cell bio notes for exam prof.pdf

Video 13:
Introduction to the
Endomembrane System
Gergely, Z.R., Martinez, D.E., Donohoe, B.S. et al. J of Biol Res-Thessaloniki
25, 15 (2018). https://doi.org/10.1186/s40709-018-0086-2

The Endomembrane
System Derived from the
cytoplasmic membrane
and forming a
coordinated unit
Include the ER, Golgi,
endosomes,
lysosomes, and
vacuoles (NOT the
mitochondria and
chloroplasts)
Allow transportation of
proteins, lipids, and
complex
polysaccharides

Transport System
Transport vesicles
allow movement
between cellular
compartments
Travel along the
cytoskeleton mediated
by motor proteins
Sorting signals within
amino acid sequences
or oligosaccharides
allow targeting to
appropriate cellular
compartment

Transport Pathways
All begin with the the
biosynthetic pathway
Proteins produced in
RER
●
Vesicles released
from RER fuse with
the cis Golgi
complex,
undergoing further
modifications
●
Transport vesicles
released from the
trans Golgi
Karp’s Cell & Molecular Biology (2020) Fig 8.2

Transport Pathways
Secretory pathways
result in exocytosis and
may be constitutive
(continuous) or
regulated (require
signal for release)
Materials are also
imported through
endocytosis and
digested within the
lysosome

Pulse-Chase &
Autoradiography
Monitor movement of silver grains (in
red) through secretory pathway

(Green)
Fluorescent
Proteins
Temperature-sensitive mutation
prevents movement of VSVG from ER
at 40OC

Other Experimental
Approaches
Subcellular fractionation (differential centrifugation)
●
Idenification of fraction containing particular enzymes or
functions
●
Now extend cell-free systems to include synthetic
liposomes
Effects of genetic mutations
●
Introduction of mutations in identify function of particular
gene
●
Newer methods include RNA interference and CRISPR

Video 14:
The Endoplasmic
Reticulum
https://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-
products.html?TablePage=106514528

The Endoplasmic
Reticulum
Likely from invagination
of cytoplasmic
membrane
Differentiated into rough
and smooth due
to presence
or absence of
bound ribosomes
on the
cytoplasmic
surface

ER Similarities
RER and SER are joined, having a
continual luminal space which
allows for free diffusion
Number of
proteins are
common to both
RER & SER
Both synthesize
some shared
lipids and
cholesterol
Karp’s Cell & Molecular Biology Fig. 8.10

Highly curved, tubular network with large numbers of
reticulons (cause bending of the membranes)
Smooth Endoplasmic
Reticulum
Detoxification of drugs
such as ethanol and
barbituates in the liver
by oxygenases
Ca2+ sequestering in
muscle cells (called
sarcoplasmic reticulum)
Synthesis of steroidal hormones in
endocrine cells

Continuous with the outer nuclear membrane and
composed of flattened sacs (cisternae) connected by
helical “ramps”
Rough Endoplasmic
Reticulum
Increased in secretory cells
(acinar cells of pancreas &
mucous cells in digestive
tract)
The site of synthesis of the
majority of phospholipids for
inclusion in cellular
membranes

Rough Endoplasmic
Reticulum
Undergo co-translational
translocation from bound
ribosomes to ER lumen
Synthesizes carbohydrate
chains and initiates N-
linked glycosylation of
proteins
Site of synthesis of approx. 1/3 of cellular proteins: those
destined for secretion or inclusion in endomembrane
organelles, integral
membrane proteins

Membrane-bound or Free
Ribosome?
Remainder of proteins are synthesized by free
(cytoplasmic) ribosomes
●
Cytosolic proteins
●
Peripheral membrane proteins for cytosolic surface)
●
Nuclear proteins (contain nuclear localization signals)
●
Proteins for other organelles (lysosomes, mitochondria,
chloroplasts)
All translation is initiated on free ribosomes
Proteins which should go to ER contain an N-terminal
signal sequence at N-terminus: results in ribosome
binding to ER surface and co-translational translocation

Co-translational Translocation
SRP binds signal sequence and guides ribosome to
translocon channel protein by binding to the SRP
receptor
SRP = signal recognition particle
Polypeptide binding to translocon opens
“plug” to allow polypeptide to enter ER
Signal removal
and folding

Synthesis of Integral Proteins
Hydrophobic “stop-transfer” sequence blocks
translocation into ER lumen
The next steps dependent upon location of positive and
negative charges at ends of the transmembrane region
●
If N-terminus side is
negative, the lateral gate
of the translocon allows
the transmembrane
region to migrate into the
membrane
●
Results in C-terminus on
cytosolic side

Synthesis of Integral Proteins
●
If N-terminus side is positive, the translocon first
reorients the transmembrane region, which is then able
to migrate through the lateral gate
●
This results in the N-terminus being located on the
cytosolic side
●
Multi-pass polypeptides utilize translocon many times in
an antiparallel
manner
●
Some proteins
utilize
alternative
pathways

Video 15:
Protein Processing
https://hms.harvard.edu/news/folding-revolution

Protein
Processing
Many secretory cells have a
polarized or directional structure
Reflects movement of
polypeptides from synthesis in the
RER, processing in the RER &
Golgi, and secretion via transport
vesicles or secretory granules
Processing is a highly regulated
multi-step process required for
the production of proteins
(biologically-active form)
Karp’s Cell & Molecular Biology Fig 8.12

Protein
Processing
Removal of signal peptide by
signal peptidase
Protein folding & molecular
chaperones
Formation and shuffling of
disulfide bonds by disulfide
isomerase
Addition of carbohydrates by
oligosaccharyltransferase

Protein Folding
Occurs spontaneously
during translation
Folding
directed by
amino acid
sequence and
the production
of favorable
interactions

Molecular Chaperones
Chaperones
redirect back to
proper folding
pathway

Membrane
Asymmetry
Cellular membranes “grow”
rather than being produced
through de novo synthesis
Protein and lipid
composition are altered as
membranes are moved from
one compartment to the next
Through the means of
membrane fusion, specific
asymmetry of membrane is
maintained

Altering Lipid
Composition
Enzymatic
conversion
Preferential
inclusion of
specific lipids
within vesicles
Movement from one
compartment to another by lipid
transfer proteins
Newly synthesized
lipids are inserted
in cytosolic leaflet,
but may be moved
by flippases

Glycosylation
Majority of proteins produced in the RER become
glycoproteins: starts in the RER & completed in the Golgi
Addition of sugars to oligosaccaride
catalyzed by specific
glycosyltransferases
●
Transfer a monosaccharide
from a nucleotide sugar
Oligosaccharide first synthesized
on dolichol phosphate
Transferred to protein by
oligosaccharyltransferase
enzyme

Glycosylation
Flippases
catalyze altered
orientation

Modification & Quality Control
Following
acquisition,
two
terminal
glucose
residues
removed
Bound by calnexin or
calreticulin (chaperone)
Final glucose removed

Conformation-sensing
enzyme (UGGT) determines
whether it is misfolded based
upon whether hydrophobic
regions are exposed

Eventually, a mannose is
removed & it is
transported to proteasome
for destruction
If it is
misfolded,
glucose
residue
again
added and
cycle
repeated

Transport from ER to Golgi
Special exit sites on edges of ER lack ribosomes and are
sites of vesicle budding
These small vesicles fuse together to form larger transport
vesicles and interconnected tubules
Vesicular-tubule carriers (VTCs) move towards the Golgi
complex and fuse with the cis-Golgi network
●
VTC transport occurs via microtubule tracks
The Golgi will allow for further modification of proteins and
sorting for final transport

