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Cell division functions in...

Reproduction, growth and development, tissue maintenance, tissue repair

Non-dividing vs dividing cells

No division: neurons, muscle cells, other terminally differentiated cellsDivision: stem cells, intestinal/skin/epithelial cells, blood cells

4 phases of the cell cycle

G1 and G2: gap phases to grow and double mass of proteins and organellesS phase: synthesis, 10-12 hours in mammalian cellsM phase: mitosis, less than one hour in mammalian cellsInterphase: G1, G2 and S phases, 23 hours in mammalian cells

Major events during the cell cycle

G1: cells grow and increase mass of proteins and organellesS: DNA replication, centrosome duplicationG2: DNA exists as CHROMATIDS, centrosome separation beginsM phase:1. Prophase- centrosome migration, mitotic spindle begins to form, chromosomes begin to condense- appear as long threads2. Prometaphase: nuclear envelope breakdown, chromosome condensation complete, chromosomes attach to spindle MTs via their kinetochores3. Metaphase: chromosomes align at the equatorial plate of the bipolar spindle4. Anaphase: chromosome separation, kinetochore MTs shorten and spindle poles move apart5. Telophase: chromosomes decondense, nuclear envelope reassembles, spindle disassembly begins6. Cytokinesis: cytoplasmic division by the actomyosin contractile ring. Initiates during anaphase, interphase MT array re-forms

Flow cytometry and the cell cycle

- One by one analysis of a large population of cells- Requires separating individual cells from culture or tissue- Cells are then stained w/ a DNA dye- Cells pass through detector which detects fluorescence emitted from each cell- DNA content can be determined for a large population of cells

Cell cycle checkpoints

Delay later events until earlier events have been completed3 main checkpoints:G1: Sufficient nutrients and signal molecules?Entering mitosis: DNA replicated and damage repaired?End of mitosis: are chromosomes properly attached to mitotic spindle?

Cdks

Key regulators of the cell cycle control system. Phosphorylate and dephosphorylate cell cycle machinery. Serine/threonine kinases. Form complexes with CYCLINS- No enzymatic activity themselves- Cdk inactive w/out cyclin but activated with it and triggers cell-cycle events by phosphorylating target proteinsEach cyclin-cdk complex phosphorylates a DIFFERENT SET of target proteins to trigger different steps

Cyclin protein concentration varies

- Cyclins undergo a cycle of synthesis and degradation in each cell cycle which drives assembly and activation of cyclin-Cdk complexes- M-cyclin (cyclin B) protein concentration increases throughout interphase and early mitosis and drops abruptly at the end of mitosis- M-Cdk (cyclin B/Cdk 1) activity is high only during mitosis- Cdk1 protein activity is constant throughout the cell cycle and exceeds the amount of cyclins

Xenopus oocytes, eggs and embryos

Oocyte= immature egg, arrested in G2Egg= female reproductive cell (arrested in M)Embryo= fertilized egg- Oocytes and eggs are large (more than 1mm) which makes them great to study- 30 min cell cycle bc they skip G1 and G2- Cell cycles are synchronous so cytoplasmic extracts can be made that represent the stage at which it was obtained

What do you predict happens when M-phase extract is injected into an oocyte? Interphase extract?

1. Instantly drives the oocyte into M-phase2. Does not drive the oocyte to M-phase"Maturation promoting factor"- MPF- found to be Cdk1 and cyclin B

Cyclin concentrations are regulated by transcription and proteolysis

- Gradual increase in cyclin concentrations is due to increasing transcription of cyclin genes- Abrupt degradation of S and M cyclins partway through M-phase is due to ubiquitin-mediated proteolysis

the activity of some Cdks is regulated by cyclin degradation

- Anaphase promoting complex (APC/C) is a ubiquitin ligase- APC/C tags M-cyclin with a chain of ubiquitin- Ubiquinated cyclins are directed to proteasomes where they are degraded. Without its binding partner, Cdk1 is inactive

Which statement is falseabout Cdk-cyclin complexes?A. Cyclins undergo a cycle of synthesis and degradation during each cell cycle.B. The protein levels of Cdks rise and fall as the cell progresses through the cell cycle.C. Cdk has little kinase activity until it forms a complex with a cyclin.D. The activities of Cdks rise and fall as the cell progresses through the cell cycle.E. Cdks phosphorylate their substrates on serine or threonine

B. The protein levels of Cdks do NOT rise and fall as the cell progresses through the cell cycle. The ACTIVITY of Cdk does rise and fall.

