CELL CYCLE

• In Unicellular species (e.g., bacteria and yeasts) – each cell division produces a complete new
organism

• S phase – “S” for DNA synthesis; when chromosome duplication occurs; occupies half of the cell-
cycle time in mammals
• GAP phases – allows for cell growth; ensures suitable conditions and complete preparation of cell
• M phase – “M” for Mitosis; when chromosome segregation (nuclear division; mitosis) and cell
division (cytokinesis) occur; occupies only a short time

4 PHASES OF EUKARYOTIC CELL CYCLE

• Cell growth occurs throughout the cell cycle, EXCEPT during Mitosis
• Interphase – refers to S Phase + G Phases

An abrupt change in the biochemical state of the cell occurs at the Metaphase-to-Anaphase Transition. A
cell can pause in metaphase before this transition point, but once it passes this point, the cell carries on
to the end of mitosis and through cytokinesis into interphase.

1. G0 Phase (G zero)
a. Resting state – cells exist in a quiescent state
b. Nerve and cardiac cells – remain permanently in this state once they reach maturity
2. G1 Phase (“Start” for yeasts; “Restriction Point” for mammalian cells)
a. In late G1
b. Cells are committed to DNA synthesis and no longer requires growth factors to complete
the cell cycle
c. Point of no return
d. Accumulation of Labile R protein to a critical amount before a cell can pass the R point
and proceed towards DNA synthesis.
3. S Phase – chromosome duplication
4. G2 Phase
5. M Phase (Mitosis and Cytokinesis)
 THE CELL CYCLE CONTROL

• Biochemical Switches that control cell cycle are…

o Binary (on/off) and Irreversible
o Robust and Reliable – system may still operate even if some components fail
o Highly Adaptable and Modifiable

• In G1 – cells have unreplicated complement of DNA

o Have greater number of cells than in G2 + M phase
• In G2 and M phases – cells have fully replicated complement of DNA (twice the G1 DNA
content)
• In S phase – cells have an intermediate amount of DNA
 CYCLIN-DEPENDENT KINASES (Cdks)

• Fluctuations in Cdks’ activities → cyclical changes in phosphorylation thru the cycle

• Increase in Cdk activity at the G2/M transition → increased phosphorylation of proteins that
control chromosome condensation, nuclear-envelope breakdown, spindle assembly, and other
events that occur in early mitosis

• Cyclins – regulates Cdks; where Cdks bind → kinase activity

o NO Cyclin → Cdk is INACTIVE

• G1-cyclins – helps govern the activities of G1/S-cyclins

• G1/S-cyclins – activate Cdks in late G1 and thereby help trigger progression through Start,
resulting in a commitment to cell-cycle entry
o G1/S-cyclins levels fall in S phase
• S-cyclins – bind Cdks soon after progression through Start and help stimulate chromosome
duplication
o S-cyclin levels remain elevated until mitosis
o S-cyclins also contribute to the control of some early mitotic events
• M-cyclins – activate Cdks that stimulate entry into mitosis at the G2/M transition
o M-cyclin levels fall in mid-mitosis

• The concentration of the 3 major cyclin types (last 3) oscillate during the cell cycle .
• The concentration of Cdks DO NOT change and DO NOT EXCEED cyclin amounts.
Cyclin-Cdk complexes of the Cell-cycle Control System

1. G1 phase starts
2. G1/S-cyclin levels rise in late G1 → leads to formation of G1/S-Cdk complexes
3. G1/S-Cdk complexes trigger progression through the Start transition
a. G1/S cyclin levels deplete
4. S-cdk complexes form at start of S phase
5. S-cdk complexes trigger DNA replication and some early mitotic events
a. M-cdk complexes form during G2 but are held inactive
b. M-cyclin levels gradually increase throughout G2
6. M-cdk complexes activated at the end of G2 → trigger entry into mitosis at the G2/M transition
a. S-cyclin levels rapidly deplete
7. Regulatory protein APC/C initiates metaphase-to-anaphase transition
a. M-cyclin levels deplete

Cdk Activation by Cyclin Attachment

T-loop – protein that blocks Cdk’s active site

1. Cyclin binds to Cdk → T-loop blocking

the active site moves out → partial
activation of Cdk

2. Cdk-activating kinase (CAK)

phosphorylates threonine residue in T-
loop → Full activation of Cdk

3. Activated Cdk transfers the phosphate

from ATP to the target serines and
threonines on target proteins
 Inhibition by PHOSPHORYLATION

• Wee 1 – phosphorylates a pair of amino acids in the roof of

the kinase active site of the cyclin-Cdk complex → INHIBITS
Cdk activity

Activation by DEPHOSPORYLATION

• Cdc25 – Dephosphorylates the a.a. in the roof of the kinase

active site → INCREASES Cdk activity

• CKIs (Cdk inhibitor proteins) – binds to cyclin-Cdk complex →

INACTIVATES the complex

Inhibition of Cyclin-Cdk Complex Activation by a CKI (Cdk inhibitor proteins)

ANAPHASE-PROMOTING COMPLEX or CYCLOSOME (APC/C)

• Anaphase-Promoting Complex or Cyclosome (APC/C) – key regulator of the metaphase-to-

anaphase transition (works by protein DESTRUCTION)
o This is the counterpart of cyclin-Cdk complexes which drives progression through the
Start and G2/M transitions (works by protein PHOSPHORYLATION)

• Member of the Ubiquitin Ligase family of enzymes – POLYUBIQUITYLATE proteins → for

destruction by proteasomes
• Catalyzes the ubiquitylation and destruction of 2 major types of proteins…
1. Securin – protects the protein linkages that hold sister-chromatid pairs together in
early mitosis
2. S- and M- cyclins – destruction of these leads to inactivation of most Cdks in cell
a. As a result, many proteins phosphorylated by Cdks from S phase to early
mitosis are dephosphorylated by various phosphatases in the anaphase cell
b. Required for the completion of M phase

• APC/C is…
o Activated in mid-mitosis by Cdc20 (or in late mitosis through early G1 by Cdh1)
o Remains active in G1 (after mitosis) – to provide stable period of Cdk inactivity
o Turned off when G1/S-Cdk is activated in late G1

• SCF – ubiquitin ligase responsible for the ubiquitylation of certain CKI proteins in late G1
o Helps control activation of S-Cdks and DNA replication
o Also responsible for the destruction of G1/S-cyclins in early S phase

REGULATED PROTEOLYSIS BY APC/ TRIGGERS METAPHASE-TO-ANAPHASE TRANSITION

1. Cdc20 activates APC/C during mitosis

2. Activated APC/C recognizes amino acid sequences on M-cyclin and other target proteins
3. E1 and E2 (ubiquitylation enzymes) help APC/C assemble polyubiquitin chains on the target
protein
4. Polyubiquitylated protein is detected by proteasome
5. M-cyclin is degraded in proteasome
 CONTROL OF PROTEOLYSIS BY SCF

1. A Kinase phosphorylates the target protein (CKI)

2. Phosphorylated target protein is recognized by the F-box protein of the Active SCF Complex
3. E1 and E2 (ubiquitylation enzymes) help APC/C assemble polyubiquitin chains on the target
protein
4. Polyubiquitylated protein is detected by proteasome
5. CKI is degraded in proteasome
 OVERVIEW OF CELL CYCLE CONTROL SYSTEM

1. Conditions for cell proliferation are right → external and internal signals stimulate the activation
of G1-Cdk
2. G1-Cdk stimulates the expression of genes encoding G1/S- and S-cyclins → G1/S-Cdk activated
3. Activated G1/S-Cdk drives progression through the Start transition
4. G1/S-Cdks unleash a wave of S-Cdk activity
5. S-Cdk activity initiates chromosome duplication in S phase and also contributes to some early
events of mitosis → M-Cdk activated
6. M-Cdk activation triggers progression through the G2/M transition and the events of early
mitosis → sister-chromatid pairs align at the equator of the mitotic spindle
7. APC/C, together with its activator Cdc20, triggers the destruction of securin and cyclins → sister-
chromatid separation and segregation → completion of mitosis
8. Multiple mechanisms collaborate to suppress Cdk activity → stable G1 period

S PHASE

• Prereplicative complex (preRC) – initiation of DNA synthesis is permitted only at origins

containing a preRC.
 OVERVIEW OF CELL CYCLE CONTROL SYSTEM

Origin recognition complex (ORC) binds to

the replication origin throughout the cell
cycle.