Video 16:
Golgi Complex &
Transport
https://www.cancer.gov/about-nci/organization/dcb/research-programs

Golgi Complex
Tubule networks on cis and trans faces are involved in
sorting of proteins:
●
cis will send back to ER or on to the cisternae
●
trans will sort to plasma membrane or intracellular
organelles

Golgi Complex
Golgi cisternae are involved in the sequential modification
of the proteins synthesized by the ER
●
Differential composition in each compartment
Osmium
tetroxide
stains
unsaturated
fatty acids
in the Cis
Cisternae
Mannosidase II enzyme
in the Medial Cisternae
Nucleoside
diphosphatase
Enzyme in the
Trans
Cisternae

Golgi Complex
Alters the N-linked oligosaccharides added within the ER
as they progress through the cisternae
Assembles O-linked oligosaccarides
Majority of
complex
polysaccharide
assembly
Different
enzymes
found in each
compartment

Glycosylation
Enzymes involved in glycosylation are integral proteins
with active sites facing the interior of the Golgi
Those involved in adding sugars are transferases
Karp’s Cell and Molecular Biology (2020) Fig. 8.23

Vesicular Transport Model
Popular in the 1980s & 90s
Vesicles move from one
cisternae to the next, while
the cisternae themselves are
“static”
In vitro evidence of budding
and fusing vesicles
Believed to occur in vivo in
an anterograde direction
Karp’s Cell and Molecular Biology (2020)
Fig. 8.24

Cisternal Maturation Model
First & currently accepted
Cisternae contents change
over time and develop in an
anterograde direction
Vesicles travel in retrograde:
are they returning enzymes?
Could both potentially be
occurring?
Fig. 8.24

Transport from ER
to Golgi:Revisited
ERGIC = ER Golgi intermediate
compartment
Comprised of the larger vesicles & tubules
formed through the fusion of transport
vesicles
3 kinds of coated transport vesicles:
●
COPI = posterograde from Golgi
●
COPII = anterograde from ER
●
Clathrin-coated = from plasma membrane
& trans Golgi network to other organelles
Fig. 8.26

Coated Vesicles
Transport vesicles are
coated in proteins which
mediate
●
Membrane curvature
●
Selective uptake of cargo
●
Selective binding to target
compartment
Fig. 8.25

COPII Vesicles
Fig. 8.25
Form outer scaffold = framework
Remainder of
proteins are
inner layer of
adaptors

COPII Vesicles
Guanine exchange factor
catalyzes Sar1-GDP to
Sar1-GTP: implants into
cytoplasmic membrane of
RER: recruits Sec23-Sec24
Fig. 8.26
Sec13-
Sec31
form outer
scaffold
Sec24 recruits
Golgi enzymes,
membrane
proteins for
docking/fusion,
and soluble
cargo receptors

Outer Vesicle
Coats
Transport vesicle shapes and sizes
are dependent upon the external
coat proteins & their interactions
Disassemble &
released into
cytoplasm to allow
docking and fusion
For COPII, catalyzed
by hydrolysis of GTP
to GDP

COPI: Retrograde Transport
●
Majority of ER-resident
proteins are retained and
unlikely to be included in
transport vesicles
●
Soluble ER-resident proteins
contain KDEL (lys-asp-glu-
leu) amino acid retrieval
sequence in C-terminus which
bind specific KDEL receptors
for return in COPI vesicle
●
Similarly KKXX signal used
for membrane proteins
●
Unique signals likely for
specific Golgi compartments

Video 17:
Trans-Golgi Transport
& Protein Sorting
http://www.nsf.gov/news/mmg/media/images/myosin_nucleus_h.jpg

Sorting to the Membrane
Both secretory proteins &
cytoplasmic membrane
proteins must be targeted
to the cytoplasmic
membrane.
Both may be transported
through secretory vesicles
in the constitutive
pathway
Some secretory proteins
also follow regulated
pathways: aggregates in
granules require hormone
or nervous stimulus
Karp’s Cell & Molecular Biology (2020) Fig. 8.2

Targeting Vesicles
4 Steps: targeted transport, tethering, docking & fusion of
membranes
Tethering may be through
fibrous or multiprotein
complexes
●
Specificity mediated
by G proteins called
Rabs
●
Rabs specific to
particular
compartments
Rabs also
recruit
motor
proteins
for
transport
through
cytoplasm

Targeting Vesicles
Docking due to interaction of v-SNARE & t-SNARE
v-SNARE
t-SNARE

Model of Synaptic Vesicle
Exocytosis
Synaptobrevin (v-SNARE) & Syntaxin (t-SNARE) both
contain transmembrane domains that anchor them to the
vesicle & plasma
membrane
Including
SNAP-25
(t-SNARE),
4 helix bundle
forms through
hydrophobic
interactions

Model of Synaptic Vesicle
Exocytosis
●
For regulated release,
may stay in interlocked
formation until
stimulus received
●
Leads to formation of
a water-filled pore as a
transition state
●
Transmembrane
domains become
embedded in single
bilayer to form the
fusion pore

Synaptic Vesicle Exocytosis
Karp’s Cell & Molecular Biology (8th
ed) Fig. 4.57
●
Binding of Ca2+
by v-SNARE triggers fusion and release
of neurotransmitters

Exocytosis
Contacts between vesicle & membrane result in pore
formation on outside of an alveolus
Fusion Pore
Fusion of vesicle and
plasma membrane
results in release of
contents into the
extracellular
environment
Luminal part of vesicle is
now on the surface of
the plasma membrane

Plant Cell Central Vacuoles
Used for storage of
many biomoleucles
including sugars,
polysaccharides, &
amino acids
Tonoplast (membrane)
site of active transport of
ions to ensure turgor
pressure
Site of cellular digestion,
in lieu of lysosomes:
contain hydrolases

Posttranslational Uptake
The proteins destined
for organelles which are
not a part of the
endomembrane system
are synthesized by free
ribosomes
Following translation,
they are transported to
the target organelle
Relies on presence of
amino acid localization
signal
Organelles with
posttranslational uptake:
●
Peroxisomes
●
Nucleus
●
Mitochondria
●
Chloroplasts
Interaction of specific
chaperones or receptors
allow directed transport
from cytoplasm
Pore proteins regulate
entry into the organelle

Mitochondrial
Uptake
Majority of polypeptides
synthesized in cytoplasm
and transported in as
unfolded polypeptides by
specific chaperones.
Different amino acid
sequences interact with
different chaperones and
protein complexes to
determine appropriate
localization
Chaperones
TOM Complex
TIM Complex

Mitochondrial Uptake
Binding of chaperones involved in targeting to TOM
complex on outer mitochondrial membrane
TOM receptor recognizes mitochondrial protein and
channel complex allows its translocation
Location of a
positively
charged
localization
signal
determines
compartment

Mitochondrial Uptake
Internal sequence results in transport through the TIM22
complex = inner mitochondrial membrane protein
A terminal
sequence
(presequence)
results in transfer
through TIM23 =
matrix protein
●
Bound by
mitochondrial
chaperones,
folded and
presequence
removed

Chloroplast Uptake
Sequential import
through Toc and
Tic complexes
for entry into
the stroma
Stroma domain
removed
Also imported in unfolded state and interact with
molecular chaperones
Targeted through presence of the N-terminal transit
peptide

Chloroplast Uptake
Chaperones assist
folding of stromal
proteins
Thylakoid transfer
domain allows
transfer into
membrane or
lumen
Some thylakoid
membrane
proteins synthesized
for direct insertion