Mechanisms that regulate activity of Cdks

1. Cyclin synthesis/destruction and association with Cdks2. Activating phosphorylation events on M-Cdk3. Inhibitory phosphorylation events on M-Cdk4. Cdk inhibitor proteins (CKIs)

Cdk is activated by Cdk-activating kinase (CAK) phosphorylation

- When a cyclin is not bound, Cdk's active site is blocked by the T loop and it is fully inactive- When a cyclin binds the T loop moves out of the active site and it is partially activated- CAK phosphorylation of the T loop is what fully activates the complex

For M-Cdk to be active, inhibitory phosphorylations must also be removed

- Cdk1 associates with Cyclin B as the levels of Cyclin B gradually rise during G2- By the end of G2, a high concentration of Cdk1/Cyclin B is phosphorylated at the activating site by CAK but is held in an inactive state due to unwanted phosphorylations by the inhibitory kinase Wee1 (hehe)- The activating phosphatase Cdc25 removes the inhibitory phosphate, leading to full activation of M-Cdk and a rapid increase in activity

The activity of a Cdk can also be blocked by the binding of a Cdk inhibitor protein (CKI)

. CKI binds both the Cdk and the cyclin and interferes with ATP or substrate binding. p27 is a CKI that binds to an activated cyclin-Cdk preventing the Cdk from phosphorylating target proteins required for progressing from G1 to S- this is an important regulatory mechanism for G1/S-Cdk and S-Cdk

The main cell-cycle "molecular break" mechanisms

G1: Cdk inhibitors block entry to S phase if the environment is not favorableG2: Inhibition of activating phosphatase (Cdc25) blocks entry to mitosis if DNA replication is not complete or there is damageM: inhibition of APC/C activation delays exit from mitosis

G1 to S transition

Are the conditions right to commit to completing another cell cycle? Once a cell passes into S, it is committed to completing the rest of the cell cycle.- An alternative is to withdraw to G0 which is a prolonged non-proliferative stage

Shutting down Cdk activity in G1

Abrupt degradation of S and M cyclins part way through M phase is due to ubiquitin mediated proteolysis.Additional mechanisms to inactivate Cdks in G1 include...- Blocking synthesis of new cyclins = no Cdk activity- CKIs bind any remaining complexes and keep them from being active

MITOGENS promote the production of CYCLINS that stimulate cell division(think: Mitogens, Mammalian cells, Cyclins, Cell division

- Mammalian cells only divide when stimulated by extracellular signals called mitogens, otherwise they are arrested in G1- If deprived of mitogens for long enough a cell will withdraw from the cell cycle altogether- One way mitogens act is by stimulating synthesis of cyclins and other proteins needed for DNA synthesis

Mitogens can ALSO stimulate cell division by inhibiting Rb

. Retinoblastoma (Rb) binds transcriptional regulators and keeps them from turning on genes needed for proliferation. Mitogens release this Rb brake by triggering activation of G1-Cdk and G1/S-Cdks which....1. Phosphorylate Rb so it releases the transcription regulators2. Transcription regulators can now activate genes required for cell proliferation

Consider the following mutations. Are they likely to cause cell-cycle arrest in G1? Why or why not?1. a mutation that removes the phosphorylation sites on the Rb protein2. a mutation that inhibits Rb's ability to bind transcriptional regulators

1. Yes, no phosphorylation sites means Rb is not inactivated by mitogens through phosphorylation, and so it will not release transcription factors to start cell proliferation2. No, if anything cells would be able to divide even in the absence of mitogens which could even lead to cancer

p53: the guardian of the genome- cancer prevention and G1-\>S regulator

. p53 is frequently mutated in human cancers. Normal cells have very little p53 protein because it is rapidly degraded by ubiquitin mediated proteolysis. Cellular stress (DNA damage, telomere shortening, hypoxia, hyperproliferative signals, etc.) in normal cells: p53 levels rise and cells undergo cell-cycle arrest, senescence or apoptosis- phosphorylation of p53 activates it and protects it from ubiquitin-mediated proteolysis- this promotes transcription of the p21 gene after p53 binds regulatory region and translation to a CKI- p21 binds G1/S-Cdk and S-Cdk and inactivates them, blocking cell cycle progression and allowing for DNA repair. Mutated cells: cells continue dividing even if DNA is damaged and cells escape apoptosis

