Professional Documents
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Cell Biology II
Course Content
Date Subject
February 19, 2024 Introduction to the course
February 26, 2024 Cytoskeleton-Part 1
March 4, 2024 Cytoskeleton-Part 2
March 11, 2024 Cell signaling Part 1
March 18, 2024 Cell signaling Part 2
March 25, 2024 Mid-term I
April 1, 2024 The cell cycle Part 1
April 15, 2024 The cell cycle Part 2
April 22, 2024 Cell death
April 29, 2024 Mid-term II
May 6, 2024 Cell junctions and the extracellular matrix Part 1
May 13, 2024 Cell junctions and the extracellular matrix Part 2
May 20, 2024 Development of multicellular organisms (NI in exam)
May 27, 2024 Overview of all topics
5.03.2024 2
The Cytoskeleton
5.03.2024 3
The Cytoskeleton
• To function properly, cells need to Cytoskeleton: a network of filaments
organize themselves in space and -Movement of chromosomes during
interact mechanically cell division
-Correct shaping, physical robustness -Intracellular trafficking of organelles
and proper internal structure are -Mechanical linkages
essential -Movement of cells
-Rearrangement of internal -Cell shape determination
components during cellular processes
Microtubules
Intermediate
filaments
Microtubules
5.03.2024 4
Function and Origin of the Cytoskeleton
5.03.2024 5
Cytoskeletal Filaments Adapt to Form Dynamic
or Stable Structures
5.03.2024 6
Actin filaments
Microtubules
Intermediate filaments
Cytoskeletal filaments adapt to form dynamic or
stable structures
Changes in cytoskeletal organization associated with cell division
Actin
-Microtubule-organizing
cytoskeleton center located in front of
Microtubule the nucleus
cytoskeleton
Duplicated
chromosomes
Cytoskeletal filaments adapt to form dynamic or
stable structures
A neutrophil in pursuit of bacteria
actin network
Actin
Intermediate
filaments
Microtubules
Filaments assemble from protein subunits that
impart specific physical and dynamic properties
5.03.2024 14
Bacterial cell organization and division depend
on homologs of eukaryotic cytoskeletal proteins
MreB/Mbl
5.03.2024 15
Bacterial cell organization and division depend
on homologs of eukaryotic cytoskeletal proteins
5.03.2024 16
Actin and Actin-Binding
Proteins
5.03.2024 17
Actin is the basic subunit of actin filaments
5.03.2024
18
Actin subunits assemble head-to-tail to create
flexible, polar filaments
• Head to tail assembly to form a tight and right-handed helix- 8nm wide filaments-
F-actin
• Polar structure with structurally different ends –
minus end (slow growing, pointed end) + -
plus end (fast growing, barbed end)
• ATP-binding cleft of actin is facing to minus-end in the filament
5.03.2024 19
Nucleation is the rate-limiting step
in the formation of actin filaments
• There are several phases in the formation of actin filaments
– Nucleation (lag phase)- small aggregates can be unstable that can need
extended time periods to make the initial “nucleated filaments”
– Elongation (growth phase)- addition of subunits to nucleated filaments on
both ends causes rapid elongation of the filament
– Steady-state (equilibrium phase)- the rate of the addition and the removal of
subunits from ends is balanced to keep a constant length
Cc= critical
concentration, the
concentration of free
subunits in solution
at steady state:
No net growth or
shrinkage
koff (rate constant for subunit loss)
Cc= [free subunits] at steady state=
5.03.2024 kon(rate constant for subunit addition) 20
The polymerization of actin and tubulin
Actin filaments have two distinct ends that grow at different rates
The polymerization of actin and tubulin
If all of the subunits are in the same nucleotide state, the addition
of subunits to either end is the same (the same kon/koff).
In reality, the kinetic rate constants for association and
disassociation are much larger for one end compared to the other
one.