1. In early G1, Cdc6 and Cdt1 associate with

ORC → bind to DNA helicase

2. Using energy from ATP hydrolysis, ORC and

Cdc6 proteins load 2 copies of inactive
DNA helicases next to origin → preRC
formed → origin is now licensed for
replication

3. S-Cdk stimulates assembly of several

initiator proteins on each DNA helicase

4. At the same time, DDK phosphorylates

subunits (Mcm proteins) of DNA helicase
→ DNA helicases activated

5. DNA helicase unwinds the DNA → DNA

polymerase and other proteins recruited
→ DNA replication begins

6. S-Cdk phosphorylates and inhibits ORC,

Cdc6, and Ct1 → prevent formation of new
preRCs at the origin until the end of mitosis

To reset cell cycle system for the next cycle…

• APC/C activation → triggers destruction of Cdt1 inhibitor (geminin), Cdt1 activation,

dephosphorylation of ORC and Cdc6
MITOSIS (M phase)
 ACTIVATION OF M-Cdk

1. M-cyclin levels gradually rise → Cdk1 associates with M-cyclin → formation of M-Cdk complex
2. M-Cdk complex is phosphorylated on…
a. an activating site by the Cdk-activating kinase (CAK) and on…
b. a pair of inhibitory sites by the Wee1 kinase → INACTIVE M-Cdk complex
3. Phosphatase Cdc25 dephosphorylates M-Cdk complex → ACTIVE M-Cdk complex
4. Cdc25 is further stimulated by active M-Cdk → Positive feedback
a. This feedback is enhanced by the ability of M-Cdk to inhibit Wee1

PRINCIPAL STAGES OF M PHASE

CONDENSIN
 MITOTIC SPINDLE IS A MICROTUBULE-BASED MACHINE

• Interpolar (polar) microtubules

o Overlap with + ends of microtubules from the other pole at the center
o Form structural basket that maintain the mechanical integrity of the spindle

• Astral microtubules
o Radiate out from the poles into the cell cortex
o Microtubules are arranged in an aster around each centrosome
o Help position the spindle apparatus in the cell
o Help determine plane of cytokinesis

• Kinetochore microtubules
o Attach sister chromatid pairs at kinetochores located at the centromere of each sister
chromatid
 MITOTIC SPINDLE IS A MICROTUBULE-BASED MACHINE

• Kinesin-related proteins – usually move toward the plus (+) end of the microtubules
• Dyneins – usually move toward the minus (-) end

• Kinesin 5
o Has 2 motor domains that interact with the ends of antiparallel microtubules in the
spindle equator causing them to slide past each other towards the spindle poles,
pushing the centrosomes/poles apart

• Kinesin 14
o Single motor domain directed to the end; cross link antiparallel interpolar microtubules
at the midzone which tend to pull poles together

• Kinesin 4 and Kinesin 10

o Chromokinesins
o (+) end directed motors
o Link microtubules to chromosome arms and push attached chromosomes away from
the pole

• Dyneins
o (-) end directed
o Link the (+) ends of the astral microtubules to components of actin at cell cortex
o Pull the spindle poles toward the cell cortex and away from each other

• Dynein and Kinesin 5

o Promote centrosome separation
o Increase spindle length

• M-Cdk and Aurora A

o Phosphorylate kinesin 5

• Aurora A and Plk

o Phosphorylate centrosome component → promotes centrosome maturation
Box 1: Motor-dependent mechanisms establish bipolarity as Eg5 (kinesin-5) motors slide antiparallel
microtubules apart with their minus ends leading and their plus ends directed toward the spindle equator.

Box 2: Minus end-directed motors such as dynein move microtubules poleward with their minus ends
leading, thereby incorporating K-fibers into the spindle, and focusing spindle poles.

Box 3: Kinetochore-associated dynein transports chromosomes along astral microtubules toward the
spindle poles from the periphery.

Box 4: Plus end-directed chromokinesins (kinesin-4 and-10) eject chromosome arms outward.

Box 5: CENP-E (kinesin-7) transports unattached kinetochores toward the equator along spindle
microtubules. MTOC, microtubule organizing center.
• G1/S-Cdk (cyclin E and Cdk2)
o Triggers cell cycle entry and initiates centrosome duplication

• S phase
o Daughter centriole begins to grow near the base of the mother centriole and at right
angle to it

• Before start of M phase

o 2 centriole pairs within a pericentriolar matrix split into 2 daughter centrosomes
• Semiconservative duplication of centrosome; once per cell cycle

MITOTIC CHROMOSOMES PROMOTE BIPOLAR SPINDLE ASSEMBLY

• Mitotic chromosomes stimulate local activation of proteins for nucleation and formation of
spindle microtubules.
• Motor proteins – orient the microtubules
• Kinesin 5 – organize microtubules into antiparallel bundles in spindle mid zone
• Movement of dynein and kinesin 14 causes the minus ends of the microtubules to converge to
form a distinct spindle pole
SLIDE 27 PPT
 CELL DEATH

APOPTOSIS

• Programmed cell death

• Cells undergo Morphological Changes
o Shrink and condense
o Cytoskeleton collapses
o Nuclear Envelope disassembles
o Nuclear Chromatin condenses → breaks into fragments
o Cell Surface bulges outward
▪ If cell is LARGE → breaks into APOPTOTIC BODIES
▪ Apoptotic bodies chemically alter cell surface → signals Macrophage to
Phagocytose the damaged cell (prevents inflammation)

• Helps in Embryonic Development (e.g., spaces bet. fingers in hands & feet; Interdigital cell death)
• Happens when…
o Structure is not needed anymore
o Abnormal, misplaced, nonfunctional, or potentially dangerous cells
▪ E.g., apoptosis of developing T and B lymphocytes that either…
• Fail to produce potentially useful antigen-specific receptors or
• Produce self-reactive receptors (makes it potentially dangerous)

• Regulation of tissues that neither grow nor shrink

o Liver
▪ Drug: Phenobarbital – induces cell division
▪ Response: Increased apoptosis until normal liver size is obtained

• Reduced or Elevated Apoptosis → Disease

o Cancer – B cell lymphoma due to excessive production of anti-apoptotic Bcl2 (reduced
apoptosis)
o Parkinson’s, Alzheimer’s, and Huntington’s diseases
o Diabetes type I – autoimmune; destruction of beta cells that produce insulin
 CASPASES MEDIATES APOPTOSIS

• Caspase – triggers apoptosis; cleave specific sequences in numerous proteins inside cell
o Have a CYSTEINE at their ACTIVE SITE
o Cleaves target cell at specific ASPARTIC ACIDS
▪ CysteinsASPartic-acid-ASE
o Synthesized as inactive precursors (activated only during apoptosis)
• 2 Major Classes of Caspase
1. INITIATOR CASPASE – begins apoptotic process
a. Inactive, soluble monomers (PROCASPASE) in cytosol
b. Activates Executioner caspase (by cleavage)
c. Protease domain at carboxy-terminal region
d. Small protein interaction domain at amino terminus
2. EXECUTIONER CASPASE