Video 18:
Lysosomes,
Endocytosis
& Phagocytosis
https://www.labroots.com/trending/immunology/5913/role-macrophages-molecular-housekeeping

Lysosomes
Are the digestive organelle
of the cell, containing
multiple acid hydrolases for
the digestion of:
●
Food particles
●
Extracellular debris
●
Pathogens
●
Biomolecule degradation
●
Autophagy
Proton transport used to
create acidic environment

Autophagy
Digestion of cellular
organelles:
●
Turnover of worn out/
defective components
●
Source of biomolecules/
energy
●
Protection from
intracellular pathogens
●
Prevents accumulation
of specific cellular
components: protective
Retained
indefinitely

Sorting of Lysosomal
Proteins
1)Mannose residue
phosphorylated in
the cis-Golgi
2)Incorporation into
clathrin-coated
vesicles
3)Mannose 6-
phosphate receptors
interact with
enzymes and
adaptors

Clathrin-coated Vesicles
Mannose-6-P
acts as targeting
mechanism for
lysosomal
enzymes
Interaction of
receptor and/or
lysosomal
membrane
proteins with
adaptor
Adaptor also
required for
assembly of
clathrin coat

Sorting of Lysosomal
Proteins
4) Clathrin coat &
receptors disassemble
5) Receptors return to
trans-Golgi
6) Develop into
endosomes, then
lysosomes
7) Secreted lysosomal
enzymes also
returned by mannose-
6-P receptors

Cellular Importation
Endocytosis
involves the
importation of
extracellular
fluid, dissolved
solutes, &
suspended
macromolecules
●
May be bulk
phase (general)
or receptor-
mediated
(specific)
Phagocytosis is the uptake of particulate matter
RECEPTOR-MEDIATED ENDOCYTOSIS

Clathrin Coat Assembly
Assembly of clathrin coats
for both endocytosis and
lysosomal vesicles
involves interaction of
clathrin subunits and
adaptor proteins

Clathrin-coated Pits
Phosphorylated
phosphoinositol
(phosophoinositides)
recruit the AP2 adaptor
proteins to the plasma
membrane
Binding allows m-subunit
to interact with tails of
receptors
b-adaptin recruits clathrin
Results in concentration
of specific receptors within
the pit

Vesicle
Formation
Dynamin proteins
polymerize to form ring
around the “stalk” of the
forming vesicle
GTP hydrolysis leads to
separation of the vesicle
from the membrane
Blocking of hydrolysis
with analog shows helical
association of dynamin

Endocytic Pathway
Fate of receptors &
ligands are dependent
on their purpose
Housekeeping
receptors (red)
Ligands include needed
substances (eg. iron
and cholesterol)
●
Receptors are recycled
to the plasma
membrane
●
Ligands to late
endosomes

Endocytic Pathway
Signaling receptors
(green)
Ligands include insulin
and growth factors
●
Both receptors &
ligands to late
endosomes
Late endosomes fuse
with additional vesicles
containing lysosomal
enzymes & fuse with
lysosomes

Phagocytosis
Food acquisition (unicellular organisms), immune cell
engulfment of pathogens
Yeast
cell
Leukocyte
Some pathogens
escape digestion:
●
M. tuberculosis
prevents fusion
with lysosome
●
L. monocytogenes
phospholipases
allow escape from
lysosome into
cytoplasm

Entrapment of particle,
followed by engulfment
to form a phagosome
Fuse with a lysosome for
digestion: usable
nutrients transferred to
cytoplasm
Residual body forms and
contents released
through exocytosis or
retained permanently as
lipofuscin pigment
granule
Phagocytosis

Public Domain doi:10.7295/W9CIL240
Video 19:
Introduction &
Microtubule
Structure

The Cytoskeleton
Divided into
three classes:
microfilaments
(actin),
intermediate
filaments (IFs),
and
microtubules
(MT)
Noncovalently
bonded protein
polymers:
highly dynamic
Microfilaments Microtubules
Merge
Intermediate
filaments

Major Functions
All three classes play
various roles in providing
structure and support
Microtubules act as
“tracks” for the movement
of organelles and
materials
Also a framework for
organization of organelles

Major Functions
Microtubule network and peroxisome transport
Peroxisomes
Peroxisomes
Peroxisomes

Major Functions
Microfilaments involved in growth
of axons
Also form the force-generating
apparatus for cell motility

Major Functions
Movement of chromosomes
Contractile ring for cytokinesis
Formation of the mitotic spindle

Microtubules
Components of cytoskeleton, mitotic spindle, cilia & flagella
Hollow tubular structures of 25 nm outer diameter
13 protofilaments shown in cross-section

Microtubules
Plus End
Minus End
Protofilaments formed of
alternating a- and b-tubulin
subunits
Offset alignment
results in helical
structure
Plus end
(b-tubulin terminus)
and minus end
(a-tubulin terminus)
results in
microtubule
structural and
functional polarity

Microtubule-Associated
Proteins (MAPs)
Multiple other proteins typically associated with
microtubules
Roles in increasing
microtubule stability and
promoting assembly of
tubulin subunits
Many regulated by
phosphorylation
Alterations in Tau function linked
to various forms of dementia

Tau & Dementia
Debate: cause or effect of
Alzheimer’s?
Brain 2006 129(11):3035-3041
Hyperphosphorylated Tau protein incapable of binding
to microtubules: found in neurofibrillary tagles/plaques

Structural Support
Organization of microtubules determines the overall shape
of the cell:
●
Radial arrangement seen in cultured animal cells results
in round, flattened
shape
●
Basal to apical
arrangement in
columnar epithelial
cells
●
Lengthwise
arrangement along
axons

Structural Organization
Association with integral membrane proteins influences
organization
Cellulose synthase
association determines
location/orientation
of cellulose
microfibrils which
determines plant
cell shape and
manner of growth
Organelle localization
eg. Golgi apparatus
Cellulose synthase Microtubules

https://foglets.com/molecular-nanomachines-kinesin-proteins/
Video 20:
Microtubules
& Transport

Intracellular Motility
Microtubule networks act as “roads” through the cell,
allowing specific transport of materials to a given target
Microtubule tracks
within for transport of
organelles, transport
vesicles and
molecules such as
mRNA & ribosomes
Neurofilaments
support
structure of
axons

Motor Proteins
Large variety of motor proteins in a given cell, each
specialized for a specific type of cargo
Three broad classes: kinesins, dyneins, & myosins
●
Both kinesins & dyneins use microtubule pathways
●
Myosins use microfilaments
Movement based on binding and hydrolysis of ATP to
cause conformational changes allowing stepwise
movement from one subunit to the next

Kinesins
Heads are highly conserved motor region: binding and
hydrolysis of ATP for movement along the microtubule
Tail sequences very diverse bind the cargo specific for that
particular type of kinesin

Kinesins
Each “step” requires the binding and hydrolysis of one
molecule of ATP
Rate of movement is dependent upon ATP concentration

Kinesins
The neck region of the molecule plays an important role in
“transmitting” the conformational changes from one head
subunit to the other
Majority of kinesins move toward the plus end of the
microtubule, but some do move toward the minus end
●
Neck region also determines the direction of movement
along the microtubule
Small group of kinesins are involved in microtubule
depolymerization instead: called microtubule
depolymerases
●
Critical in cell division processes

Kinesins & Organelle Transport
Normal Kinesin mutant
Normal
Microtubu
les
Mitochond
ria

Cytoplasmic Dynein
Dynein first discovered as the motor protein associated
with cilia and flagella
Composed of two identical
heavy chains
Involved in positioning of
spindle & chromosome
movement
Transport towards
minus end of the microtubule:
positioning of centrosomes &
Golgi, and in various transport

