The active caspase is a _____ What are your 3 initiator caspases and your 3 executioner caspases?

The active caspase is a tetramer Initiator caspases (caspase-8, -9 and -10) - extrinsic pathway leads to activation of caspase-8 and -10 Executioner caspases (caspase-3, 6 , and 7) Intrinsic pathways leads to activation of initiator caspase-9

The following is the sequence of changes in apoptotic cells: •Cell ____ and cytoplasm becomes ____ . •Chromatin ____ against the ____ membrane. •____ breaks up into rounded fragments •Cell emits ____ that break off as small, rounded apoptotic bodies. •Macrophages or neighboring cells phagocytize apoptotic bodies

The following is the sequence of changes in apoptotic cells: •Cell shrinks and cytoplasm becomes dense. •Chromatin aggregates into dense masses against the nuclear membrane. •Nucleus breaks up into rounded fragments •Cell emits protrusions that break off as small, rounded apoptotic bodies. •Macrophages or neighboring cells phagocytize apoptotic bodies

Which pathway is the major mechanism of apoptosis?

The intrinsic, mitochondrial initiated pathway

The process of apoptosis may be divided into two phases: the initiation phase, and the execution phase. Initiation phase occurs in response to signals from two distinct pathways: -The extrinsic, _____-initiated pathway: signals originate outside of the cell. -The intrinsic, _____ initiated pathway: signals originate from within the cell.

The process of apoptosis may be divided into two phases: the initiation phase, and the execution phase. Initiation phase occurs in response to signals from two distinct pathways: -The extrinsic, death-receptor-initiated pathway: signals originate outside of the cell. -The intrinsic, mitochondrial initiated pathway: signals originate from within the cell.