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Toad Lilies
Elephant Frog
Cell- cell interaction
• Cells may communicate by direct contact.
Immune
connection:
Macrophage
will use direct
contact to alert
T cells that
invaders are
present
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 11.4
Epithelial Cell Junction Types
Structures in the B7/CD28
family. Structures are
modeled on the crystal
determinations. Loops have
been added to one end of
the IgV domains to
emphasize the orientation
of the CDR-like loops and
their interaction with ligand
or lack thereof.
General Schemes of Intercellular Signalling
Extracellular signaling
molecules released by
cells occurs over distances
from a few microns - autocrine (c)
and paracrine (b) signaling to
several meters in endocrine (a)
signaling. In some instances,
receptor proteins attached to the
membrane of one cell interact
directly with receptors on an
adjacent cell (d).
© 2000 by W. H. Freeman and Company. All rights reserved.
Representative (typical) Neuron
http://www.horton.ednet.n
s.ca/staff/selig/Activities/n
ervous/na1.htm
1. cell body or soma
-single nucleus with prominent nucleolus
-Nissl bodies
-rough ER & free ribosomes for protein
synthesis
-proteins then replace neuronal cellular
components for growth
and repair of damaged axons in the PNS
-neurofilaments or neurofibrils
give cell shape and support bundles of
intermediate filaments
-microtubules move material
inside cell
-lipofuscin pigment clumps
(harmless aging) - yellowish
brown
Anatomy of a Synapse
Copyright © 2008 Pearson Education, Inc.,
publishing as Benjamin Cummings.
Figure 8.2a
Propagation of an Action
Potential
Why is the
resting
potential a
negative
number?
What is the significance of the
refractory period?
Refractory Period
Neurotransmitters
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•
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•
•
More than 100 identified
produced by neurons and stored
within the neuron
secrete these NTs in response to
generation of an electrical signal
(action potential) by the neuron
bind onto target neurons (synapse)
or target muscle cells
(neuromuscular junction)
Some bind receptors on the target
and cause channels to open in the
target (sodium channels)
–
–
•
•
e.g. binds to receptors on target
neuron – causes generation of
another action potential by target
neuron
e.g. binds to receptors on target
muscle cells – causes contraction
Others bind receptors on the target
and result in a second messenger
system
Results in either excitation or
inhibition of the target
Neuromuscular junction
Neuromuscular junction
EPSPs: excitatory post-synaptic potentials
IPSPs: inhibitory post-synaptic potentials
What kinds of hormone are there?
Known Hormonal Classes
• Proteins & peptides
chemcases.com/olestra/
images/insulin.jpg
• Lipids (steroids, eicosanoids)
• Amino acid derived
(thyronines, neurotransmitters)
chem.pdx.edu/~wamserc/
ChemWorkshops/ gifs/W25_1.gif
• Gases (NO, CO)
website.lineone.net/~dave.cushman/
epinephrine.gif
What is a hormone receptor?
Hormone Receptors are cellular proteins that
bind with high affinity to hormones & are
altered in shape & function by binding; they
exist in limited numbers.
Binding to hormone is noncovalent &
reversible.
Hormone binding will alter binding to other
cellular proteins & may activate any receptor
protein enzyme actions.
What are the main types of receptors?
Membrane Receptors
Imbedded in target cell membrane; integral
proteins/
glycoproteins; penetrate through membrane
For protein & charged hormones (peptides or
neurotransmitters)
3 major groups: Serpentine = 7 transmembrane
domains, Growth factor/cytokine = 1
transmembrane domain, Ion channels
•
•
•
•
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easily travels through the blood - hydrophilic
but cannot diffuse through plasma membrane!
therefore absolutely requires the expression of receptors
on the cell surface – integral membrane proteins that act
as first messenger
the receptor protein activates a series of signaling events
within the cells
– e.g. epinephrine binds to receptor and activates an
adjacent G-protein in membrane
– G-protein activates adenylate cyclase to convert ATP
to cyclic AMP (cAMP) in the cytosol
– cAMP acts as a 2nd messenger
– cAMP activates a series of proteins in the cytosol
called kinases
– kinases act to phosphorylate their targets – either
activating them or inhibiting them
– this speeds up/slows down physiological responses
within the cell
– phosphodiesterase inactivates cAMP quickly
many second messengers are made in cells in response
to specific hormones
–
•
•
e.g. calcium, IP3, DAG
Cell response is turned off unless new hormone
molecules arrive
this mechanism allows for amplification – one H-R
combination can activate two G proteins which activates 4
kinases which activate 16 more kinases etc…….
What are the main types of receptors?