Why is the increase in M-Cdk activity at the start of M phase so abrupt?A. Association of Cyclin B with Cdk1B. CAK phosphorylation of Cdk1C. Wee1 phosphorylation of Cdk1D. Cdc25 phosphorylation of Cdk1E. None of the above

E.- None of the above. It is abrupt because Cdc25 phosphatase REMOVES the inhibitory phosphorylations on Cdk1 that were placed by Wee1

Errors in DNA replication can arrest the cell cycle in G2

. S-Cdk provides the signal to initiate DNA (and prevent re-replication) in S phase. If errors occur or replication is delayed the cell can arrest the cell cycle in G2, delaying entry to M phase. Cdc25 is inhibited so that Wee1 inhibitory phosphorylations aren't removed and M-Cdk remains inactive

Once activated, M-Cdk indirectly activates even more M-Cdk through a positive feedback loop

. By the end of G2 a high concentration of Cdk1/Cyclin B is phosphorylated at the activating site by CAK but is still held in an inactive state due to Wee1 phosphorylations. Phosphatase Cdc25 removes the phosphates and M-Cdk is active promoting two positive feedback loops via phosphorylation:1. activates the activator, Cdc252. Inhibits the inhibitor, Wee1This rapidly drives the cell from G2 to M

When a population of control cells and mutant cells wereanalyzed using a flow cytometer (remember this sorts cellsaccording to the amount of DNA they contain), the graphs shownon the right were obtained (one graph is a typical flow cytometry graph and the mutant has no cells in 2n)Which of the following would not explain the results with themutant?A. inability to initiate DNA replicationB. inability to begin M phaseC. inability to activate proteins needed to enter S phaseD. too much production of a signal that causes the cells toremain in G1

B. inability to begin M phase- cells in M phase have completed S phase so they would be expected to have 2n

Key regulatory events in mitosis

1. Abrupt increase in M-Cdk activity at G2/M triggers the events of early mitosisM-Cdk phosphorylates target proteins leading to.... Chromosome condensation. Increase in dynamic instability of MTs. Mitotic spindle assembly. Nuclear envelope breakdown2. APC/C triggers the metaphase-to-anaphase transition

cohesins and condensins help configure duplicated chromosomes for separation

. Cohesins and condensins are structurally related protein complexes that are both thought to form ring structures around chromosomal DNA. Cohesins link the two sister chromatids together (cohesion) and condensins helps single sister chromatids coil up into a more compact form (condense)- Immediately after a chromosome is duplicated in S phase, the two sister chromatids remain tightly held together by cohesin ring structures- Chromatids separate during anaphase when the ring is proteolytically cleaved by separase- Cohesion is important to avoid mis-segregation- When cells enter M phase, condensins help condense each sister chromatid into a smaller, more compact structure- M-Cdk phosphorylates condensin to trigger chromosome condensation- Condensed chromosomes can be more easily segregated

The mitotic spindle: sister chromatids and centrosomes are separated into the two daughter cells

. Before M phase begins:- DNA must be fully replicated into sister chromatids and centrosomes must be duplicated. Mitotic spindle assembly- dynamic instability, MAPs, MT motors are required. Microtubules make stable linkages with the sister chromatids via kinetochores and separate at the beginning of anaphase

The centrosome duplicates to form the two spindle poles of the mitotic spindle

. Centrosome duplication begins in S phase at the SAME TIME as DNA replication. Both copies of the replicated centrosome remain together in S and G2 phases but in M phase the two centrosomes separate to form the two spindle poles of the mitotic spindle even before nuclear breakdown occurs. The centrosomes nucleate an array of MTs called an aster, and MT motor proteins help organize these MTs into a bipolar spindle. Upon nuclear envelope breakdown (NEBD), the spindle MTs are able to interact with the chromosomes

A bipolar mitotic spindle is formed by the selective stabilization of interacting MTs

. The mitotic spindle begins to form in prophase. The minus ends of the MTs are anchored in the centrosomes/spindle poles (center), whereas the free plus ends exhibit dynamic instability. At the start of mitosis, MTs become more dynamic- M-Cdk phosphorylates microtubule associated proteins (MAPs) that normally stabilize MTs (phosphorylation releases MAPs from MTs). When MTs from one spindle pore interact with MTs from the opposite spindle pole, MT motor proteins and different MAPs crosslink and stabilize the interpolar MT plus ends