Cellular
Transport
Model for use of
kinesins and dyneins
in cellular transport
based upon
microtubule polarity
Vesicles may have
both types bound at
one time: inactive vs
active forms, or a tug-
of-war?

https://foglets.com/molecular-nanomachines-kinesin-proteins/
Video 21:
Microtubule
Dynamics

Microtubule-Organizing
Centers (MTOCs)
Specialized structure where the initial formation (or
nucleation) of a small section of the microtubule occurs,
followed by its rapid elongation
MTOCs include:
●
Centrosomes
●
Basal body: associated with cilia/flagella & similar to
centrosomes
●
Plant MTOC: more dispersed on outer surface of
nuclear envelope

The Centrosome
Contains two barrel-shaped
centrioles surrounded by electron
dense pericentriolar material (PCM)
Major site of microtubule initiation in
animal cells & is the centre of the
cell’s microtubule network

Nucleation
Microtubules always assembled with the minus end
associated with the centrosome
Growth occurs
through the
addition of
tubulin dimers
to the plus end
May remain
associated or
severed and
anchored in
other regions
of the cell

Nucleation
Ring of 13 g-tubulin monomers in complex
with additional proteins at site of nucleation
g-tubulin, a/b-tubulin, DNA

Microtubule Dynamics
Noncovalent association of dimers allows rapid
reorganization or polymerization/depolymerization
as needed
Interphase
Preprophase
band: future
division plane
Mitotic
spindle
formation
Phragmoplast:
cell wall
formation
between cells

Role of GTP
b-tubulin is a GTPase and
must be bound to GTP for
polymerization to occur
GTP is hydrolyzed shortly
after polymerization
GDP-bound tubulin lacks
stability on its own, & will
rapidly depolymerize in the
absence of MAPs
Structural cap model
describes effect

Dynamic Instability
Plus-end tracking
proteins (+TIPs) bind to
ends of microtubules to
regulate rate of growth or
shrinkage
+TIPs mediate
attachment to structures
(eg. kinetochores, actin)
Growing & shrinking microtubules found within the same
region of the cell
Rapid shift between growing & shrinking phases

Video 22:
Cilia & Flagella
https://www.mdpi.com/2313-7673/3/2/5/htm

Cilia & Flagella
Hair-like cellular projections which are usually motile
Both are constructed from microtubules in the same
fashion
Both may be used for cellular motility by unicellular
organisms, and flagella present on animal gametes
Differentiation based upon cell type and type of motion

Cilia
Single, non-motile cilium
on almost all cells used
as sensory “antennae”
Used for cellular motility, but also found on
surface of non-motile cells for the purpose of
moving extracellular materials
Use a coordinated oar-like
motion
& 9.26

Flagella
Found in singles or pairs
for locomotion
Variety of patterns of
motions, including
assymetric waveforms and
symmetrical propellar-like
movements

Cilia/Flagella Structure
Membrane is continuous with the plasma membrane
Basal body is an
MTOC identical to
the centriole
Primary cilium
derived from
centriole, new basal
bodies in secondary
cilia
A and B tubules of
basal body form
doublets of the
axoneme

Axoneme is the core structure of
longitudinal microtubules and associated
proteins
Axoneme Structure
9 peripheral doublets
surrounding 2 central
single microtubules
(9 + 2 array)
Central sheath connects
to A tubules by radial
spoke proteins

Karp’s Cell & Molecular Biology (2020) Fig 9.28 & 9.29
Axoneme Structure
Nexin-dynein
regulatory
complex
(N-DRC)
Movement of outer & inner
axonemal dynein arms result in
bending motion of flagella/cilia

Intraflagellar
Transport
Growth occurs only at the
(+) end
Transport uses IFT
particles (protein
complexes) to carry
tubulin or other cargo
Kinesin transports
outward, and cytoplasmic
dynein inward

Karp’s Cell & Molecular Biology (2020) Fig 9.32 & 9.33
Mechanism
Dynein stem is
anchored to the
A tubule
Movement due to binding
& release of dynein heads
1) Heads bind to B tubule
2) Conformational change in dynein
causes sliding of anchored doublet
3) Heads release B tubule allowing
return to original position
4) Cycle repeats
Nexin bridges results in limited
movement and bending

Sliding Mechanism
Central position occurs when outer
doublets are all at the same level
Sliding of doublets in relation
to the position of its
neighbours will
determine the direction
of bending
Inner tubules slide towards base
of the cilium/flagellum
*Different positional relationships can
result in different stroke patterns

Video 23:
Intermediate Filaments
https://micro.magnet.fsu.edu/primer/techniques/fluorescence/gallery/cells/mdok/
mdokcellsexlarge8.html

Intermediate Filaments
Provide mechanical strength to the cells, particularly those which
undergo large amounts of stress (nerves, muscles, epithelia)
Only in animals so far, but other insoluble fibres of diverse
protein sequences found in other eukaryotes
Chemically heterogeneous, with approx. 70 different genes in
humans
●
All of similar structure: solid & unbranched with 10-12 nm
diameter
●
5 classes based on cell type of expression, and biochemical,
genetic, and immuonologic criteria
– Cytoplasmic filaments (Types I – IV)
– Lamins (Type V): support nuclear membrane

IF Structure All IFs share same basic
structure:
●
a-helical central fibrous
domain: homologous
sequences
●
Terminal globular domains
of variable size/sequence
●
Different N- and C-terminal
sequences and their
association together results
in polarity within the dimers
●
Anti-parallel association of
dimers into nonpolar
tetramer = basic unit

IF Structure ●
Subunits associate in
groups of 8 to form a unit
length
●
End-to-end association to
form the polymerized IF
●
Growth through
intercalation within
existing IF
●
Polymerization &
depolymerization
regulated through
phosphorylation
●
Only cytoskeletal fibres
that lack polarity
~60 nm

Cytoplasmic IFs
Include proteins
such as keratin
(epithelia),
desmin (muscle),
and the
neurofilaments
(CNS and PNS
nerves)
Interconnected to
other cytoskeletal
filaments by
plectin cross-
bridges

Epithelial Cells IFs (Types I and II) are
composed of keratin
Found radiating
throughout the cell
Often terminate on
desmosomes
(communication points
between cells)
Karp’s Cell & Molecular Biology Fig. 9.43 & 7.27

At least three different
proteins (Type IV Ifs)
Unique in structure
due to presence of
sidearms : ensure
appropriate spacing
between parallel fibres
Increasing amounts of
IFs as neuron matures
Karp’s Cell & Molecular Biology Fig. 9.43 & 7.27
Neurofilaments

Lamins
Form a strong,
mesh-like grid on
the nucleoplasmic
side of the inner
nuclear membrane
called the nuclear
lamina
Disassembled
during prophase of
mitosis/meiosis

Video 24:
Microfilaments and
Myosin
By Howard Vindin - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?
curid=39436133

Microfilaments
= Actin filaments = F-actin
Roles:
●
Cellular motility
●
Intracellular motile processes
(vesicle transport,
phagocytosis, cytokinesis)
●
Muscle contraction
Highly conserved, with different
isoforms for specialized roles

Microfilaments
Assembled from monomers into
polar fibre with “barbed” and
“pointed” ends
Organized into:
●
Ordered arrays
●
Highly branched networks
●
Tightly anchored bundles
(+) “barbed”
end
(-) “pointed”
end

Assembly &
Disassembly
Regulated through
presence of ATP/ADP with
hydrolysis after
incorporation
Critical concentration for
the addition of ATP-actin
to either end is different:
lower concentrations
required for addition to
(+) end
At 0.3 mM (-) end removal
= (+) end addition