Nuclear Receptors
Nuclear proteins that usually act in pairs & bind to
specific Hormone Recognition Elements (HREs) =
sequences on the DNA in the promoter regions of
target genes
For small, hydrophobic molecules (steroids,
thyroid hormones)
Action of Lipid-Soluble Hormones: Endogenous
signaling
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Hormone must be carried by a
transport protein that allows it to
dissolve within the aqueous
(watery) environment of the blood
plasma
Hormone diffuses through
phospholipid bilayer & into cell
the receptor is located within the
cell (cytoplasm or the nucleus)
binding of H to R results in its
translocation into the nucleus
the H then binds directly to specific
sequences within the DNA =
response elements
this binding turns on/off specific
genes – activates or inhibits gene
transcription
if turned on - new mRNA is formed
& directs synthesis of new proteins
new protein alters cell’s activity
if turned off – no new protein results
and the cell’s activity is altered
Signal transduction: Adenyl cyclase system
Gs/i/o/x – G proteins, CaM – calmodulin, GTP – guanosine triphosphate, ATP –
adenosine triphosphate, ADP – adenosine diphosphate, cAMP – cyclic adenosine
monophosphate, 5´-AMP - 5´-adenosine monophosphate, PKA – protein kinase A,
PKC – protein kinase C
Signal transduction: Phosphoinositide system
PI-PLC - phospholipase C specific for phosphoinositides, PIP2 - phosphatidylinositol4,5-biphosphate, IP3 - inositol-1,4,5-triphosphate; DG - diacylglycerol; PKC – protein
kinase C
AR – adrenoceptor, G – G protein, PI-PLC – phosphoinositide specific
phospholipase C, IP3 – inositoltriphosphate, DG – diacylglycerol, CaM –
calmodulin, AC – adenylyl cyclase, PKC – protein kinase C
Gs/i/o/x – G proteins, CaM – calmodulin, GTP – guanosine triphosphate,
ATP – adenosine triphosphate, ADP – adenosine diphosphate, cAMP –
cyclic adenosine monophosphate, 5´-AMP - 5´-adenosine
monophosphate, PKA – protein kinase A, PKC – protein kinase C
•
Models of estrogen action. In the
“classical” pathway of estrogen action
(i), estrogen or other selective
estrogen receptor modulators (SERMs)
bind to the estrogen receptor (ER), a
ligand-activated transcription factor
that regulates transcription of target
genes in the nucleus by binding to
estrogen response element (ERE)
regulatory sequences in target genes
and recruiting coregulatory proteins
(CoRegs) such as coactivators. Rapid
or “nongenomic” effects of estrogen
may also occur through the ER
located in or adjacent to the plasma
membrane (ii), which may require the
presence of “adaptor” proteins, which
target the ER to the membrane.
Activation of the membrane ER leads
to a rapid change in cellular signaling
molecules and stimulation of kinase
activity, which in turn may affect
transcription. Lastly, other non-ER
membrane-associated estrogenbinding proteins (EBPs) may also
trigger an intracellular response (iii).
•
Risk factors can be
distinguished in terms of
their ability to cause breast
cancer directly, through
genetic damage, or by
altering hormonal
metabolism. Vulnerability
factors prolong the
duration of breast cell
growth, while contributing
factors can distort
hormone levels.
•
Bifunctional pathways to
breast cancer. Abbreviations:
E2, 17ß-estradiol; E1, estrone;
OHE1, hydroxyestrone; ER,
estrogen receptor. In the
bifunctional pathway, the E2
metabolites affect cell
proliferation and breast
cancer development either
directly via receptorindependent mechanisms
involving structural/functional
alterations in DNA, or
indirectly via receptordependent mechanisms
involving phenotypic growth
regulation. Both mechanisms
eventually upregulate
aberrant proliferation and
development of breast cancer
(37).
• The response of a particular cell to a signal
depends on its particular collection of receptor
proteins, relay proteins, and proteins needed
to carry out the response.
Figure 19-34 Fluctuating Levels of Mitotic Cyclin and MPF
During the Cell Cycle
Figure 19-38 Role of the Rb Protein in Cell Cycle Control
Role of the p53 Protein in Responding to DNA Damage
Apoptosis
• Apoptosis (1972)
– Greek word “falling off”
• Built-in (programmed)
mechanism)
• or self-destructionsuicide
• Type of programmed
cell death based upon
morphological features
Programmed cell death during development. Programmed cell death is involved in
forming structures such as the digits of the hand (a), deleting structures such as nearly all
of an insect's larval components (b), controlling cell numbers in, for example, the nervous
system (c) and eliminating abnormal cells such as those that harbour mutations (d).
Studies on the
development of the
nervous system showed
that in the process of
assembling sensory
fields, neurons are
eliminated by orderly
cell death in order to
tailor sensory input to
environmental stimuli
(elimination or
transplantation of limbs
as key examples).