CASPASE ACTIVATION DURING APOPTOSIS

1. Apoptotic signal triggers assembly of adaptor proteins

2. Initiator caspases bind to multiple binding sites of adaptor proteins → forms large complexes
a. Either through extrinsic or intrinsic pathway, or mitochondrial pathway
 3. Within the complex, Initiator caspases DIMERIZE → Activated Initiator Caspase → individual
caspases in dimers cleave each other at a specific site in their protease domain → stabilized
complex
a. In some cases, protease domain of Initiator caspase is also cleaved
4. Protease domains rearranged into large and small subunits
5. Initiator caspase cleaves Executioner Caspase in protease domain → conformational change →
Activated Executioner Caspase
6. Executioner caspase then cleaves variety of key proteins → controlled cell death (APOPTOSIS)

Caspase Cascade is IRREVERSIBLE (destructive and self-amplifying)

Initiator Caspase is first activated in response to an apoptotic signal via Extrinsic or Intrinsic Pathway

DNA Fragmentation during Apoptosis

In healthy cells, endonuclease CAD (Caspase Activated DNAse)

associates with its inhibitor iCAD…

1. Executioner caspase (either 3, 6, or 7) activated →

cleaves iCAD (nuclease released)
2. Activated CAD cuts chromosomal DNA between
nucleosomes
3. DNA fragments formed

Protein Kinases (e.g., FAK) cause apoptotic cell detachment from

its neighbors

Nuclear lamins: disassembly of nuclear lamina and nucleus

shrinkage

Activation of Puma and Noxa in response to DNA Damage (important in cancer treatment)

1. DNA damage detected → tumor suppressor protein p53 accumulates (lack of or mutated P53 →
cancer cells survive)
2. p53 activates gene transcription of genes that encode BH3-only proteins Puma and Noxa
3. Puma and Noxa triggers the intrinsic pathway of apoptosis → damaged DNA eliminated

EXTRINSIC PATHWAY OF APOPTOSIS

• Activated by Cell-surface Death Receptors – belong to Tumor Necrosis Factor (TNF) receptor
family (homotrimers)
o Has an extracellular ligand-binding domain, a single transmembrane domain, an
intracellular death domain (required to activate apoptosis)
• Recruits the intrinsic pathway to amplify the caspase cascade to kill cells
o Bid – a BH3-only protein that links the extrinsic and intrinsic pathways of apoptosis
▪ Is normally inactive (activated by cleavage of caspase-8 → Bid inhibits anti-
apoptotic Bcl2 family proteins → amplified death signal)
EXTRINSCIC PATHWAY OF APOPTOSIS ACTIVATED THROUGH FAS DEATH RECEPTORS

1. Trimeric FAS ligands (FasL; or Tumor Necrosis Factor <TNF>) on surface of a killer lymphocyte
attaches to trimeric FAS receptors (TNF receptor; TNFR1 or TNFR2) on surface of target cell
2. Several ligand-bound receptor trimers cluster → activates Death Domains on the FAS receptor
tails
3. Death domains of activated FAS receptor interact with similar death domains on the adaptor
protein FADD (FAS-Associated Death Domain)
4. Each FADD protein recruits an initiator caspase (caspase-8) via death effector domain on both
FADD and the caspase → DISC (Death-Inducing Signaling Complex) formed
5. Within DISC, 2 adjacent initiator caspases CLEAVE one another → activated protease dimer
formed
6. Protease dimer cleaves itself in the region linking the protease to the death effector domain →
stabilized active caspase dimer
7. Caspase dimer is released into cytosol → active caspase dimers activate executioner caspase (by
cleaving them) → apoptosis

“Activated caspase-8 may activate Bid to amplify death signal, or activate intrinsic pathway.”
Regulation of Apoptosis through Bid (Amplification of Caspase Cascade)

1. Caspase-8 cleaves Bid → Bid become activated → tBid (truncated Bid) generated
2. tBid binds to Bax
3. Bax inserts into the Outer Mitochondrial Membrane → Caspase cascade amplified
a. tBid also inhibits anti-apoptotic Bcl2

INTRINSIC or MITOCHONDRIAL PATHWAY OF APOPTOSIS

• Depends on Mitochondria
• Often done due to DNA damage or in response to development signals (in vertebrate cells)
o Irreparable DNA
o High cytosolic Ca2+ concentration
o Viral infection
• Cytochrome c – binds to the adaptor protein Apaf1 (Apoptotic Protease Activating Factor-1)
o Apaf1 oligomerize into a wheel-like heptamer called Apoptosome
o Apoptosome recruits caspase-9 proteins (only those in proximity)

• Bcl2 Family Proteins – regulate intrinsic pathway of apoptosis

o Pro-apoptotic Bcl2 – promotes apoptosis by enhancing the release of cytochrome c
▪ Effector Bcl2 Family Proteins – structurally similar to Bcl2 but NO BH4 domain
• BAX – mainly located in cytosol; translocated to the mitochondrial outer
membrane only in the presence of apoptotic signal
• BAK – bound to the mitochondrial outer membrane even with NO
apoptotic signal

“Mutation in BAX and BAK leads to resistance to pro-apoptotic signals.”

“Only 1 of the 2 effector Bcl2 proteins is needed to activate intrinsic
pathway of apoptosis.”
“Activation of BAX and BAK depends on activated pro-apoptotic BH3-only
proteins.”

▪ BH3-only protein – share sequence homology with Bcl2 in only the BH3 domain
• Inhibits anti-apoptotic Bcl2 family proteins
• Promote apoptosis mainly by inhibiting anti-apoptotic Bcl2 family
proteins
o Some may directly bind to BAX and BAK to help stimulate their
aggregation

o Anti-apoptotic Bcl2 – inhibit apoptosis by blocking the release of cytochrome c

▪ Bcl2 – has 4 BH domains (BH1-4); located on the cytosolic surface of outer
mitochondrial membrane
▪ BclX1 – has 4 BH domains (BH1-4); located on the cytosolic surface of outer
mitochondrial membrane

“At least 1 of the 5 anti-apoptotic Bcl2 protein is needed for a mammal to survive.”
The Intrinsic Pathway of Apoptosis

1. Intracellular Apoptotic Stimuli activates effector Bcl2 family proteins (BAK or BAX) → BAK or BAX
proteins aggregate on the outer mitochondrial membrane → causes mitochondria to release
Cytochrome c and other proteins
2. Cytochrome c binds with Apaf1 → Apaf1 unfold partly → domain that interacts with the same
domain in other activated Apaf1 molecules exposed
3. Seven activated Apaf1 proteins form a large ring complex called the Apoptosome
a. Each Apaf1 protein contains a caspase recruitment domain (CARD), and these are
clustered above the central hub of the apoptosome.
b. CARD is related in structure and function to the death effector domain of caspase-8
4. CARDs of Apaf1 bind to CARDs in multiple caspase-9 molecules → caspase-9 recruited into the
apoptosome → caspase-9 activated
5. Activated caspase-9 cleaves and thereby activates downstream Executioner caspases
6. Caspase cascade → apoptosis
• Inhibitors of Apoptosis (IAPs)
o Have 1 or more BIR (Baculovirus IAP Repeat) domains – enables them to bind to and
INHIBIT activated caspases
o Some polyubiquitylate caspases → to be destructed by proteasomes
• Anti-IAPs – promotes apoptosis
o Smac, Diablo, Omi
o Bind to IAPs to prevent their binding to caspases for apoptosis to occur