Nuclear envelope breakdown is required for completion of spindle assembly in animal cells

. Prometaphase begins with nuclear envelope breakdown, releasing sister chromatids and MT motor proteins from the nucleus so they can interact with spindle microtubules. M-Cdk phosphorylates nuclear lamins triggering disassembly of the nuclear lamina. M-Cdk phosphorylates nuclear pore complexes, promoting their disassembly

The bipolar mitotic spindle

. Each duplicated chromosome has two kinetochores- one on each sister chromosome. Kinetochores (proteins) assemble on centromeres (DNA) during late prophase. The kinetochores face in opposite directions and attach to MTs from opposite spindle poles- "bi-orientation". The cell cycle control system monitors tension across the kinetochores to ensure bi-oriented chromosomal attachments have been made before going into anaphase

Kinetochores attach sister chromatids to the mitotic spindle

. The kinetochore is a multilayered protein structure built upon the centromere region of each chromosome with plus ends of kinetochore MTs embedded in MT attachment sites at the kinetochore. Each duplicated chromosome has 2 kinetochores (1 for each sister chromatid, multiple MT attachment sites per kinetochore. Chromosomes begin to attach to kinetochore MTs at prometaphase when the nuclear envelope breaks down. MTs use a search and capture mechanism to find and attach to kinetochores. Kinetochore MTs attach to chromosomes at kinetochores

8 chromatids in G1 phase, how many _____________ during mitotic prophase?

a) sister chromatids- 16b) kinetochores- 16c) centrosomes- 2d) centromeres- 16e) centrioles- 4 (2 in each centrosome)

Stages of mitosis and cytokinesis

Mitosis= nuclear division- duplicated chromosomes are separated by microtubule-based mitotic spindle into what will become the two daughter cellsCytokinesis= CYTOPLASM division- actomyosin-based contractile ring pinches pinches one cell to two cells

The APC/C triggers the metaphase-to-anaphase transition

. APC/C triggers the separation of sister chromatids by promoting degradation of Securin, thus promoting cleavage of Cohesin. APC/C promotes the degradation of S and M-cyclins, thus inactivating most of the Cdk in the cell. M-Cdk activity rapidly declines leading to:- Stabilization of MTs- Reorganization of the mitotic spindle and formation of the central spindle. Reformation of the nuclear envelope

M-Cdk triggers the assembly of the mitotic spindle

. When prophase activities increase, spindles begin to form. M-Cdk phosphorylates MAPs (microtubule associating proteins) which reduces the MAP ability to stabilize MTs. Increased MT dynamic instability along with increased MT nucleation due to gamma-Turc at centrosomes leads to dense MT asters that are ideally suited for capturing sister chromatids. MTs use a search and capture mechanism to find and attach to kinetochores

Cohesin

Protein complex that links sister chromatids together after DNA has been replicated

Centromere

Specialized DNA sequence that allows duplicated chromosomes to be separated during mitosis

Kinetochore

Protein complex that assembles on the centromere of a condensed mitotic chromosome

the site to which spindle microtubules attach.

Centrosome/centrioles

MTOC that sits near the nucleus and duplicates to form the two poles of the mitotic spindle.

Three classes of MTs make up the mitotic spindle

Kinetochore MTs- MT bundles that attach the chromosomes to the spindle poleInterpolar MTs- MTs whose plus ends overlap with the plus end of MTs from the other poleAstral MTs- MTs that radiate outward from the poles and contract the cell cortex

Chromosomes line up at the "metaphase plate"

. During metaphase, chromosomes align at the metaphase plate, halfway between the two spindle poles. Chromosomes at the metaphase plate oscillate back and forth- Tug-of-war between the two spindle poles- Chromosomes are under tension- In anaphase, this tension will help pull the sister chromatids apart

Sister chromatids separate at anaphase

. Chromosomes chillin at the metaphase plate await a signal to separate. Once all kinetochore-MT attachments are bi-oriented APC/C is activated (serves as ubiquitin ligase) and Separase cleaves cohesins. This sudden and coordinated loss of sister chromatid cohesion allows anaphase to begin