Apoptosis plays in an important role in
normal developmental processes
Jacobson et al (1997) Cell, Vol. 88, 347–
A cancer cell (mauve) undergoing apoptosis
Comparison of cell death by necrosis and apoptosis
Known Apoptotic Stimuli
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Withdrawal of NGF
Etoposide
Actinomycin D
UV radiation
Staurosporin
Enforced m-Myc expression
Glucocorticoids
Physiological Relevance of Apoptosis
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Embryonic Development
Regulation of/by Immune System
Negative Selection
CTL Killing (eg. Immune surveillance, viral infections)
Terminating Active Immune Response
Tight Regulation of Cell Number (eg. BM, GI, Uterus, Skin)
Compensatory Response to Cell Stress
Intrinsic Pathway (e.g. GF removal, XRT, Chemo.)
Extrinsic Pathway (e.g. FAS.Ag, TNF-R Activation)
Senescence (ageing)
Morphology of Apoptosis
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cell shrinkage
extracellular exposure of phosphatidylserine
shows boiling and blebbing
chromatin condenses*-most characteristic feature
DNA is degraded into oligonucleosomal fragments
disassembly into apoptotic bodies
• membrane bound, contains portions of the nucleus and
various organelles
– phagocytosis by neighboring cells
Does not elicit inflammation-hallmark of
apoptosis
MORPHOLOGICAL FEATURES OF APOPTOSIS
• Cell shrinkage
• Chromatin condensation and
fragmentation.
• Formation of cytoplasmic blebs and
apoptotic bodies.
• Phagocytosis of apoptotic bodies by
adjacent healthy cells or macrophages.
• Lack of inflammation.
Morphological and biochemical
characteristics of apoptosis
 Morphologi changes:
Early : Chromosome condensation, cell body shrink
Later : Blebbing and Nucleus and cytoplasm fragment—
Apoptotic bodies
At last: Phagocytosed
Biochemical features of
apoptosis
1.PROTEIN CLEAVAGE:
Caspases (cysteine protease)
Nuclear scaffold
Cytoskeletal protein
2.PROTEIN CROSS-LINKING:
Transglutaminase
Cytoplasmic proteinshrunken shalls
apoptotic bodies
Biochemical features of apoptosis
3. DNA breakdown: 50-300 kb pieces
Ca2+, Mg2+ dependent endonucleases
DNA oligonucleosomes
DNA ladders (also seen in necrosis)
4. PHAGOCYTIC RECOGNITION
Receptors on macrophages for the
surface components
(phosphatidylserine, thrombospondin)
on apoptotic bodies.
DNA fragmentation: biochemical
hallmark
• DNA cleaved into non-random fragments
• Targets of endonuclease attack: linker regions between nucleosomes
• 180-200 bp fragments & multiples of this unit
Agarose gel
electrophoresis
Chromatin
Fragmented
Chromatin
mitochondria
Caspase-3 activation via tumor necrosis factor (TNF) family receptors (for
example, Fas), FADD (Fas-activated death domain protein) and caspase-8
represents the extrinsic pathway (blue), whereas caspase-3 activation via the
mitochondrial release of cytochrome c and Apaf-1–mediated processing of caspase9 represents the intrinsic pathway (red)3.
For clarity, not all of the players are shown. Procaspase-3 is shown as a PAC-1–
sensitive dormant single-chain precursor with an N-terminal prodomain (Pro).
During apoptosis, caspase-3 assembles as an active p17-p12 heterotetramer after
proteolytic processing between the p17 and p12 subunits (at Asp175) and removal
of the prodomain2.
PAC-1 is proposed to regulate the Asp-Asp-Asp (DDD) safety catch at amino acids
179–181 in procaspase-3, consequently inducing a conformational change that
leads to proteolytic processing into the active p17 and p12 subunits1.
Cys163 is the catalytic cysteine in the active site of caspase-3; the sequence shown
illustrates its proximity to the DDD safety catch and DDM motif. Although
caspase-7 (not shown) is believed to be a downstream caspase, its position relative
to caspase-3 in apoptosis pathways is unclear.
Functional homologues of caspases and caspase regulators across species are
indicated by the same colour.
Caspase-9 in mammals and Dronc in the fruitfly Drosophila melanogaster are
initiator caspases, whereas caspase-3 and -7 in mammals and Drice in fruitflies
belong to the class of effector caspases.
CED-3 (cell-death abnormality-3) in the nematode worm Caenorhabditis elegans
functions both as an initiator and effector caspase.
The inhibitor of apoptosis (IAP) proteins suppress apoptosis by negatively
regulating the caspases, whereas SMAC (second mitochondria-derived activator
of caspases)/DIABLO (direct IAP-binding protein with low pI) in mammals and
the RHG proteins Reaper, Hid, Grim and Sickle in fruitflies can remove the
IAP-mediated negative regulation of caspases.