Myosin
Superfamily of motor proteins which
associate with actin filaments
Minimum of 17 classes
identified, with about 40
different myosins in humans
Type II (conventional)
myosins first identified
in muscle
Actin subunit
comprised of 6
polypeptide
chains
ATP-binding
cleft & motor
function
associated with
the head

Myosin Type II Filaments
Tails associate with
heads on either end to
form a bipolar filament
Myosin moves towards
(+) end of the
microfilament
Bipolar arrangement
results in
microfilaments being
pulled towards one
another

Unconvensional Myosins
Do not form filaments
and generally operate
independently
Myosin I has a single
head and acts as
attachment point for
microfilaments to the
plasma membrane
●
Potential involvement
in the alteration of
membrane shape

Unconvensional
Myosins
Many involved in the transport of
vesicles and organelles, or as
organelle tethers
Myosin V shows movement
similar to kinesin, with one head
bound to actin at all times

Unconvensional Myosins
Believed that long distance
transport occurs on
microtubules, with
microfilaments used for local
movement

Video 25:
Muscle Contraction
https://www.oldschoollabs.com/what-is-hypertrophy/

Muscle
Organization
Skeletal muscle cell
(fiber) formed through
fusion of mononucleated
cells early in
development
Each myofibril is
composed of repeating
contractile units called
sarcomeres

Sarcomeres
Visible striations due to
the presence of thin and
thick filaments and their
regions of overlap
Z-lines are borders
between sarcomeres

Sarcomeres
Composed of actin (thin)
filaments and conventional
myosin (thick filaments)
Actin
Myosin

Sliding
Filament
Model
Contraction results
in decrease in width
of the I and H bands
as actin moves
inward, shortening
the sarcomere
Karp’s Cell & Molecular Biology
(2020) Fig 9.51

Composition & Organization
Thin filaments are the microfilaments and associated
proteins
●
Tropomyosin fits within the goove of the microfilament,
7 actin subunits in length
●
Troponin globular protein which interacts with the end
of tropomyosin and with actin

Composition & Organization
Thick filaments are composed of several hundred myosin
molecules with bipolar orientation
Titin is an elastic protein stretching from M line to Z line
●
Maintains overall structure, aids relaxation

Molecular Basis of
Contraction
Each myosin filament head undergoes conformational
change to move actin filament 10 nm.
Each filament interacts with approx 100 myosin
molecules, resulting in continuous contraction of several
hundred nm

Swinging Lever Mechanism
1) Myosin tightly bound in absence of ATP
2) ATP binding results in release
3) ATP hydrolysis results in conformational change
4) Rebinding to actin 5) ADP realeased and repeat
http://www.esrf.fr/UsersAndScience/Publications/Highlights/2003/MX/MX03/
Lever arm
swings

Excitation-Contraction
Coupling
Transverse tubules
transmit impulses
from action
potentials to interior
of cell
Sarcoplamic
reticulum stores
calcium ions
needed to induce
muscle contraction
Muscle fibers organized into motor
units stimulated by a single nerve

Excitation-Contraction
Coupling
https://schoolbag.info/biology/mcat/53.html
Action potential results in release of
calcium ions from SR
Binding of calcium to
troponin causes change in
conformation

Video 26:
Cellular Motility
http://entertoys.blogspot.com/2016/09/

Cellular Motility
Relies upon the presence of
actin networks in the cell
cortex (below the cell
membrane)
Processes include:
●
Endocytosis &
phagocytosis
●
Extension of processes
●
Cytokinesis
Organization/function
dependent on actin binding
proteins Karp’s Cell & Molecular Biology (2020) Fig 9.39

1) Nucleating proteins
●
Spire contains multiple actin-binding domains to form
cluster for nucleation
●
Formins used to create and lengthen unbranched
filaments at the (+) end
●
Arp 2/3 complex is
template for addition
of actin monomers to
add branches
Actin Binding Proteins
Karp’s Cell & Molecular Biology (2020)
Fig 9.66

2) Monomer-sequestering proteins (thymosins)
●
Bind actin-ATP monomers to prevent
polymerization
●
Regulates rate of polymerization/depolymerization
3) End-blocking (capping)
proteins
●
Prevent addition or loss
of actin
●
May be associated with
the (+) or (-) end

4) Monomer polymerizing proteins
●
Profilin binds to same site as thymosin
●
Promotes removal of ADP, allowing ATP to bind
and polymerization to occur
5) Depolymerizing proteins (cofilin family)
●
Bind to pointed end to cause depolymerization
7) Filament-severing proteins
●
Cause fragmentation (eg. cofilin and gelsolin)
●
May create additional free barbed ends for growth,
or may act as capping proteins

Karp’s Cell & Molecular Biology
(2020) Fig 9.60
6) Cross-linking and bundling
●
Flexible rod-like proteins
for forming mesh-like
network for support
(eg. filamin)
●
Globular bundling proteins
for parallel groupings
as seen in microvilli
(eg. villin & fimbrin)
8) Membrane-binding proteins
●
Anchorage for changes in
membrane shape

Cell Locomotion: “Crawling”
Protrusion formed
on leading edge
Lower surface of
protrusion attaches
to substratum
Majority of cell is
pulled forward
Rear contacts
broken

Lamellipodia
Dynamic fan-like or “ruffled”
protrusion on leading edge
of motile cells

Directed Motility
Stimulus results in activation of filament nucleating
proteins (Arp 2/3 complex) by WASP/WAVE
Polymerization of actin-ATP mediated by profilin

Directed Motility
Capping of older filaments occurs,
while allowing new branches to
continue growing, resulting in
the outgrowth of the
lamellipodium

Directed Motility
Older filaments are
comprised of actin-
ADP
Depolymerization of
the (-) ends of
capped
microfilaments
mediated by cofilin
Profilin allows
release of ADP to
allow formation of
actin-ATP for further
use

Traction Forces
Focal adhesions are
formed between the
leading edge of the
lamellipodium and
the substratum
These focal
adhesions allow for
the bulk of the cell to
be moved forward
by myosin and actin
Vinculin staining at
focal adhesions

Axonal Outgrowth
Growth cone at tip resembles a highly motile fibroblast
●
Broad lamellipodium
●
Short microspikes towards edge of lamellipodia
●
Elongated filipodia
Actin
Tubulin

Axonal Outgrowth
Direction of growth is determined by physical and chemical
stimuli detected by receptors in the growth cone
Tubulin Ephrin
f
●
Response to
chemoattractants
(eg. netrin) which
diffuse through
extracellular
environment
●
Interacting with
membrane-bound
molecules (eg. ephrin)
to encourage growth in
that particular direction.