APOPTOSIS INHIBITION BY EXTRACELLULAR SURVIVAL FACTORS

• Survival Factors – extracellular signals that inhibit apoptosis

Role of Survival Factors in Adjusting the Number of Developing Nerve Cells to Amount of Target Tissue

1. Survival factors bind to cell-surface receptors

2. Bcl2 family protein regulated
a. Stimulate synthesis of anti-apoptotic Bcl2 family proteins (Bcl2 and BclX1)
b. Inhibits function of pro-apoptotic BH3-only proteins (Bad)
c. Phosphorylates and inactivates anti-IAP proteins (Hid)
3. Intracellular signaling pathways that suppress apoptotic program activated

A. Increased Production of Anti-apoptotic Bcl2 family protein

1. Survival factor binds to cell-surface receptor

2. Transcription regulator activated → gene encodes for Bcl2 protein
3. Bcl2 protein blocks apoptosis

B. Inactivation of Pro-apoptotic BH3-only protein

1. Survival factor binds to cell surface receptor → activated cell receptor

2. Activated receptor activates Akt (serine/threonine protein kinase)
3. BH3-only protein Bad is phosphorylated and inactivated → Bcl2 activated
4. Bcl2 suppresses apoptosis

C. Inactivation of anti-IAPs (in Drosophila)

1. Survival factor binds to cell-surface receptor → activated receptor

2. Activated receptor activates MAP kinase cascade
3. Anti-IAP protein Hid is phosphorylated → IAP activated
4. IAP blocks apoptosis
 CASPASE-DEPENDENT APOPTOSIS (ACTIVATION OF CASPASE 3)

“Activation of caspase 3 and caspase-dependent apoptosis are caused by the initiator caspases, caspase
8 and caspase 9, as part of the extrinsic and intrinsic apoptotic signaling pathways, or by granzyme B.”
CASPASE-DEPENDENT APOPTOSIS

Starting from the Extrinsic Pathway

1. Fas Ligand (FasL) binds to Fas receptors → activated Fas receptors

2. Activated receptors, FADD, and pro-caspase 8 form a DISC → caspase 8 is activated
3. Activated caspase 8 activates caspase 3
4. Activated caspase 8 cleaves BID → truncated BID (tBID) formed
5. tBID activates the intrinsic apoptotic pathway

In the Intrinsic Apoptotic Pathway

1. Pro-apoptotic BCL-2 family members BAX and BAK1 (BCL-2 Antagonist Killer 1) form pores in the
mitochondrial membrane
2. Pro-apoptotic mitochondrial factors, including DIABLO (also known as SMAC) and Cytochrome c
are released
a. DIABLO inhibits IAPs which inhibits Caspase 9 and Caspase 3
b. Cytochrome C activates Caspase 9
3. Caspase 9 activates Pro-caspase 3 (turns into Caspase 3) → induces apoptosis

• Granzyme B – can directly cleave and activate pro-caspase 3; it can also cleave BID, resulting in
granzyme tBID, which can activate the Intrinsic apoptotic pathway.
• cFLIP (cellular FLIP/caspase 8 inhibitor protein) can block the activation of caspase 8.

Clearance of Apoptotic Cells

1. During blood coagulation, intracellular Ca2+ level is elevated → stimulates scramblase activity
on the PM of platelets (left)
2. Phospholipid scrambling causes exposure of PHOSPHATIDYLSERINE on the surface
3. Coagulation protein complexes assemble on phosphatidylserines
During Apoptosis…

1. Caspases (3 and 7) activate scramblases (mediated by Xkr8) and inactivate flippases (Atp11C)
a. Done by cleaving at their caspase-recognition sites (right)
2. Phospholipid scrambling by scramblase → Phosphatidylserine move to the outer leaflet (on
the surface of the apoptotic cell where it acts as an “eat me” signal for macrophages)
3. Macrophages detect phosphatidylserines → apoptotic cell PHAGOCYTOSED

NECROSIS

• Cell death in response to an acute insult (damage or trauma or lack of blood supply)
• Most of the time caused by ENERGY / ATP DEPLETION → dysregulation of ion homeostasis across
cell membrane

• Cells undergoing Necrosis…

o Swell and Burst → contents spill over neighboring cells → release of DAMP (damage-
associated molecular patterns) → elicits and sustains Inflammatory response for
immune cells

• Necroptosis – a form of necrosis; highly regulated and programmed; triggered by specific

regulatory signal
o “Inflammatory Cell Death”
o Mediated by Proteases (in contrast to Apoptosis which is mediated by...?)

MOLECULAR MECHANISM OF APOPTOSIS AND NECROPTOSIS

 • Death receptor – mediates both extrinsic apoptosis and necroptosis
o Activation of caspase-8 – drives the pathway towards apoptosis
o Inhibition of caspase-8 – leads to necroptosis.

Necroptotic Pathway

1. RIPK1 and RIPK3 interact with each other → necrosome is formed

2. Necrosome (complex) phosphorylates MLKL (Mixed Lineage Kinase domain-Like) → promotes
oligomerization of MLKL
3. Oligomeric form of MLKL form plasma membrane channels for cytoplasmic leakage of the target
cell
a. Oligomeric MLKL translocate towards the plasma membrane from cytosol → pore
formation → inflammatory response
 CELL JUNCTIONS AND THE EXTRACELLULAR MATRIX

• Connective tissues – (e.g., bone or tendon) are formed from an extracellular matrix (ECM)
o ECM – produced by cells that are distributed sparsely in the matrix
▪ It is the MATRIX—rather than the cells—that bears most of the mechanical
stress to which the tissue is subjected

• Direct attachments between one cell and another are relatively rare. But the cells have
important attachments to the matrix.
o Cell–matrix junctions – link the cytoskeleton to the matrix, allowing the cells to move
through the matrix and monitor changes in its mechanical properties.

• In Epithelial Tissues
o ECM is less pronounced (only a thin mat of basal lamina)
o Unlike in usual connective tissue, the CYTOSKELETONS serve as the main stress-bearing
component rather than the ECM
 • 2 Types of ANCHORING JUNCTIONS that Link Cytoskeletons of Adjacent Cells

1. Adherens Junctions – anchorage sites for actin filaments

2. Desmosomes – anchorage sites for intermediate filaments

• 2 Types of ANCHORING JUNCTIONS that Link Cytoskeletons of Cells to Basal Lamina

1. Actin-linked cell-matrix junctions – anchor actin filament to the matrix

2. Hemidesmosomes – anchor intermediate filaments to the matrix

2 Other Types of CELL-CELL JUNCTIONS

• Tight junctions – hold the cells closely together near the

apex, sealing gap between the cells
o Prevent molecules from leaking across epithelium
• Gap junctions – are channel-forming junctions
o Create passageways that link cytoplasms of
adjacent cells

TRANSMEMBRANE ADHESION PROTEINS – span plasma membrane; one links to cytoskeletons inside
cell; one links to other structures outside cells

1. Cadherin – superfamily; mediate attachment of cells to cell → cadherin-based anchoring

junctions:
a. If linked to actin → forms Adherens junctions
b. If linked to intermediate filaments → forms Desmosomes
2. Integrin – superfamily; mediates attachment of cells to matrix (as in Hemidesmosomes)
 CADHERINS (ADHERENS JUNCTION AND DESMOSOMES)