The polymerization of actin and tubulin
T (ATP/GTP) form
monomer: Adds
D (ADP/GDP) form
monomer: Leaves
The polymerization of actin and tubulin
The polymerization of actin and tubulin
+ +
-
+
-
Actin filament elongation is mediated by formins
NPF:
nucleation-
promoting
factor
5.03.2024 33
Nucleation and branching of actin filaments by
the Arp2/3 complex
5.03.2024 34
Profilin competes with thymosin for binding to actin
monomers and promotes assembly
Thymosin: binds free actin and prevents its addition to the filaments
5.03.2024 35
Actin-binding proteins influence
filament dynamics and organization
+ +
-
+
-
Actin-binding proteins influence
filament dynamics and organization
+
-
-
Actin filament-binding proteins alter filament
dynamics
• Two main protein groups regulate the behavior of the filament:
-first group binds to the sides of the filament
-second group binds to the ends of the filament
• Trompomyosin- bind 6-7 actin subunits in the filament
– Stabilization and stiffening of the filament
– Prevent its interaction with other proteins
• Capping proteins at the plus end (CapZ) decrease the rates of filament growth
and disassembly at plus end due to inactive plus end
5.03.2024 38
Severing proteins regulate actin filament
depolymerization
• Severing proteins: break a filament into smaller portions
– Can increase the nucleation
– Can speed up the dissociation
• Gelsolin superfamily
– Ca2+ dependent
– bind exposed sites on the filaments and sites in between adjacent subunits
• Cofilin
• Bind to both monomeric and
filament bound actin
• Provide more twisting è
weakening the contacts between
subunits
• Make it much easier for an ADP- • Cofillin has higher affinity for D-form
actin subunit to dissociate from (-) actin filaments rather than T-form
end • Efficient disassembly of old filaments
5.03.2024 39
Higher order actin filament arrays influence
cellular mechanical properties and signaling
• Dendritic networks: Arp
nucleating
• Bundles: formin nucleating
• Web-like (gel-like) networks
• Accessory proteins help these
arrays of organization
– Crosslinking proteins:
bundling proteins (parallel
bundles) and gel-forming
proteins (angled attachment)
– Crosslinking proteins have
two similar actin binding sites
– The spacing and arrangement
of bundles are determined by
the interaction of the actin
with the crosslinking
40
Higher order actin filament arrays influence
cellular mechanical properties and signaling
• Bundling proteins: straight Organization of actin filaments into bundles by
stiff connections two different accessory proteins
– Fimbrin or α-actinin
• Crosslinking proteins- bent
connections
– Filamin in lamellopodia
– Filamin interact with many
proteins –connect and
coordinate cellular events
with the actin cytoskeleton
• The ERM family (Ezrin,
radixin and moesin)-
connection of cortical actin
cytoskeleton to the plasma
membrane
5.03.2024 41
Bacteria can hijack the host actin cytoskeleton
• Viscous environment of cytosol prevent diffusion of large particles
• Listeria monocytogenes- recruits and activates Arp2/3 complex at
their surface to push itself within cytosol
5.03.2024 42
https://youtu.be/VVgXDW_8O4U
5.03.2024 43
Myosin and Actin
5.03.2024 44
Actin-based motor proteins are members of the
myosin superfamily
• Myosin II is the first discovered motor protein
– 2 heavy and 2 light chains
– Coiled-coil wrapping of alpha helices mediates
dimerization
– Long heavy chain tails bundles with other tails to
form oppositely oriented bipolar filaments in
muscle
– Head groups bind and hydrolyze ATP as they are
moving toward plus end
5.03.2024 45
Myosin generates force by coupling ATP
hydrolysis to conformational changes
5.03.2024 46
Sliding of Myosin II along actin filaments causes
muscles to contract
• Three muscle types in animals contract during
functioning by using actin filaments and myosin
filaments: skeletal, smooth and cardiac
Myofibrils
Multinucleated muscle cells
- Contains long cylindrical myofibrils
- Myofibrils are formed from repeated contractile units
Sarcomere
called sarcomeres
Thick filaments formed from myosin
Thin filaments formed from actin
Hexagonal lattice
5.03.2024 47
Sliding of Myosin II along actin filaments causes
muscles to contract
(+) end anchorage (-) end stabilization