Video 27:
Video 27:
Introduction to
Introduction to
Cell Signalling
Cell Signalling
https://www.kurzweilai.net/using-cells-chemical-signaling-to-control-cancer-or-detect-toxins

Cell Signalling &
Communication
Cells receive signals from the environment, which results
in a particular response:
●
Alterations in cell behavior (eg. Chemotaxis)
●
Alterations in gene expression (eg. Gene expression)
Multicellular organisms need to coordinate their behavior
●
Embryonic development
●
Cell division

Vocabulary
Signal molecules: extracellular molecules which act as
messengers between cells
Signalling cell: the cell which produces the signal molecule
Target cell: the cell receiving the signal
Receptor protein: a protein in the target cell which
recognizes and responds specifically to that signal
Signal transduction: means by which the message is
translated into a response

Receptors may be found
on the cell surface or
within the cell
Hydrophilic signals bind
to surface receptors
Hydrophobic signals
bind to intracellular
receptors
Receptor
Proteins

Autocrine Signalling
Receptors on the surface of the cell allow it to respond to a
signalling molecule it produces itself

Paracrine Signalling
Messenger produced by signalling cell acts on nearby
target cells: signal travels short distance through
extracellular space
Limited due to
their stability,
digestion by
enzymes, or
through
interactions
with the
extracellular
matrix

Endocrine Signalling
Messenger produced for long
distance signalling: signal
travels through bloodstream
Many hormones are
produced in organs distant
from the site of the target
cell

GAP JUNCTIONS
●
Hydrophilic channels
allow communication
between cells that form
during development
CNX OpenStax / Wikimedia Commons
Other Forms
of Cell
Signalling
https://projects.ncsu.edu/project/bio183de/
Lab/cells_celldivision/cells_celldivision1.html
SYNAPTIC

Signalling Overview
Diverse signalling molecules ranging from small soluble
compounds and proteins to large cell surface bound
glycoproteins
Specific receptors on
the target cell will bind
to their ligand
(signalling molecule)

Diversity of Extracellular
Messengers
Amino acids & their derivatives (eg. Epinephrine &
dopamine) act as neurotransmitters and hormones
Gases (NO, CO)
Cholesterol-derived steroid hormones
Eicosanoids (derived from fatty acids) act in regulating
various systemic responses (inflammation, blood
pressure)
Polypeptides/proteins on membrane of signalling cell or
secreted into extracellular environment

Cell Signalling Receptors
G protein-coupled receptors (GPCR): binding of ligand
results in activation of GTP-binding protein
Receptor protein-tyrosine kinases (RTK): binding of
ligand results in activation of kinase activity and
phosphorylation of target proteins
Ligand-gated channels: alterations in ion concentrations
lead to altered cellular activities (neurotransmission,
muscle contraction, calcium as a second messenger)
Steroid hormone receptors: intracellular, act as
transcription factors

Signalling Overview
Ligand binding to receptor results in conformational
change: transmits signal across membrane
Effector enzyme
generates small molecule
second messenger Karp’s Cell & Molecular Biology (2020) Fig 15.2
Recruitment of
signalling proteins
OR

Signalling
Overview
Sequential
interaction of
proteins, resulting in
conformational
changes which affect
their activity
Final target protein is
activated leading to
alterations in cellular
responses
RESPONSE

The addition (kinases) or removal
(phosphatases) of phosphate
groups alter protein
conformation and
therefore activity
Termination of signalling
requires the destruction
or inactivation of
receptors, second
messengers, and
transduction proteins Karp’s Cell & Molecular Biology (2020) Fig 15.2
Signal Transduction
Pathways
Phosphorylation occurs
on serine, threonine
or tyrosine
Proteins are cytoplamic
or membrane-bound

Video 28:
G Protein-Coupled
Receptors
http://www.aureliabio.com/news/gpcrs-significance-pharmaceutical-world/

Superfamily of receptors that mediate signalling through
interaction with G proteins
G Protein-Coupled
Receptors (GPCR)
G proteins are
trimeric proteins that
bind to GDP/GTP
Following activation,
G proteins interact
with specific
effectors to initiate
signalling

Largest group of proteins in animals, with highly varied
roles in the cell
G Protein-Coupled
Receptors
Karp’s Cell & Molecular Biology (2020) Table 15.1

G Protein-Coupled Receptor
Action
Consist of 7 a-helical
transmembrane
domains (also called
7TM receptors)
Extracellular loops form
binding site for ligand
Ligand binding will
results in
conformational change
in transmembrane
domains
N-term
C-term

G Protein-Coupled Receptor
Action
Conformational change
results in strong affinity for
the G protein
Heterotrimeric G proteins
consist of three subunits:
●
a (contains GDP/GTP
binding site), b, and γ
●
Binding to receptor
results in exchange of
GDP for GTP
N-term
C-term
a
GDP/GTP
binding site

GPCR
Action
GTP binding results in
dissociation of a from
b/γ and its interaction
with the effector
molecule to initiate the
signalling cascade
Hydrolysis of GTP to
GDP results in a
dissociation and the
effector being “turned
off”

GPCR Action
GDP-bound a loses affinity
for effector molecule and
associates with b/γ again
4 different types of G
proteins with different a
subunits which interact with
different effectors:
● Gs activate adenyl cyclase
● Gi inhibit adenyl cyclase
● Gq with phospholipase C b
(PLCb)
● G12/13 not characterized
b/γ also involved in
signalling and interact
with adenyl cyclase
PLCb, and K+ and Ca2+
channels

Regulation of G Protein
Activity
G proteins with GTP bound interact with the effector
molecule to activate them
G proteins are slow-acting GTPases, resulting in their
eventual hydrolysis of bound GTP to GDP + Pi
Acceleration of this process occurs through the action of
regulators of G protein signaling (RGSs)

Termination of Response
●
G protein-coupled
receptor kinase (GRK)
are serine-threonine
kinases which
phosphorylate activated
GPCR
●
Phosporylated residues
are bound by arrestin,
which compete with G
proteins for binding to
GPCR Karp’s Cell & Molecular Biology (2020) Fig. 15.6
Effectors are no longer active after dissociation of the a
subunit, but the receptors need to undergo desensitization
in order to deactivate them:

Arrestin & Internalization
●
Inclusion in
signalling
complexes &
activation of
transcription
factors (3)
●
Digestion (4)
●
Recycled to
cell surface
(5/6)
Arrestins associate with AP2 in clathrin-coated pits, which
results in GPCR accumulation within the pits
Endocytosis occurs, resulting in 3 potential roles:

GPCRs and Bacterial Toxins
Some bacterial toxins target the functioning of GPCRs
through targeting the function of the a subunits
●
Cholera toxin modifies a subunits by inhibiting GTPase
activity, leading to continual activation of adenylate
cyclase.
– This is the cause of diarrhea due to altered water
retention by intestinal cells.
●
Pertussis toxin inhibits function of a subunits, interfering
with signalling required for immune defense

Interact with multiple targets, resulting in a coordinated
rapid response to external stimuli
Most are soluble (except phosphoinositides & DAG) and
will rapidly diffuse through the cell
Include:
●
cyclic AMP/GMP (cAMP/cGMP)
●
phosphoinositides, diacylglycerol (DAG), inositol
triphosphate (IP3)
●
Ca2+
●
NO
Second Messengers

Cyclic AMP
Production catalyzed by adenylyl cyclase
●
Interacts with GTP-bound G protein a
subunits
●
Recall that G proteins may either
activate adenylyl cyclase (as) or
inhibit its function (ai)
Each activated
adenylyl cyclase
allows a rapid
amplification of the
signal initiated through
ligand binding

Reg
ulati
on
of
Bloo
d
Gluc
ose
In response to
glucagon (from
pancreatic a-cells)
or epinephrine
●
Through G protein-
coupled receptors
●
Also cause inhibition of
glycogen synthase
Activated by
insulin from
pancreatic b-cells
●
Mediated by a
receptor
protein-tyrosine
kinase

Glucose Mobilization
2) Adenylyl cyclase in cell membrane
is activated & cAMP is produced
●
Downregulation may occur
through action of
phosphodiesterase
1) Ligand binding
results in G
protein activation
& mobilization of
the as subunit
3) cAMP then binds to &
activates protein kinase A
(PKA)

PKA
https://www.creative-enzymes.com/resource/protein-kinase-a_15.html
Inactive form is
tetramer of 2 catalytic
and 2 regulatory
subunits
Following binding of
cAMP, regulatory
subunits dissociate,
releasing 2 active PKA
PKA phosphorylates
targets within the cell,
regulating their activity