• Fungi and Plants – LACK cadherins (also in some bacteria and archaea)
o Cadherins seem to be part of the essence of being an ANIMAL
• CALCIUM-DEPENDENT
o NO Ca2+ in extracellular medium = NO adhesion by cadherins
• Glycoproteins; mediate cell-cell adhesion with 5 TANDEM REPEAT DOMAINS (extracellular)
o Cis dimers – (lateral); between cadherins from same cell surface (via N-terminus)
o Trans dimers – (perpendicular); between monomers from opposing cell surface
▪ Mediate cell-cell adhesion (trans dimerization)

CLASSICAL CADHERINS
• E-cadherin – in epithelial cells
• N-cadherin – on nerve, muscle, and lens cells
• P-cadherin – on cells in the placenta and epidermis

NONCLASSICAL CADHERINS
• Protocadherins – in the brain
• Desmocollin – forms desmosomes
• Desmoglein – forms desmosomes

• Cadherins – mediate HOMOPHILIC adhesion (like-to-like connection, e.g., actin-to-actin)

o N-terminal tip of cadherin – where homophilic binding occurs
▪ Lies furthest from the membrane (terminal domain)
▪ Forms a KNOB and a nearby POCKET
• Knob of a cell INSERTS to the Pocket of another cell for them to connect
• Hinge – connects 2 cadherin domains which form a more-or-less rigid unit
• CALCIUM – bind to sites near hinge; PREVENTS it from FLEXING → cadherin strings remain
RIGID and SLIGHTLY CURVED
o With Ca2+ = cadherin strings RIGID; no flexing of hinge
o Without Ca2+ = hinge can flex; cadherin strings FLOPPY
▪ Without calcium, N-terminus weakens binding affinity to other cadherin

• Cadherins bind to partners with relatively LOW AFFINITY

o Low affinity = stronger attachment to other cadherins
o High affinity or Strong attachment = weaker bonds of parallel cadherins

• Cadherins assemble side-to-side

o VELCRO PRINCIPLE – many weak attachments → stronger adhesions
▪ Allows for easier regulation (assembly and disassembly)

• Cadherins are important in cell sorting during embryonic development

o Essential for TISSUE SEGREGATION

CADHERINS CONTROL EPITHELIAL – MESENCHYMAL TRANSITIONS

• Assembly of cells into an epithelium = REVERSIBLE process

o Mesenchymal cells switch to express adhesion molecule → epithelial cell (tight assoc.)
o Epithelial cells change character and disassemble → mesenchymal cell (loose assoc.)

• Controlled by transcription regulatory proteins…

o Slug
o Snail
o Zeb
o Twist – increased expression → converts epithelial cell to mesenchymal cell
▪ Does this by inhibiting expression of cadherins

• Important in Cancer invasiveness and treatment

o Mutated E-cadherin (loss) → less adhesion → cancer cells escape from epithelium →
metastasis
o Twist overexpression → may turn epithelial cells into malignant cancer cells
▪ Enhances cell migration → metastasis
o Treatment = BLOCKING of Twist expression (cells return to nonmalignant EC)

ADHERENS JUNCTIONS RESPOND TO FORCES GENERATED BY THE ACTIN CYTOSKELETON

• Adherens junctions BALANCE force at both sides of the junction

o DIRECT RELATIONSHIP → Increased pulling tension at one cell = increased contractile
force in the other cell (increased actin filaments)
 MECHANOTRANSDUCTION IN ADHERENS JUNCTIONS

Mechanism 1

1. Catenin activates GTPase Rac

2. Rac promotes additional actin protrusion → expanded contact zone
3. Rac Inactivation → recruitment of GTPase Rho
4. Rho promotes assembly of myosin II → contractile activity → tension generation → junction
expansion → actin recruitment

Mechanism 2

1. Cell tension sensed by α-catenin → α-catenin stretched (folded to extended conformation)

2. Unfolding exposes binding sites for vinculin → vinculin attaches
3. Vinculin recruits more actin filaments to the junction → strengthened junction linkages
• Large actin contractile force → reduced cell-cell adhesion (important in tissue remodeling)

• ADHESION BELT (Zonula adherens) – surrounded by contractile bundles of actin filaments and
myosin II (in apical domain)
• In Animal Morphogenesis
o Actin-dependent contraction + loss of adherens junction = cell insertion
(INTERCALATION)
o Increased degradation of β-catenin (due to phosphorylation) → loss of adhesion

DESMOSOMES GIVE EPITHELIA MECHANICAL STRENGTH

 • Structural Components of a Desmosome
o Dense Plaque – at the cytoplasmic Surface; mixture of intracellular adaptor proteins
(Plakophilin, Plakoglobin, and Desmoplakin)
▪ Attachment sites for Keratin IF
o Nonclassical cadherins (e.g., Desmogleins and Desmocollins)
▪ Connects cytoplasm (bind to plaques) to the ECM (hold adjacent cells)
• Desmoglein and Desmocollin – cytoplasmic tails bind PLAKOGLOBIN (γ-
catenin) and PLAKOPHILIN
o PLAKOPHILIN – binds DESMOPLAKIN
o DESMOPLAKIN – binds to sides of intermediate filament (ties desmosomes to IFs)

• Pemphigus vulgaris – antibody made against desmogleins (autoimmune) → loss of cell-cell

contact in between keratinocytes → blisters

TIGHT JUNCTIONS

• Epithelia (and Endothelia)

o only have connections to other tissue (basal side) and NOT in apical surface
o POLARIZED
▪ Apical side (bathed by extracellular fluid)
▪ Basal side (attached to basal lamina)
• Tight junctions mediate PERMEABILITY
o Tight sealing prevent leaking back of nutrients into the gut lumen
o Selectively permeable (water and solutes freely diffuse)
• Paracellular transport
o Some are permeable to specific ions or solutes (e.g., Na+)

• Claudins – essential for tight junction formation and function

o No Claudin-1 gene → no tight junctions → rapid loss of water
o Form paracellular pores
▪ Claudin-16 – in kidney tubule; for reabsorption of Mg2+
• If mutated → impermeable to Mg2+ → excessive EXCRETION of Mg2+
▪ Claudin-1 – mutation leads to UNCONTROLLED WATER LOSS
• Occludin – essential for limiting junctional permeability
• Tricellulin – required to seal cell membrane together; this prevents transepithelial leakage at
points where 3 cells meet

SCAFFOLD PROTEINS at cytoplasmic domain

• Zonula occludens – ZO-1, ZO-2, ZO-3 (for structural support)

o Each Zo has 3 PDZ domains (1 for claudin-attachment)
o SH3 domain – attaches signaling protein
o GK domain – attaches occluding or actin

Anticadherin antibodies that block the formation of adherens junctions also block the formation of tight
junctions.
 GAP JUNCTIONS

• Consist of Channel-forming Proteins

o Connexin – predominant in vertebrates
o Innexin and Innexon – predominant in invertebrates

• 1.4 nm pore size = exchange of inorganic ions and other water-soluble molecules
o Macromolecules such as proteins and nucleic acids CANNOT pass
• ELECTRICAL COUPLING VIA GAP JUNCTIONS
o Allows for rapid transmission of action potentials

• Switches from Open to Close conformation via…

o Membrane potential, pH, Ca2+ concentration

• Connexins – 4-pass transmembrane protein

o 6 connexins = 1 connexOn (hemichannel)
• ConnexOn – pairs in parallel form → complete gap junction of ~2-4 nm

• Are TRANSIENT – half-life = few hours

REMOVAL OF OLD CONNEXON

• Happens at Middle of Plaque

1. Inserted into the plasma membrane via EXOCYTOSIS
2. Diffuse in the membrane plane
3. Bump into periphery of connexon plaque → trapped
a. Corollary – there should be CONNEXONS that HAVE NOT YET PAIRED
i. Unpaired Connexons stay at closed conformation