Myosins walk toward both (+) ends of the thin filaments- each of the myosin head interacts
with the actin filament for a short period of time; low processivity
Accessory proteins determine the filament organization, length and spacing in the sarcomere
5.03.2024 48
A sudden rise in cytosolic Ca2+ concentration
initiates muscle contraction
• Muscle cells need signal from outside to initiate the contraction process
• The contraction is a very rapid process after signal is received
• Specialized organization of the muscle cells enables them to respond to external
signals very rapidly
Signal received by muscle cells
Spreading of electrical
excitation by T tubules
5.03.2024 50
A sudden rise in cytosolic Ca2+ concentration
initiates muscle contraction
• Smooth muscles
– Activated by calcium
– Calmodulin gets activated
– Calcium/calmodulin binds to
MLCK
– Phosphorylation of myosin light
chain
– Bundling and interaction with actin
5.03.2024 51
Actin and myosin perform a variety of functions
in non-muscle cells
• Non-muscle cells: small amounts of contractile actin-myosin II
bundles
• Regulated by myosin phosphorylation
• Provide mechanical support to cells- stress fibers at focal
adhesions, circumferential belt in epithelial sheet, contractile ring
during cytokinesis, pulling of the rear end of cell during movement
5.03.2024 52
Myosin superfamily members
• Myosin types- highly conserved motor domain followed by a diverse tail domains
• Diversification of tails facilitate the recognition of other proteins and carriage of
different cargoes
• > 37 myosin families
• All but myosin VI move toward plus end on actin filaments with different
speeds Intracellular organization eg
microvilli
Contractile activity,
cytokinesis, forward
movement
Vesicle and organelle
transport
5.03.2024 53
Microtubules
5.03.2024 55
Microtubules
• Alpha and beta tubulin are tightly bound with non-covalent bonds
• Each one binds to one molecule of GTP
GTP: is hydrolyzed
and is important for
microtubule
dynamics
GTP at the
interface, intrinsic
part of the protein
5.03.2024 56
Microtubules are hollow tubes made of
protofilaments
• 13 protofilaments are arranged in
hollow configuration to form a
microtubule
• Organization into protofilaments cause
directionality; alpha tubulin is at one
(minus) end and beta is on the other
(plus) end
• Protein interactions give stability to the
structure
• Longitudinal interactions: Between α
and β subunits
• Lateral interactions: Between β-β and
α-α subunits
• Addition or removal of subunits almost
exclusively happens at the microtubule
ends
5.03.2024 57
Microtubules undergo dynamic instability
5.03.2024 58
Direct observation of the dynamic instability of
microtubules in a living cell
Microtubules undergo dynamic instability
• One end grows by adding T-form subunits for a certain time point as the addition
of subunit rate is larger than the hydrolysis rate
• At some point, there is a sudden change into D form that can cause rapid shrinkage
• Dynamic instability is the rapid interconversion between growth and shrinkage
states
C: If GTP hydrolysis
proceeds more rapidly
than subunit addition, cap
is lost and MT begins to
shrink
R: If GTP-containing
subunits are added to the
shrinking end, a new cap
forms and MT growth
resumes
60
Model for the structural consequences of GTP
hydrolysis in the microtubule lattice
5.03.2024 62
A protein complex containing Ɣ-tubulin nucleates
microtubules
• γ-Tubulin is in less amount than alpha and beta tubulins and they are important
for the nucleation of microtubule growth
• Nucleation happens at specific locations known as microtubule-organizing
center (MTOC) and γ-tubulin is the marker for MTOC
• MTs nucleate at their minus end and the plus end is growing out from MTOC
• γ-Tubulin ring complex (γ-TuRC) is a multi-protein complex that serve as a
template to form a microtubule with 13 protofilaments
lock washer
γ-TuSC:
γ-tubulin
small complex
5.03.2024 63
Microtubules emanate from the centrosome in
animal cells
• Most animal cells have a single MTOC known as centrosome near the nucleus
• Microtubules radiate from centrosomes in a star-like appearance with minus
end in the centrosome and plus end pointing outward
• Centrosome = centrosome matrix (> 50 γ-TuRC) + a pair of centrioles (arranged