AIF, apoptosis-inducing factor;
APAF1, apoptotic-protease-activating factor-1;
Cyt c, cytochrome c;
EndoG, endonuclease G;
HTRA2, high-temperature-requirement protein A2.
Role of Caspases
• Effectors (cell disassembly) (caspases 2,3,6,7)
and initiators (caspases 8,9)
• 14 identified mammalian caspases- 12 in humans
• Cysteine protease that has an absolute
requirement
requirement for cleavage after
aspartic acid
• High specificity for which proteins are digested
– PARP (116 kDa) nuclear polymerase that repairs DNA is
cleaved by caspase 7.
Types of Caspases
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Nedd2/Ich-1/caspase2
YAMA/CPP32/apopain/caspase-3- most characterized
TX/Ich-2/ICErelII/caspase-4
TY/ICErelIII/caspase-5
Mch2/caspase-6
ICE-LAP-3/Mch-3/CMH1/caspase-7
FLICE/MACH/caspase-8-most proximally activated caspase
ICE-LAP-6/caspase-9
Mch-4/FLICE 2/caspase-10
Ich3/caspase-11
Caspases (cysteine aspartic acid-specific proteases) are highly
specific proteases that cleave their substrates after specific
tetrapeptide motifs (P4-P3-P2-P1) where P1 is an Asp residue.
The caspase family can be subdivided into initiators, which are
able to auto-activate and initiate the proteolytic processing of
other caspases, and effectors, which are activated by other
caspase molecules. The effector caspases cleave the vast
majority of substrates during apoptosis.
All caspases have a similar domain structure comprising a propeptide followed by a large and a small subunit (see figure).
The pro-peptide can be of variable length and, in the case of
initiator caspases, can be used to recruit the enzyme to
activation scaffolds such as the APAF1 apoptosome. Two
distinct, but structurally related, pro-peptides have been
identified; the caspase recruitment domain (CARD) and the
death effector domain (DED), and these domains typically
facilitate interaction with proteins that contain the same motifs.
Caspase activation is usually initiated through proteolytic
processing of the caspase between the large and small subunits
to form a heterodimer. This processing event rearranges the
caspase active site into the active conformation. Caspases
typically function as heterotetramers, which are formed
through dimerization of two caspase heterodimers. Initiator
caspases exist as monomers in healthy cells, whereas effector
caspases are present as pre-formed dimers.
Not all mammalian caspases participate in apoptosis. For
example, caspase-1, caspase-4, caspase-5 and caspase-12 are
activated during innate immune responses and are involved in
the regulation of inflammatory cytokine processing (for
example, IL1
and IL18). Interestingly, caspase-12 is
expressed as a truncated, catalytically inactive protein in most
humans (caspase-12S*). However, a subset of individuals of
African descent express full-length caspase-12 (caspase-12L*)
and these individuals appear to be more susceptible to
inflammatory diseases. To date,
400 substrates for the
mammalian caspases have been identified, but the significance
of many of these cleavage events remains obscure.
Caspase 3
• Caspase 3 (CPP32/apopain/YAMA)
– shares similarity to CED 3.
– Protein substrates include:
• PARP (poly ADP ribose) polymerase
• PKC
• sterol-regulatory element-binding protein
• DNA dependent protein kinase (DNA repair)
• U1-associated 70 kDa protein (mRNA splicing)
• MEKK
• DNA fragmentation factor- cytosolic factor that induces
nuclear fragmentation
•
DNA damage-apoptosis model structure. The diagram depicts the general
structure of the model. The model contains mathematical equations describing the
individual protein-protein interactions and catalytic reactions for over 80 species that
fall within the broad outlines shown. The equations are solved in specialised software
including MatLab, and are run multiple times to simulate multi-cell environments.
p53 Dependent Apoptotic Pathways
Schematic representation of the
p53-dependent apoptotic
pathways by transcriptional
activation of BAX, PUMA and
APAF-1.
Fertilization in sea urchin.
1. The acrosome releases
enzymes to digest the jelly
coat.
2. Actin filaments bind to
receptors in the vitelline
layer.
3. The sperm and egg
plasma membranes fuse
and become depolarized,
preventing polyspermy the ability of other sperm to
fertilize the egg.
4. The sperm nucleus
enters and fuses with the
egg's nucleus.
5. The vitelline layer swells
to form a fertilization
envelope that also blocks
polyspermy.
Releases ZPdegrading
enzymes
(from Gilbert, Developmental Biology)
The Formation of
Primary Germ Layers
Figure Stages of prenatal development
Frill-necked Lizard
Squid lizard