PLANT PLASMODESMATA PERFORM MANY SAME FUNCTIONS AS GAP JUCNTIONS

• Plasmodesmata – DIRECTLY connects cytoplasms of adjacent cells

o Desmotubule – a narrow cylindrical structure that runs through the center of
plasmodesmata; connects Smooth ER of connected cells
▪ Derived from Smooth ER
• Plasmodesmata is formed during Cytokinesis
• Allows passage of molecules of weight ~800 daltons (~1000 daltons in Gap Junction)
o Annulus – where molecules pass from cell to cell

SELECTINS

• Unlike Ig superfamily, selectins REQUIRE Calcium for adhesion

• Cell-surface carbohydrate-binding proteins (lectins) that mediate a variety of transient cell–cell
adhesion interactions in the BLOODSTREAM
• Mainly governs WHITE BLOOD CELL traffic
o Enable white blood cells to attach to epithelial cells and tissues (migration of leukocytes
out of the bloodstream)

• Each selectin has a LECTIN domain (extracellular) – binds to specific oligosaccharide of other
cells
o L-selectin – on white blood cells (e.g., on peripheral lymph nodes)
o P-selectin – on blood platelets and endothelial cells locally activated by inflammatory
response
o E-selectin – on activated endothelial cells

NEUTROPHIL MOVEMENT DURING INFLAMMATION

• Selectins and Integrins act in sequence to let white blood cells leave the bloodstream and
enter tissues
o Selectin – for weak adhesion (due to low affinity of lectin domain to carbohydrate
ligand); ROLLING – carried by blood flow until it activates integrin
o Integrin – for strong adhesion and emigration of white blood cells

1. Inflammation detected → Endothelial Activation

a. Neutrophils attach to P- and E- selectins of the activated endothelial cells
2. Neutrophil trapping
a. Neutrophils interact with inflamed venule endothelium
3. Neutrophil Activation
a. Platelet Activating Factor (PAF) is displayed → PAF sends a signal to increase binding
activity of some integrins
4. Neutrophil Adhesion
a. Activated integrins cause neutrophils to stop rolling and adhere firmly to vessel wall
5. Neutrophil Invasion

IMMUNOGLOBIN SUPERFAMILY (IGSF) MEDIATE CA2+-INDEPENDENT CELL-CELL ADHESION

• NCAM – Neural Cell Adhesion Molecule; homophilic binding to integrins; in neurons

• VCAM – Vascular Cell Adhesion Molecules; heterophilic binding to integrins
• ICAM – Intercellular Cell Adhesion Molecules; heterophilic binding to integrins; endothelial
 o ICAM is recognized by White Blood Cells
• NCAM – contains repeats of SIALIC ACIDS → negative charge repels → Cell adhesion INHIBITED
o A Non-immune cell adhesion
• Integrins + Ig superfamily = mediate cell-cell adhesion

ANIMAL EXTRACELLULAR MATRIX

• Fibroblasts – produce most of the matrix macromolecule in connective tissues

o Chondroblasts – produce cells of cartilage
o Osteoblast – produce cells of bone

• 3 Major Classes of Macromolecules that Form the ECM

1. Glycosaminoglycan (GAGs) – large and highly charged polysaccharides (negative);
covalently linked to proteins in the form of proteoglycans
2. Fibrous Proteins – primary members of collagen
a. Collagen – strengthen and help organize ECM
b. Elastin – rubberlike; provides ECM resilience
3. Noncollagen Glycoproteins - carry conventional asparagine-linked oligosaccharides

• Proteoglycans form a GEL-LIKE GROUND SUBSTANCE – where collagen and glycoproteins are
embedded
o Polysaccharide Gel – resists compressive forces on matrix while permitting rapid
diffusion of nutrients, metabolite, and hormones between the blood and tissue cells

GLYCOSAMINOGLYCAN (GAG)

• Forms hydrated gels

• Unbranched polysaccharide chains composed of repeating disaccharide units of 2 different
sugars, namely
o Amino sugar – N-acetylglucosamine or N-acetylgalactosamine
o Sulfated or carboxyl groups on their sugars – Uronic acid (glucuronic or iduronic)
• Highly negatively charged (anionic); hydrophilic

• 4 Main GAG groups

1. Hyaluronan
2. Chondroitin Sulfate and Dermatan sulfate
3. Heparan sulfate
4. Keratan sulfate
• Highly extended conformation
• Occupy a huge volume relative to their mass
• Low GAG concentrations → formation of Hydrated Gels
• Attract cations esp. Na+ → large amount of water Into the matrix → swelling pressure or
turgor → enables ECM to withstand compressive forces
o Sodium – resist compressive forces
o Collagen and Fibrils – resist stretching forces

• Found in cartilage matrix of knee joints

o Dermatan sulfate deficiency – short, prematurely aged appearance, generalized defects
of skin, joints, muscles, and bones

HYALURONAN

• “Hyaluronic acid” or “Hyaluronate” – simplest GAG

o Contains NO sulfated sugars
o NOT covalently linked to any core protein (all other GAGs have covalently-linked
protein)
o Spun out directly from cell surface (unlike other GAGs synthesized inside cell and
released by exocytosis)

• Hyaluronan – resists compressive forces in tissues and joints

o Constituent of joint fluid → serves as a LUBRICANT
o Space filler during embryonic development → helps in conformational changes
▪ E.g., helps in the formation of heart valves and septa
• HYALURONIDASE – degrades hyaluronan

PROTEOGLYCAN

• Protein polysaccharide complex with a core protein covalently attached to GAGs

• Resist crushing/compressive forces
o cushion cells; acts as lubricant
o Provide binding sites for growth hormones to protect from proteases
o Regulate diffusion of small signaling molecules in developing embryo

• Decorin – binds to collagen fibrils and regulates fibril assembly and fibril diameter
o NO DECORIN → fragile skin (due to reduced tensile strength)
• Aggrecan – found in cartilage for resilience and toughness; > 100 GAG chains

• Syndecan – found in cell–matrix adhesions; modulates integrin function by interacting with

fibronectin on the cell surface and with cytoskeletal and signaling proteins inside the cell

COLLAGEN

• Rich in proline and glycine (vital in formation of triple-stranded helix)

• Has α chains – each consists 3 amino acids per turn
o Glycine X Y sequence
▪ X commonly proline
▪ Y commonly hydroxyproline
 • Provides HIGH TENSILE STRENGTH
• Most Abundant protein in human body
• Produced by Fibroblasts

• SCURVY – failure to hydroxylate collagen chains (lysine and proline)

o Ascorbic acid as coenzyme
• FIBRILLAR COLLAGEN
o Collagen I, II, II, V, and IX – strengthened by covalent cross-links bet. Lysine,
hydroxylysine residues of adjacent molecules
o Provides INSOLUBLE FRAMEWORK → determines MECHANICAL PROPERTIES of ECM

• Pulmonary Fibrosis and Liver Cirrhosis – abnormalities in collagen

• Type I – most common; in skin and bone; a FIBRILAR COLLAGEN (forms fibrils)
o COLLAGEN FIBRILS – resist tensile forces
o As compared to GAGs – resist compressive forces
• Type IX and XII – are FIBRIL-ASSOCIATED COLLAGENS; link fibril to one another and to other
components in ECM
o Important in organizing collagen fibrils
• Type IV – a NETWORK-FORMING collagen; forms major part of basal lamina
o Non-fibrillar
o Restricted to the basement membrane
• Type VII and X – for stabilization of membranes
o In angiogenesis, and interactions with other ECM molecules
• Type VII – form dimers that assemble into specialized structures called ANCHORING FIBRILS
o Anchoring Fibrils – help attach basal lamina to underlying connective tissue