4) Inactivates glycogen synthase
5) Activates phosphorylase
kinase
6) Phosphorylase kinase
activates glycogen
phosphorylase
7) Glycogen
phosphorylase
depolymerizes
glycogen
Phosphatases
involved in
downregulation
PKA

9) PKA which enters the nucleus
phosphorylates the cAMP response
element-binding protein (CREB)
10) CREB binds response elements
within the promoter of genes to
upregulate their transcription

Regulation of PKA Targeting
PKA is known to
phosphoylate over 100
different substrates
Which responses occur
in a given cell type due
to:
●
Differential
expression of these
targets
●
Presence of PKA-
anchoring proteins
(AKAPs)

Regulation of PKA Targeting
Different AKAPs found within specific membranes in
different cell types
Sequester PKAs to certain cellular
regions to coordinate their
interactions with targets

Phosphatidylinositol (PI)
Numerous lipid derivatives function as second messengers
Enzymatically altered by phospholipases, phospholipid
kinases, and phospholipid phosphatases
Those derived from PI called phosphoinositides
Hbf878, CC0, via Wikimedia Commons

Phosphoinositides
Number of derivatives are created through the actions of
kinases which remain associated with the membrane
PI(4)P, PI(4,5)P2, and PIP3 (not shown) all act though
interaction of the phosphorylated inositol ring with lipid-
binding domain containing proteins
Domain acts
to recruit
these
proteins to
the membrane
for protein-
protein
interactions

Phospholipase C (PLC)
GPCR binding may also lead to
activation of phosphatidylinositol-
specific phospholipase C-b, which
cleaves PI(4,5)P2
Diacylglycerol
Inositol 1,4,5-
triphosphate

Diacylglycerol (DAG)
https://www.mdpi.com/2072-6694/6/2/860
DAG remains associated with the membrane and binds to
proteins containing the DAG-binding domain
Protein kinase C (PKC) associates with DAG and
phosphorylates various targets:
●
Extracellular
signal molecule
release
●
Cell growth &
differentiation
●
Metabolism
●
Apoptosis
●
Immunity

Inositol 1,4,5-triphosphate
IP3
is a soluble second messenger which will rapidly
diffuse through the cell
IP3
receptor on SER is a calcium channel, which opens
upon binding
Ca2+
also a second
messenger,
interacting with
various targets:
●
Muscle
contraction
●
Exocytosis
●
Cell shape

Video 30: Receptor
Protein-Tyrosine Kinases
By Emw - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=8762948

Receptor Protein-Tyrosine
Kinases
Large number of kinases expressed by different cell types
Approximately 2/3 of tyrosine kinases are membrane-
bound receptors which are activated through ligand
binding and dimerization
●
Ligands inclued growth factors & hormones
Regulate diverse processes:
●
Cell growth & proliferation
●
Differentiation
●
Cellular motility

Kinases
Monomeric
receptors undergo
dimerization due to
ligand binding

Kinases
In the absence of
phosphorylation, the
ATP-binding active site
of the kinase domain is
blocked by the
activation loop
Phosphorylation results
in activation loop being
in stable conformation
away from the active
site, allowing trans-
autophosphorylation of
adjacent cytoplasmic
domains

Kinases
The phosphorylated
tyrosines and their
surrounding amino acid
sequences determine
the specificity of
interactions with
signalling proteins
Most characterized
domains:
●
Src-homology 2
(SH2)
●
Phosphotyrosine-
binding (PTB)

RTK Signal
Transduction
May be bound by adaptor
or scaffolding proteins
which allow the
recruitment of other
proteins
Docking proteins may
themselves be
phosphorylated by the
receptor to allow
interaction with signalling
molecules

RTK Signal Transduction
Many transcription
factors are able to bind
to activated RTKs
Binding results in
phosphorylation, leading
to dimerization
Following dimerization,
the transcription factor is
transported into the
nucleus, where it binds
specific DNA sequences
to allow transcription

RTK Signal Transduction
Activation of enzymes
through:
●
Recruitment to membrane
& co-localization with
substrate
●
Conformational change
due to binding to receptor
●
Phosphorylation
Regulation of enzymatic activity by RTKs is also common
●
Kinases, phosphatases, lipases

Turning RTK Signals Off
Ubiquitin ligases contain
SH2 domains which allow
them to associate with
RTKs and label them for
internalization
Ubiquitination results in
interaction with the AP2
adaptor which localizes
the receptors into clathrin-
coated pits for endocytosis
●
Destruction in the lysosome
●
Recycling back to cell surface
●
Use in endosomes for
intracellular signalling

Signalling
Cascades
Most well known RTK pathway is
the Ras-MAP kinase pathway:
●
Activation of Ras (a small G
protein) through GTP binding
●
Sequential activation of protein
kinases
●
Phosphorylation of targets
including transcription factors,
other kinases, cytoskeletal
components, and more

https://erc.bioscientifica.com/view/journals/erc/26/6/ERC-19-0098.xml
Small G Proteins
Monomeric G proteins, similar in function to the
heterotrimeric G proteins (see GPCRs)
●
Interaction with adaptor (Grb2/Sos) makes nucleotide
binding site of Ras available
●
Ras active when GTP is bound
●
GTPase function, resulting in
hydrolysis to GDP and inactivity
Ras functions by recruiting
Raf to the membrane,
leading to its phosphoryation
and activation

Regulation of
Small G Proteins
3 types of accessory proteins are
involved in the regulation of small G
proteins
1) Guanine nucleotide
dissociation inhibitors (GDIs)
●
Prevent the release of GDP
●
Results in maintenance of
inactive G proteins

Regulation of
Small G Proteins
2) Guanine nucleotide-exchange factors
(GEFs)
●
Binding allows the exchange of GDP for
GTP, resulting in activation of the G
protein
●
Binding of the G protein to its target
results in GEF release, activation of
the target & signal transduction

Regulation of
Small G Proteins
3) GTPase-activating proteins (GAPs)
●
Binding to G protein/target complex results in
acceleration of GTPase activity
●
Once GTP hydrolysis occurs,
complex disassociates

Regulation of MAP Kinase
Pathways
Scaffolding proteins
allow specific
assembly of
components for
mediating particular
processes
Kinases only
phosphorylate specific
substrates
Inactivation of MAP
kinases by
phosphatases (MKP) https://erc.bioscientifica.com/view/journals/erc/26/6/ERC-
19-0098.xml

a and b chains
produced from single
polypeptide
a chain contains the
insulin binding domain
b chain is
transmembrane protein
with tyrosine kinase
domain
Exists as stable dimer
even in the absence of
insulin
Insulin binding causes
confromational change
in kinase domains
Insulin Receptor

Autophosphorylaed insulin receptor binds to
PTB domain of insulin receptor sustrates (IRS-1
& IRS-2)
IRSs also have PH domain (interact with
phospholipids on membrane)
Phosphorylated tyrosines interact with signal
molecules with SH domains
Insulin Receptor

Following activation by binding to IRS, PI3K produces
phosphoinositide PIP3 which leads to the recruitment of PKB
& PDK1
Role of PI 3-kinase
PDK1 &
mTOR
activate PKB
for signal
transduction
PKB mediates
glucose uptake by
causing fusion of
GLUT4 receptor
vesicles with
membranes

https://www.cellsignal.com/pathways/insulin-receptor-signaling-pathway
Video 31: More Second
Messengers & Interactions