Targeted protein degradation drives the metaphase to anaphase transition

. Progression through the G2/M checkpoint is dependent on activation of M-Cdk. Progression through the metaphase to anaphase transition is promoted by ubiquitin-mediated proteolysis. The APC/C catalyzes the ubiquination and destruction of two key proteins in order to promote the metaphase to anaphase transition1. Destroying S and M cyclins inactivates most of the Cdk in the cell2. Destroying Securin allows the sister chromatids to separate in anaphase

APC/C triggers the separation of sister chromatids by promoting cleavage of Cohesin

. Cohesin complexes encircle sister chromatids, holding them together. Securin normally holds Separase in an inactive state (bound together). Activation of APC/C leads to the ubiquination and proteasomal degradation of securin, freeing separase. Separase can now proteolytically cleave Cohesin allowing the sister chromatids to be pulled apart by the mitotic spindle

Transitioning to anaphase

. Every chromosome must be attached properly to the mitotic spindle before anaphase initiates. Unattached kinetochores send a "stop" signal to the cell-cycle control system which blocks the activation of APC/C. Without active APC/C the chromosomes can't be pulled apart. If anaphase were to begin before all chromosomes are properly attached, this would lead to aneuploidy (two many or two few chromosomes)

Forces that separate sister chromatids at anaphase

. Anaphase A- chromosomes are pulled poleward due to depolymerization of kinetochore MTs. Anaphase B- spindle poles are pushed and pulled apart due to...1. Kinesin mediated sliding of interpolar MTs past each other2. Dynein attached to the plasma membrane near the spindle poles pulling the poles apart

The nuclear envelope re-assembles at telophase

. During TELOPHASE a nuclear envelope re-assembles around each group of chromosomes to form two daughter nuclei. When M-Cdk in inactivated due to M-cyclin destruction, phosphatases dephosphorylate nuclear pore proteins and nuclear lamins, allowing them to re-assemble

Cytokinesis

Cytoplasm is divided in two by a contractile ring of actin and myosin filaments which pinches the cell into two daughters, each with one nucleus1. Signaling btwn the anaphase mitotic spindle and the cell cortex to generate an equatorial zone of active RhoA (small GTPase)2. Active RhoA directs assembly of the contractile ring3. The contractile ring is made primarily of F-actin and Myosin II4. The contractile ring constricts until it compacts the MTs into a narrow intracellular bridge with a midbody at the center5. During abscission, membrane scission occurs

The MTs of the mitotic spindle separate the chromosomes AND specify the position of the actomyosin contractile ring

. The MT-based mitotic spindle assembles first to separate the duplicated chromosomes. The actomyosin-based contractile ring then assembles to divide the cell in two during cytokinesis. MTs of the mitotic spindle determine plane of cleavage AND timing of cytokinesis which ensures that the cleavage furrow cuts btwn the two groups of segregated chromosomes and avoids aneuploidy

How does the mitotic spindle specify the position of the contractile ring?

1. The central spindle (interpolar microtubules) generates a furrow-inducing signal2. Overlapping interpolar MTs recruit proteins that activate RhoA at the cell equator3. RhoA controls the assembly and contraction of the actomyosin contractile ring4. The signal originates during anaphase, so this mechanism helps regulate proper timing of cytokinesis

Taurus experiment

Shows importance of overlapping microtubules. Process is not DNA-dependent as once believed

Active RhoA promotes the formation of contractile actomyosin

. RhoA is activated at the equatorial-cell complex. RhoA-GTP promotes F-actin assembly by activating formins. RhoA-GTP promotes Myosin II activation and assembly by activating ROCK (Rho kinase)

What happens if Rho is inhibited?

Chromosome separation is successful (Rho not involved), and cytokinesis is unsuccessful

The contractile ring divides one cell into two

. During cytokinesis cleavage furrow becomes visible on cell surface, deepens, and spreads around cell until it divides in two. The contractile ring generates the force for cleavage furrow formation. Composed primarily of F-actin and Myosin II as well as additional structural and regulatory proteins. Assembles on cytosolic face of plasma membrane and tethered to plasma membrane

Abscission

. Central region of the anaphase spindle is remodeled to form the midbody, densely packed region of MTs and proteins in the center of the narrow intracellular bridge between the daughter cells. Contractile ring constricts until it reaches the MTs at the midbody. The midbody directs abscission

What controls cell number and size?

1. Cell death2. Cell proliferation3. Cell growth

Apoptosis helps regulate animal cell numbers

Cells kill themselves in response to:• Developmental signal• Abnormal proliferation• Abnormal metabolism• Pathogen infection• DNA damage• Lack of survival signalKills the cell but saves the organism!