COLLAGEN-BASED DISEASES

1. Fibril collagens : mutations

a. Type I (osteogenesis imperfect fragile bones)
b. Type II (cartilage; dwarfism, skeletal deformities).
2. Ehler Danlos syndromes (hyperflexibility of joints and highly extensible skin)
3. Fibrosis (not genetic) – scarring due to overproduction of collagen in lung (pulmonary fibrosis)
or liver (cirrhosis)
4. Non fibrillar (type IV)
a. Alport syndrome – kidney disease of the glomerular basement membrane
• FIBROBLASTS – influence alignment of collagen fibers
• COLLAGEN FIBERS – affect distribution of fibroblasts
o Embryonic tissue containing fibroblasts placed apart on a collagen gel → collagens
organize into compact bands → fibroblasts migrate out the explants

ELASTIN

• Gives tissues ELASTICITY

o Skin, blood vessels, lungs
o Dominant in Arteries
o Mutation – narrowing of aorta → excessive proliferation of smooth muscle cells in the
arterial wall
• Elastin – hydrophobic protein about 750 Amino acids long rich in glycine and proline
o Elastin + Microfibril = Elastic Fibers
• Like collagen but NOT glycosylated

• Tropoelastin – is secreted into the extracellular space, where it forms lysine cross links to other
tropoelastin molecules → generate a large network of elastin fibers and sheets.

• Marfan’s syndrome – mutation in fibrillin gene;

o ruptured aorta, displacement of eye lens, bone and joint abnormalities
 FIBRONECTIN

• Serve as large scaffold proteins containing multicopies of specific protein interaction domains
with multiple repeat domains
o Interaction depends in specific TRIPEPTIDE SEQUENCE (Arg-Gly-Asp or RGD)
▪ Peptides that also contain RGD sequence competes with fibronectin in binding
sites
• Binds with HEPARIN – an anti-coagulant

• 3 different fibronectin repeats

o FN1 – binds fibrin
o FN2 – binds heparin
o FN3 – binds collagen

• Fibronectin attaches with integrin → linkage transmits tension (detects it) → fibronectin
stretches → type III fibronectin repeats unfold → binding sites exposed → other fibronectin
molecules attach → formation of fibrils → withstands tension

BASAL LAMINA

• Thin, tough, flexible sheet of matrix molecules; an essential underpinning of all epithelia
• Determine cell polarity; influence cell metabolism; organize proteins in adjacent plasma
membranes
• Promotes cell survival, proliferation, or differentiation
o Tissue regeneration after injury by providing scaffold for migration of regenerating cells
as in the regeneration of neuromuscular junction
• Highway for cell migration
o Selective barrier to the movement of underlying fibroblasts to the epithelium

• Connects epidermis to dermis

o JUNCTIONAL EPIDERMOLYSIS BULLOSA – genetic defects in basal lamina
▪ Epidermis detaches from dermis → blisters

MAJOR COMPONENTS OF BASAL LAMINA – LAMININ and TYPE IV COLLAGEN

• Epithelial cells and Stroma (underlying connective tissue under epithelial cells)
o Each contribute one set of basal lamina cells

• LAMININ – organize basal laminae and anchor them to cells

o Makes up the early basal laminae during development
o Composed of 3 long polypeptide chains (α, β, and γ) held together by disulfide bonds
▪ NO γ1 chain = unable to make basal lamina
▪ Heterotrimers
o Help cell migration (primordial germ cell) during development and for cell
differentiation
 o Laminin mutation – disrupt kidney filter function by interfering cell differentiation that
contact it and support it.
• TYPE IV Collagen – consist of three separately synthesized long protein chains (α chains) that
twist together to form a ropelike superhelix
o Assemble into flexible, feltlike network → gives basal lamina TENSILE STRENGTH
o Alport syndrome – type IV collagen mutation; dysfunctional glomerular filter
• Other Components of Basal Lamina – collagen, nidogen, perlecan . Also, fibronectin and type
XVIII collagen

ECM PROTEASES (DEGRADATION OF MATRIX)

• Cells MUST be able to CUT THROUGH MATRIX

o Enables them to Divide while embedded in ECM
o Enables them to Travel through ECM
• NO Enzymes that CUT Matrix = cells unable to divide and migrate
o Crucial in White Blood Cells’ response to injury
o Important in cancer cell metastasis → invasion

• MATRIX METALLOPROTEASES (MMPs)

o Are Zinc- and Calcium- dependent endopeptidases
o Crucial for cells to be able to divide when embedded in matrix
o For tissue remodeling, embryonic cell proliferation, migration, wound healing,
angiogenesis, apoptosis, immunity
o Collagenases, gelatinases, stomelysins, matrilysins, membrane-type MMPs
• SERINE PROTEASES
o Highly reactive serine in their active site
MMPs and Serine proteases cooperate to degrade matrix proteins such as collagen, laminin, and
fibronectin.

• ECM Fibrils – NOT static; in constant remodeling; can be stretched during tension OR contracts
when relieved

MATRIC METALLOPROTEINASES (MMPs)

• Increased activity of Growth Factor and Cytokines → triggers activity by MMPs

• Heparan sulfate of matrix proteoglycans – Interact with the growth factors → cell proliferation

• MMPs DEGRADES Type I and Type II Collagen, proteoglycans, and GAGs → skin wrinkles
• Associated Diseases
o Arthritis, tumor progression, blood clots, heart attacks, metastasis (in cancer cells)

Summary

Cells are embedded in an intricate extracellular matrix, which not only binds the cells together but also
influences their survival, development, shape, polarity, and migratory behavior. The matrix contains
various protein fibers interwoven in a network of glycosaminoglycan (GAG) chains. GAGs are negatively
charged polysaccharide chains that (except for hyaluronan) are covalently linked to protein to form
proteoglycan molecules. GAGs attract water and occupy a large volume of extracellular space.
Proteoglycans are also found on the surface of cells, where they often function as co-receptors to help
cells respond to secreted signal proteins. Fiber-forming proteins give the matrix strength and resilience.
The fibrillar collagens (types I, II, III, V, and XI) are ropelike, triple-stranded helical molecules that
aggregate into long fibrils in the extracellular space, thereby providing tensile strength. They also form
structures to which cells can be anchored, often via large multidomain glycoproteins, such as laminin and
fibronectin, that bind to integrins on the cell surface. Elasticity is provided by elastin molecules, which
form an extensive cross-linked network of fibers and sheets that can stretch and recoil.

INTEGRINS and HEMIDESMOSOMES

• Form CELL-MATRIX junctions

• Mostly connect to actin filaments; those at hemidesmosomes bind to intermediate filaments
• Consist of α and β subunits
o Noncovalently-attached glycoproteins
o Short intracellular C-terminal tail
▪ C-terminal tail of the β subunit binds to adaptor proteins (e.g., talin) which
interact with actin filaments
o Large N-terminal extracellular domain
▪ Divalent cation-binding domains (Calcium and Magnesium)
• Directly binds to specific amino acid sequence motifs in extracellular
matrix proteins (e.g., fibronectin) or even in surface proteins of cells
▪ RGD Sequence and Leu Asp Val (LDV) sequence = Integrin-binding sequences
found in ECM proteins
• Ca2+ and Mg2+ in the extracellular domain
o Concentration of these affect binding of integrins to matrix ligands
o Needed to modulate integrins’ binding activity as one integrin can have different ligand-
binding specificities in different cell types.