at right angles to each other, that become the basal bodies of flagella and cilia in
motile cells)
• Duplicate during cell division and move to opposite sides to form the mitotic
spindles
• In fungi and diatoms, MTOC is embedded in the nuclear envelope
• Higher plant cells nucleate MTs all around the nuclear envelope
• No centrosome in plants and fungi
5.03.2024 64
The center-seeking behavior of a centrosome
5.03.2024 65
Microtubules emanate from the centrosome in
animal cells
• Microtubules emanating from centrosome can survey the outlying regions of cells
and position the centrosome at the center of the cells
• A microtubule array can find the center of a cell even in the absence of a
centrosome
• MTs establish a general coordinate system that can position many organelles
within cells
5.03.2024 66
Microtubule organization in different cell types
Some of the major accessory proteins of the microtubule
cytoskeleton
+ +
-
Some of the major accessory proteins of the microtubule
cytoskeleton
- +
- +
Microtubule-binding proteins modulate filament
dynamics and organization
• Proteins that bind along the sides of
filaments affect their stability and
mechanical properties
• MAPs (microtubule-associated proteins)
bind along the MTs
– Stabilization of microtubules against
disassembly
– Facilitate interaction with other cellular
components
– Two interacting domains, one bind to MT
and the other project outward
• Packing of MTs depend on the length of
MAPs
• MAPs are regulated by kinases that define
their localization and activity
5.03.2024 71
Tubulin-sequestering and microtubule-severing
proteins destabilize microtubules
• Stathmin: bind to tubulin heterodimers
• Prevent their addition onto the ends of
microtubules è reduced MT elongation
• Stathmin enhances the occurrence of
shrinkage state during dynamic instability
• Phosphorylation of stathmin prevents its Katanin action
interaction with tubulin è increase
elongation and decrease dynamic instability
• Severing proteins-destabilize
microtubules (e.g. katanin)
two subunits- small subunit: ATP hydrolysis
and severing; large one: centrosome
localization
-release of microtubules from MTOC for rapid
depolymerization
5.03.2024 72
Motor proteins generate force by coupling ATP
hydrolysis to conformational changes: Kinesins
• Two head groups are bound to MT, leading head with ADP
loosely attached while lagging head with ATP tightly
bound
• Exchange of ADP with ATP in the leading head causes the
throwing of the lagging head 8nm forward to a new
binding site
Tilting of the
lagging head
determines the
directionality
The
orientation of
the unbound
head
determines the
5.03.2024 directionality 73
Motor proteins generate force by coupling ATP
hydrolysis to conformational changes: Dyneins
• When bound to ATP, dyneins detach from the
MT
• ATP hydrolysis causes dynein to bind to the
MT
• When ADP and Pi are released from dyneins, it
causes a large conformational power stroke
• Move cargo 8nm at a time towards minus end
5.03.2024 74
Motor proteins mediate the intracellular
transport of membrane enclosed organelles
– Motor proteins transport and position
membrane enclosed organelles Dynein interacts with a
– Kinesins carry secretory vesicles, organelles large protein complex while
along the MT carrying their cargo
Nocodazole
- Dynactin complex
– MT has minus end near the center and plus
end at the periphery
– Dyneins move molecules toward the center
and kinesins carry them to the periphery
– MT and motor proteins organize the ER
(kinesins ) and the Golgi apparatus (dyneins)
within cells
– The tails and their associated light chains
determine the specificity of the cargo
– Membrane associated receptors on the
organelles/vesicles interact with the tails, eg.
Amyloid precurcor protein interacts with
kinesin-1
5.03.2024 75
Cells regulate motor protein function
• The activity of motor proteins are regulated during changing the position of
membrane-enclosed organelles, cell movement
Bidirectional movement
5.03.2024 76
Cilia and flagella are motile structures built from
microtubules and dyneins
• Cilia and flagella are formed from microtubules and dyneins Flagella Cilia
• Flagella- on sperm and many protozoa; swimming through
liquid media.