Apoptosis can remove structures that are no longer needed

Cells in the tail of a tadpole undergo apoptosis as it grows legs

Developmentally mediated apoptosis

. Unwanted cells removed by apoptosis. Hands and feet start out as spade-like structures and individual digits are separated through apoptosis

Apoptosis can help adjust the number of developing neurons to match the number of target cells

. Target cells produce a limited amount of survival signal. If more neurons are produced than can be supported by the limited survival signal, then some will undergo apoptosis. This ensures all target cells are contacted by neurons and all others are eliminated. Similar dependence on survival signal helps control cell numbers in other tissues during development and in adulthood

Necrosis vs apoptosis

Apoptosis: Programmed cell death by activating a regulated intracellular death program• Regulated process for cell to destroy itself by biochemical and morphological changes.• Produces cell fragments that phagocytic cells engulf and remove before the contents of the cell can spill outonto surrounding cells and cause damage.
Necrosis: : Accidental cell death caused by injury, radiation, chemicals, or lack of nutrition.• Not engulfed by phagocytic cells

often causes inflammation.

Characteristic features of apoptosis

• Cytoplasmic condensation (cell shrinkage) • Cytoskeleton collapse • Cell rounding • Chromatin condensation • DNA fragmentation • Membrane blebbing • Cell fragmentation • Formation of apoptotic bodies • Phagocytosis by macrophages or neighboring cells

Apoptosis is mediated by caspases

• Inactive initiator procaspases are activated in response to signals that induce apoptosis.• Activated adaptor proteins bring procaspases together to activate cleavage.• Pairs of cleaved initiator procaspases associate to form dimers, resulting in protease activation andcleavage of its partner at a specific site in the protease domain, thus forming an active initiator caspase.

Proteolytic caspase cascade during apoptosis

Active initiator caspases activate executioner caspases by cleavage - activating conformational change.• Executioner caspases cleave multiple target substrates in the cell -\> resulting in apoptosis.• Each initiator caspase can cleave many executioner caspases, which can in turn cleave many substrates = caspase cascade.
In short: initiator caspase -\> executioner caspase -\> apoptosis

Key targets of caspases involved in apoptosis

• Nuclear lamins - breakdown of nuclear lamina and nuclear envelope• Proteins that normally inhibit endonucleases - leads to fragmented DNA• Mitochondrial proteins - disrupts electron transport• Cytoskeletal, cell adhesion, and signaling proteins - causes cell to round upand detach from its neighbors

Intrinsic vs extrinsic apoptosis pathways

Intrinsic- Activation from INSIDE the cell, such as DNA damage or other stressors- Cytochrome C release from mitochondria and apoptosome assemblyExtrinsic- Activation from OUTSIDE the cell, such as a killer lymphocyte killing a cancer cell- Death receptor and DISC assemblyThese pathways use their own initiator caspase activation systems, but both use the same executioner caspases

The extrinsic apoptosis pathway: activated by cell-surface receptors

. Membrane bound ligands (such as Fas) on the signaling cell bind to the cell-surface death receptor (such as Fas death receptor) on the target cell. Death receptor proteins contain an extracellular ligand-binding domain and an intracellular "death domain". Separate adaptor proteins bind death receptors and recruit initiator caspases, forming a DISC, which activates the initiator caspases. Initiator caspases activate executer caspases to induce apoptosis

The intrinsic apoptosis pathway: depends on release of cytochrome C (ETC protein) from the mitochondria

1. BCl2 proteins control the release of cytochrome C.2. Cytochrome C binds a procaspase-activating adaptor protein, causing it to oligomerize into a pinwheel-like structure called the apoptosome3. Apoptosome recruits initiator procaspases bringing them into close proximity so they can activate one another4. This activates executioner procaspases, leading to apoptosisThe balance between pro and anti-apoptotic BCl2 proteins determines whether a cell undergoes the intrinsic pathway or not. They can heterodimerize in various ways and inhibit each other's activity

Pro-apoptotic BCl2 proteins Bax or Bak release cytochrome C from the mitochondria

1. Before an apoptotic stimulus they are just inactive monomers2. Afterwards, they aggregate in the mitochondrial outer membrane and form openings allowing cytochrome C to be released into the cytosol

Anti-apoptotic BCl2 proteins

Inhibit apoptosis by binding pro-apoptotic effector BCl2 proteins and preventing them from aggregating so cytochrome C is not released

Pro-apoptotic BH3 only proteins (such as Bad) promote the intrinsic pathway

They either...1. Bind and inhibit anti-apoptotic BCl2 proteins2. Directly bind pro-effector BCl2 proteins to trigger their aggregation on the mitochondrial outer membraneThe cell can both produce BH3-only pro-apoptotic proteins or activate ones already made

Would treatment of cells with an inhibitor of Bcl2 (disrupts its protein-protein interactions) promote apoptosis or promote cell survival? Why?