• Intracellular portion of Integrin = binds to a complex of several different proteins which,

together, form a linkage to the cytoskeleton
o 23 out of 24 integrins bind to actin filaments

• Talin – a giant adaptor protein bound by C-terminal intracellular tail of β subunit

o Contains a string of multiple domains for binding actin and other proteins that help
reinforce and regulate the linkage to actin filaments
• α6β4 integrin – spans the membrane, attaching to keratin filaments intracellularly and to
laminin extracellularly
o Plectin and BP230 – adaptor proteins that connect α6β4 integrin to keratin filaments

• β2 subunit – exclusively expressed on the surface of white blood cells

o β2 integrins mainly mediate cell-cell interactions (e.g., in endothelial cells)
• α1β2 (LFA1)
o Found on white blood cells
o Enables white blood cells to attach to the Ig family protein ICAM1 on vascular
endothelial cells at sites of infection
• Leukocyte adhesion deficiency (genetic disease)
o Fails to synthesize functional β2 subunits (suffer repeated bacterial infections)
• Β3 integrins – found on blood platelets
o Binds to blood clotting factor fibrinogen
• Glanzmann’s Disease
o Genetic deficiency in β3 integrins
o Suffer from defective clotting and bleed excessively (platelets fail to interact with
fibrinogen and mediate normal blood clotting)
Schematic model of leukocyte adhesion cascade involved in immune cell infiltration. A step-by-step
process involving rolling, firm adhesion, and transmigration moves the leukocyte in the circulatory system
towards the tumor site. Tis cascade is mediated by various molecules interacting with each other

• β2subunit of integrin αLβ2( LFA1) on the surface of WBC binds to ligand ICAM1 of the endothelial
cell for cell-cell adhesion at the site of infection
• Leukocyte adhesion deficiency (LAD) – β2 subunits mutation → results to repeated bacterial
infections

INTEGRINS SWITCH BETWEEN AN ACTIVE AND AN INACTIVE CONFORMATION

• Inactive state
o External segments – folded together into a compact structure that cannot bind matrix
proteins
o Cytoplasmic tails – hooked together to prevent interaction with cytoskeletal linker
proteins.
• Active state
o External domains – unfold and extend to expose a high-affinity matrix-binding site at
the tip of the subunits
o Cytoplasmic tails – unhooked at the membrane to expose intracellular binding sites for
cytoplasmic adaptor proteins
 INSIDE-OUT INTEGRIN ACTIVATION MECHANISM

Activation of Integrins by Intracellular Signaling (in platelets)

1. Extracellular signal protein THROMBIN binds to thrombin receptor (GPCR – PAR1) → activates
GPCR on the cell surface → signaling cascade (via IP3 pathway)
2. Rap1 (GTPase) activated
3. Activated Rap1 interacts with the protein RIAM → RIAM recruits TALIN to the plasma
membrane
4. Talin head domain interacts with PI(4,5)P2 → C-terminal rod domain of talin dissociates from its
N-terminal head domain → talin’s binding sites for integrin and other proteins exposed
5. Together with another protein called KINDLIN, talin interacts with the Integrin β chain → trigger
integrin activation
6. Talin then interacts with adaptor proteins such as VINCULIN → formation of an actin linkage

Talin regulation depends in part on an interaction between its flexible C-terminal rod domain and the N-
terminal head domain that contains the integrin-binding site. This interaction is thought to maintain talin
in an inactive state when it is free in the cytoplasm.

• Talin – competes with the integrin α chain for its binding site on the tail of the β chain
o When talin binds to the β chain, it blocks the intracellular α–β linkage, allowing the two
legs of the integrin molecule to spring apart
 OUTSIDE-IN INTEGRIN ACTIVATION MECHANISM

1. External matrix protein bind to low-affinity inactive integrin

2. Binding sites for talin and other cytoplasmic adaptor proteins are exposed on the tail of the β
chain
3. Adaptor proteins bind to integrin
4. Actin filaments attach to the intracellular end of the integrin

CRUCIAL in BLOOD CLOTTING

 THE VELCRO PRINCIPLE

• Following activation of integrins…

o Integrins CLUSTER together → creates a dense PLAQUE in which many integrin molecules
are anchored to cytoskeleton filaments

• Critical components of Cell-Matrix Junctions

o Integrin-linked kinase (ILK)
▪ Binding partners: pinch + parvin = form a trimeric complex with ILK
o Actin-binding proteins
▪ E.g., vinculin, zyxin, VASP, and α-actinin – promote assembly and organization of
actin filaments
o Focal Adhesion Kinase (FAK) – interacts with multiple components in the junction ∎

• Anchorage Dependence – dependence of cell growth, proliferation, and survival on attachment

to a substratum
o For some cells (e.g., epithelial, endothelial, and muscle) cell survival depends on
attachments to the ECM
▪ Loss of contact → Apoptosis
o Mutation that disrupts this control → Cancer cell invasive behavior (metastasis)

FOCAL ADHESION KINASE

• Focal Adhesions
o Prominent sites of tyrosine phosphorylation
• Focal Adhesion Kinase
o A major tyrosine-phosphorylated protein found mostly in focal adhesions
 1. Integrins cluster at cell-matrix contacts
2. FAK is recruited to the integrin β subunit by intracellular adaptor proteins such as Talin or Paxillin
3. Clustered FAK molecules phosphorylate each other on a specific tyrosine → phosphotyrosine
docking sites
a. Members of Src family of cytoplasmic tyrosine kinases phosphorylate FAK on additional
tyrosines → docking sites for a variety of additional intracellular signaling proteins created
b. Observed in Outside-in signaling of integrins ∎

INTEGRIN-MEDIATED MECHANOTRANSDUCTION

“TALIN is a TENSION SENSOR at cell-matrix junctions”

Tension across cell–matrix junctions stimulates the

local recruitment of vinculin and other actin-regulatory
proteins, thereby strengthening the junction’s
attachment to the cytoskeleton.

The long, flexible, C-terminal region of talin is divided into a series of folded domains, some of which
contain vinculin-binding sites (dark green lines) that are thought to be hidden and therefore inaccessible.
One domain near the N-terminus, for example, comprises a folded bundle of 12 α helices containing five
vinculin-binding sites.

1. Talin (adaptor protein) senses tension (mechanical forces) at cell-matrix junctions

2. N-terminal end of talin binds integrin while C-terminal end of talin binds actin
3. Actin filaments are pulled by myosin motors → talin is stretched → binding sites of vinculin in C-
terminal region of talin exposed
4. Vinculin is recruited →vinculin organize additional actin filaments → increased strength of cell
junction

• If cells are attached to rigid matrix → integrins sense high tension → recruits additional integrins
and other proteins to increase junction’s ability to withstand tension
 PLANT CELL WALL

• Cell walls arise as a cell plate that forms between the plasma membranes of newly formed
daughter cells.
o Cell Plate – forms during cytokinesis
o The walls of growing cells are primary walls
▪ Primary Walls – allow flexibility lacking in the thicker…
▪ Secondary Walls – present in mature cells; with lignin (LACKS flexibility)

• Plant Cell wall is HYPOTONIC

o Primary Cell Wall – built from Cellulose
Microfibrils interwoven with a network of
PECTIC Polysaccharides
• CELLULOSE MICROFIBRILS – influence orientation of
cell elongation
o Cell elongates only in a direction
PERPENDICULAR to the orientation of the
innermost layer of microfibrils
• Cellulose – gives it tensile strength → allows for the
development of TURGOR PRESSURE

• Matrix of the cell wall contains…

o Hemicelluloses
o Pectins (negatively charge; hydrated gel)
o Proteins (Expansins – for cell growth)