Karp’s Cell and Molecular Biology (2020) Fig 4.56
Calcium plays important
roles in cell functions
●
Synaptic
transmission
●
Muscle
contraction
https://schoolbag.info/biology/mcat/53.html
Calcium as an Intracellular
Messenger

Inositol 1,4,5-triphosphate &
Calcium
IP3
receptor on SER
is a calcium channel,
which opens upon
binding
GPCR binding may
also lead to activation
of Phospholipase C-
b, which cleaves PIP2
to produce DAG and
IP3
Cytoplasmic calcium concentration normally low (10-7
M)

IP3-mediated signalling
due to oscillation in
cytoplasmic calcium
levels
IP3
and Calcium Oscillation
Karp’s Cell & Molecular Biology (2020) Fig. 15.11 & Table 15.2

Opening of voltage-gated Ca2+
channels (1) leads to entry of
some Ca2+ ions
Binds to ryanodine receptors
(RyR) (2) on the surface of the
SER
Triggers opening of these
channels and release of stored
Ca2+ from ER (3)
Calcium-induced
Calcium
Release

Cytoplasmic concentration restored
through action of:
●
Ca2+ pumps in ER (4)
●
Na+/Ca2+ exchanger in plasma
membrane (5)
Response may be localized in small
region or may result in increasing,
spreading wave throughout cell
Calcium-induced
Calcium
Release

Sperm contain a phospholipase, which cleave PIP2
contained within the egg to produce PI3
Calcium wave in Fertilization
PI3 binds
receptors on
the SER,
resulting in
calcium wave
Image
produced here
every 10
seconds

Store-Operated Calcium
Entry
STIM1 are calcium-sensing proteins which are normally
dispersed throughout the ER membrane

Store-Operated Calcium
Entry
STIM1 become clustered
near plasma membrane &
recruit Orai1
Cellular stores may become depleted
through repeated responses
Orai1 Ca2+ ion
channels allow
import of Ca2+ &
storage in ER

Cell-type specificity due to
ability to:
●
Activate/inhibit enzymes
●
Alter transport
●
Change membrane
permeability
●
Cause membrane fusion
●
Impact structure and
function of the cytoskeleton
Cellular
Effects of Ca2+

Mediate effects of calcium through
interacting with it and target proteins
Calmodulin most widely studied and found in
all eukaryotes
●
Low affinity for calcium, so only binds
when calcium increased in cytoplasm
●
Alters conformation and binds to cell-
specific targets
●
Targets include the Na+/Ca2+ exchanger,
thereby acting to bring a halt to further
signalling as well.
Calcium-binding Proteins

Nitric Oxide
Acts as both an extracellular and a
second messenger to regulate
various processes
Acetylcholine signalling (1) results
in the influx of Ca2+ (2),
which activates nitric
oxide synthase (NOS)
NOS produces NO
from arginine (3), &
NO diffuses from the
endothelial cell into
neighbouring smooth
muscle cells (4)

Nitric Oxide
In the muscle cell, NO binds and
activates guanylyl cyclase, which
produces cGMP (5)
cGMP is a second messenger,
which mediates muscle
relaxation through the
activation of a cGMP-
dependent protein
kinase, which results
in blood vessel
dilation
Nitroglycerine is
treatment for angina:
catabolized to NO
Converted to
GTP by cGMP
phosphodiesterase

Multiple different ligands can
result in the activation of a single
effector = convergence
Convergence, Divergence &
Crosstalk
eg. Both GPCR & TKR
activate PLCs

Signals from the same ligand
can result in the activation of
more than one effector =
divergence
Crosstalk
eg. PLC, PI3K, & GAP all
activated by TKR

Different pathways may
regulate each other = crosstalk
Crosstalk
eg. Calcium can inhibit
PKC activity

Video 32: Apoptosis
https://www.thoughtco.com/apoptosis-372446
Credit: Steve Gschmeissner/Science Photo Library/Getty Images

Apoptosis
(Programmed Cell Death)
Ordered destruction of the cell which is essential in the
process of development and maintenance of health in
multi-cellular organisms

https://www.novusbio.com/research-areas/apoptosis
Apoptosis
(Programmed Cell Death)
External or
internal signals
initiate apoptosis
Apoptotic bodies
are removed by
phagocytes

Apoptosis vs Necroptosis
Apoptosis results in safe
clearance of cellular
components
Rupturing of cellular
membranes during
necroptosis results in
release of cellular
components, resulting in
inflammation
https://www.creative-diagnostics.com/
necroptosis-signaling-pathway.htm

Apoptosis in Development
Cells which are located between the eventual digits
undergo apoptosis, with macrophages (blue fluorescence)
ingesting the apoptotic bodies.

Apoptosis in Maintaining
Health
●
Prevention of autoimmunity: eliminate T cells and B cells
with interact with “self” antigens.
●
Down-regulation of immunity following clearance of
pathogens.
●
Removal of cells with irreparable DNA damage
– Loss of appropriate triggering of apoptosis is a central
feature of cancerous cells
●
Inappropriate triggering of apoptosis leads to disease
(eg. neurodegeneration)

Intrinsic Pathway
Regulated by members of the Bcl-2 family of mitochondrial
proteins upon receiving internal stimuli
●
Both pro-apoptotic and anti-apoptotic members with
several BH domains are normally expressed and
associate with one another in the membrane
●
Proapoptotic BH3-only members act as initiators of
apoptosis by inhibiting the anti-apoptotic members and
activating the proapoptotic members
●
Following activation, pro-apoptotic Bax/Bak form pores in
the mitochondrial outer membrane, allowing the release
of cytochrome c (& other mediators) from the
intermembrane space

Intrinsic Pathway
Cytochrome c then interacts
with various cytoplamic proteins
and procaspase-9
This is the activated initiator or
apoptosome
Caspase-9 cleaves numerous
other “executioner”
procaspases, leading to their
activation

Caspases
Family of cysteine proteases which are central in the
activation of cell death pathways.
Targets are varied cellular components including:
●
Proteins regulating cell adhesion: isolates cell from its
neighbours
●
Proteins involved in regulating anti-apoptotic signal
pathways
●
Cytoskeletal proteins, including the lamins
●
Inhibitors of DNA digestion: initiates the destruction of
the genome

Extrinsic Pathways
Tumor-necrosis factor (TNF) is a
cytokine produced in response
to infection by intracellular
pathogens and other
environmental stresses.
Following activation, the TNF
receptor death domains bind to
an adaptor protein (TRADD) for
the recruitment of effector
proteins (RIP1K).
Signalling results in apoptosis,
necroptosis, or cell survival?

Extrinsic Pathways:
Apoptosis
RIP1K can associate with
FADD to recruit 2 molecules of
procaspase-8
Procaspases cleave each
other to produce activated
caspase-8
Caspase-8 acts of various
“executioner” procaspases to
activate them and initiate the
apoptotic pathway

Extrinsic Pathways:
Necroptosis
Much less understood then
apoptotic pathway
Association of RIP1K with
RIP3K leads to their
phosporylation and formation
of the necrosome
Phosphorylation of MLKL and
its insertion in the membrane
interferes with phospholipid
structure and membrane
stability

Extrinsic Pathways:
Cell Survival?
TNF binding to the TNFR also results in activation of the NF-kB
transcription factor
●
First identified in B cells, but utilized in all animal cells as a
positive regulator for cell survival and proliferation
●
Normally sequestered in cytoplasm through association with
IkBs (inhibitor proteins), which become phosphorylated
following TNF binding and inactivated
●
Following release, NF-kB relocates to the nucleus to initiate
transcription.
●
Inappropriate expression linked to the development of
cancer

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