It would promote apoptosis because Bcl2 would not be able to interact with and inhibit Bax and Bak, so they would aggregate and allow cytochrome c release from mitochondria, thus promoting intrinsic apoptosis.

Survival signals inhibit apoptosis

. Survival signals bind cell-surface receptors, activating intracellular signaling pathways that inhibit apoptosis, often by regulating BCl2 family proteins. Some signals increase production of anti-apoptotic BCl2 proteins, others inhibit the function of pro-apoptotic BH3-only proteins such as BadWhen cells are DEPRIVED of survival signals they can produce and/or activate pro-apoptotic BH3-only proteins which activates apoptosis

The PI 3-Kinase/Akt Signaling Pathway Promotes Cell Survival

1. Extracellular survival signal activates an RTK, which recruits and activates PI 3-kinase2. PI 3-kinase produces an inositol phospholipid, which serves as a docking site for the kinase Akt3. Activated Akt dissociates from the PM and phosphorylates pro-apoptotic BH3-only protein Bad- releases anti-apoptotic protein BCl2 which was held in an inactive state by binding Bad4. BCl2 inhibits Bak/Bax proteins from causing cytochrome C release, inhibits apoptosis

Comparing the two apoptosis pathways - intrinsic and extrinsic - which of the followingstatements is true?A. The intrinsic pathway is receptor-mediated, while the extrinsic pathway is mitochondria mediated.B. Bcl2 family proteins are involved in regulating only the extrinsic pathway.C. The intrinsic pathway involves the release of cytochrome c into the cytoplasm, while the extrinsic pathway requires Fas ligand/Fas death receptor interactions.D. The intrinsic pathway uses executioner caspases, while the extrinsic pathway uses initiator caspases.E. Apoptosome formation is required to activate initiator caspases for the extrinsic pathway.

C. The intrinsic pathway involves the release of cytochrome c into the cytoplasm, while extrinsic pathway requires Fas ligand/Fas death receptor interactions.

Compare similarities and differences between the formation of the deathinducing signaling complex (DISC) for the extrinsic apoptosis pathway with the formation of the apoptosome for the intrinsic apoptosis pathway

DISC: Fas ligand binding Fas death receptor is what initiates complex formation. Adaptor proteins that bind to Fas death receptor recruits procaspases. Downstream of procaspases is activation of executioner caspasesApoptosome: release of cytochrome C from the mitochondria initiates complex formation. Adaptor protein complexed with cytochrome C recruits procaspases. Downstream of procaspases is activation of executioner caspases

Mitogens

Stimulate cell division by overcoming molecular brake mechanisms in the cell cycle control system

Growth factors

Stimulate cell growth by promoting protein synthesis and inhibiting degradation of proteins and other macromolecules

Cells are organized into tissues

Epithelial, connective, nervous, muscle, etc.Tissues can be an organized assembly of:1. cells held together by cell-cell adhesions such as in the skin- little ECM, cellular cytoskeleton carries the mechanical load2. ECM- in bone or tendon ECM is plentiful and carries the mechanical load3. Both cells and ECM, such as connective tissue in the intestinal wall

Epithelial cells form sheets that separate different compartments in the body

Epithelial tissues are present in the esophagus, air sacs of lungs, mammary glands, kidneys, intestines and skin

Cells can be packed together in different ways to form an epithelial sheet

. There are four basic types of epithelia: squamous, columnar, cuboidal and stratified. Epithelial cells sit on a thin mat of extracellular matrix- the basal lamina, which may be associated with underlying connective tissue