• Cilia- shorter, beat with a whip-like motion , in cells lining the
respiratory tract
• Bending of the core (axoneme) produces movement
Axoneme bending
5.03.2024
79
Primary cilia perform important signaling
functions in animal cells
• Primary cilium- shorter, nonmotile counterpart of cilia and flagella in many cells
• They are generated from basal bodies during interphase
• Basal bodies are the roots of cilia and flagella at the cell surface in eukaryotes
• At the core of each basal body is a centriole
• Primary cilia sense and respond to the exterior environment
5.03.2024 80
Intermediate Filaments and
Septins
5.03.2024 81
Intermediate filaments and septins
• Vertebrates, mollusk and nematodes contain intermediate filaments
• IFs mainly found in cells that encounter mechanical stress
• Septins are able to polymerize into long apolar filaments
• Different families of IFs are diverse and are encoded by 70 different human genes
with distinct and cell-type specific functions
5.03.2024 82
Intermediate filament structure depends on the
lateral bundling and twisting of coiled-coils
• Elongated proteins with a
conserved central α-helical
domain
• A pair of parallel dimers
associate in an anti-parallel
fashion to form staggered
tetramer
• No binding site for a
nucleotide
• No structural polarity
• Lateral packaging of 8
tetramers to form the filament-
32 individual α-helical coils
• Rope-like character- easy
bending, streching
5.03.2024 83
Intermediate filaments impart mechanical
stability to animal cells
• Keratins- 54 distinct keratins in human
– Equal mixing of type I (acidic) and type II
(neutral/basic) keratins
– Disulfide bonds between keratin networks can survive
in nature even after the cell death
– Keratins impart mechanical strength to epithelial cells
through desmosomes and hemidesmosomes
5.03.2024 84
Intermediate filaments impart mechanical
stability to animal cells
• Neurofilaments are found in high concentrations along the axons of
vertebrate neurons
Neurons Glial Cells Cross-section of a neuron
5.03.2024 87
Cell Polarization and
Migration
5.03.2024 88
Many cells can crawl across a solid substratum
5.03.2024 89
Actin polymerization drives plasma membrane
protrusion
• Protrusion of leading edge depends on forces generated
by actin polymerization that pushes membrane outward
• There are different types of protrusions:
• Filopodia: one-dimensional organization of actin
cytoskeleton formed from long bundled actin filaments,
in migrating growth cones of neurons and some
fibroblasts
• Lamellipodia: epithelial cells, fibroblasts and some
neurons form these two dimensional, sheetlike structures.
They contain a cross-linked mesh of actin filaments most
lie in a plane parallel to the solid substratum
• Invadopodia and podosomes: Extend in 3D and
Blebbing
important for cells to cross barriers. Contain the same
regulatory components as filopodia and lamellipodia-
degrade the ECM
• Blebbing: local detachment of cells from the underlying
actin cortex cytoplasmic flow push the membrane
outward
90
Lamellipodia contain all of the machinery
required for cell motility
5.03.2024 91
Lamellipodia contain all of the machinery
required for cell motility
• Lamellipodia- unidirectional
movement- coordinated activity of
several factors
• Cofilin at the back for
depolymerization of D-form
filaments
• Delayed hydrolysis of ATP at the
leading edge provides
unidirectionality
5.03.2024 92
Myosin contraction and cell adhesion allow cells to
pull themselves forward
• For the cells to advance, the
protrusion is followed by attachment
of the cell to the substratum at the
front
• De-adhesion and contraction happens
at the back
• Myosin II
– help to connect the actin cytoskeleton to
the substratum through integrin-mediated
adhesions- focal adhesions
– Bipolar myosin II associate with actin at
the back and pull them into a new
orientation
5.03.2024 93
Myosin contraction and cell adhesion allow cells to
pull themselves forward
• For the cells to advance, the
protrusion is followed by attachment
of the cell to the substratum at the
front
• If attachment does not happen
properly, retrograde flow happens
where actin filaments move rearward
• When cells are moving on substratum
they exert a significant pull on the
surface
5.03.2024 94
Cell polarization is controlled by members of the
Rho protein family
• Functionally and structurally distinct front and back
• Locomotion promotes the establishment of cell polarity
• External signals result in the local reorganization of the actin cytoskeleton
• These signals converge on members of the Rho protein family, Cdc42, Rac and
Rho
• External signals can activate the three Rho protein family members
5.03.2024 95
External signals can dictate the direction of cell
migration
• Chemotaxis- movement of a cell toward or away from a source of a
diffusible chemical
• Rho family members are the target of these signals
5.03.2024 96
Harvard MCB - BioVisions Lab: The Inner Life of the Cell (Narrated)
• https://www.youtube.com/watch?v=QplXd76lAYQ
• https://www.youtube.com/watch?v=WM7uw8cRUp0
5.03.2024 97