The ECM of animal connective tissues

. The connective tissue underlying an epithelium contains a variety of cells and ECM components. The predominant cell type is fibroblasts, which secrete the ECM. Collagen and fibronectin are key fibrous components of the connective tissue ECM. Polysaccharides and associated proteins form a hydrated gel that fills the spaces in the fibrous network and resists compressive forcesConnective tissue ECM can take on many forms:- ECM underlying an epithelium- Calcified ECM forms bone and teeth- Transparent substance of the cornea- Ropelike organization of tendons. The tensile strength in animal connective tissue is not generated by the cytoskeleton, rather by collagen

Collagen is the major protein of connective tissue ECM

. Long, stiff, triple-strand helix- 3 polypeptide chains (alpha chains) wound around each other- Collagen is secreted into the extracellular space, then 3-stranded collagen helices assemble into higher-order polymers called collagen fibrils- Collagen fibrils often aggregate into huge cable-like bundles several microns in diameter called collagen fibers, which help resist tensile forces. Collagen is rich in glycine and proline- Glycine regularly spaced every 3rd residue to allow alpha chains to pack tightly- Proline ring structure helps stabilize helical conformation in each alpha chain

Procollagen precursors are cleaved to form mature collagen and assemble into fibrils outside the cell

. Fibroblasts secrete collagen and the other extracellular matrix molecules- Synthesized internally- Secreted by exocytosis- Assembles into huge collagen fibrils outside the cell. Collagen assembled initially as a procollagen molecule with unstructured terminal peptides on either end, which keep it from assembling into fibrils inside the cell. An extracellular enzyme removes the terminal peptides and the mature collagen molecules can now assemble into collagen fibrils

Which of the following statements about animal connective tissues is true?A. Collagen synthase enzymes embedded in the plasma membrane synthesize the collagen in the extracellular matrix.B. In connective tissue, the intermediate filaments within the cells are important for carrying the mechanical load.C. Collagen is synthesized as procollagen and secreted to the outside of the cell in a secretory vesicle.D. A Collagen synthase enzyme organizes the mature collagen molecules into ordered collagen fibrils.

C. Collagen is synthesized as procollagen and secreted to the outside of the cell in a secretory vesicle.

Fibroblast cells also organize the ECM

Fibroblasts organize collagen fibers by tugging on them- This generates parallel bundles of collagen fibers in ligaments and tendons

Collagen fibers in bone

. The bone matrix occupies most of the volume in the tissue. The cells (osteoblasts) appear as small, dark objects in the bone matrix. Layers of bone matrix containing oriented collagen fibers form striped bands. Calcium phosphate crystals fill the space between the collagen fibers to make the bone matrix resistant to compression and tension

Collagen fibers are arranged in a "plywood-like" pattern in some tissues

. Collagen fibrils in tadpole skin are arranged in a "plywood-like" pattern- alternating layers with different orientations in order to resist tensile stress in multiple directions. This arrangement is also found in mature bone and the cornea

Abnormally stretchable skin is one example of the defects that arise in genetic syndromes due to defects in collagen assembly. (a)What types of mutations would you predict disrupt collagen fibril assembly?(b)Would you expect collagen mutations to be detrimental if only one of the two copies of a collagen gene is defective?

a) mutations in glycine (which is present every 3rd position and necessary for assembly) or a mutation in the enzyme that converts procollagen into collagenb) 3 collagen alpha chains come together to form a helix, so even one defective mutant will impair assembly

How do animal cells interact with and migrate on the extracellular matrix?

1. Protrusion- actin polymerization at the leading edge2. Adhesion/Traction- focal adhesions assemble at the leading edge and disassemble at the trailing edge3. Retraction- trailing edge retraction requires actomyosin contractility

Cell-matrix adhesion is necessary for cell migration

. For a cell to move, it must adhere to an ECM and exert traction force. Cell-matrix adhesions (focal adhesions) occur at specific places where the actomyosin cytoskeleton on the inside of the cell is linked via transmembrane integrins to the ECM on the outside of the cell

Formation of focal adhesions in a migrating goldfish fibroblast

Some focal adhesions rapidly assemble and then disassemble, others make stable connections with actomyosin stress fibers to generate traction force allowing the cell to migrate

Integrins link the ECM to the actin cytoskeleton

. Fibronectin (ECM) is a dimer of two large subunits joined by two disulfide bonds at one end- Made up of a series of domains separated by flexible linkers- Has cell attachment sites and extracellular matrix-binding sites. Integrin is single-pass transmembrane protein- forms a heterodimer (alpha and beta subunits)- Extracellular domain binds to fibronectin- Intracellular domain binds to actin filaments