Lecture 01: Cytoskeleton
The eukaryotic cytoskeleton is a set of dynamic filamentous networks whose assembly/disassembly controls cell shape, migration, intracellular
transport and cell division. Two major dynamic systems covered here are actin filaments (F-actin) and microtubules (MTs). Both are built from
globular subunits that bind and hydrolyze nucleotides (ATP for actin, GTP for tubulin). Nucleotide hydrolysis is not required to make the polymer
but it is what makes the polymers dynamic (treadmilling / dynamic instability).
• Actin: major driver of membrane protrusions, cell migration, contractility (with myosin), and cortical stiffness. Rapid turnover allows fast
cellular shape changes.
• Microtubules: provide long-range intracellular tracks for motors (kinesin/dynein), organize the mitotic spindle during cell division, and
determine major cell polarity axes by dynamic probing of space.
• The different kinetics and regulatory proteins (nucleation factors, capping proteins, severing proteins, stabilizers) tune filament behaviour
to cellular tasks.
Structure & polarity:
• Actin:
o Monomer = G-actin (binds ATP). Polymer = two long, staggered
protofilaments forming a right-handed helix (F-actin).
o Filament is polar: barbed (plus) end grows/shrinks faster; pointed
(minus) end grows/shrinks slower.
o ATP is bound in monomers and hydrolysed after incorporation
→ ATP-actin (T form) versus ADP-actin (D form).
o Polarity can be visualized experimentally (e.g., “decoration” with myosin
S1 fragments).
• Microtubules:
o Building block = α/β tubulin heterodimer (both bind GTP; GTP on β-
tubulin is hydrolysable after polymerization; α-tubulin’s GTP is structural).
o Microtubules are hollow cylinders typically made of 13 protofilaments
(in most eukaryotic cells).
o MTs are polar: plus end (β-tubulin exposed) is the rapidly growing end;
minus end (α-tubulin exposed) is the slower end and often anchored
(e.g., at centrosome).
Kinetics: on/off rates and the critical concentration:
• Kinetic constants:
o kon (M-1 s-1) = rate constant for subunit addition
o koff (s-1) = rate constant for subunit loss
• If the plus and the minus ends are both in (“T”) form:
o Plus-end: koff is high, kon is high
o Minus-end: koff is low, kon is low
• At equilibrium (steady state): kon [subunit] = koff
• Critcal concentration (CC) for an end: CC = koff / kon
o If [subunit] > CC → net growth at that end
o If [subunit] < CC → net shrinkage at that end
• Because kon and koff differ between barbed/pointed (or plus/minus) ends, each end has different kinetic behaviour even though the
thermodynamic free-energy change per incorporation is the same.
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,Actin dynamics: nucleation, elongation, treadmilling:
• Nucleation (formation of the first oligomer) is rate-limiting for polymerization. Adding pre-formed
filament “seeds” shortens the lag and speeds up reaching steady state. Actin polymerization
occurs more rapidly in the presence of pre-formed filaments.
• Treadmilling: when [G-actin] is between the critical concentrations for ATP- and ADP-states, you
observe net addition at the barbed (+) end and net loss at the pointed (–) end.
o Mechanism: newly added subunits are ATP-bound (T form) and hydrolysis to
ADP lags behind incorporation → barbed end stays T-rich and stable, pointed
end tends to be D-rich and unstable.
o Condition for classic treadmilling: CC (T) < [actin] < CC (D)
o Plus-end: Actin-ATP – “T form” (Koff is low, Kon is high, Koff/Kon=Cc (T) is low)
o Minus-end: Actin-ADP – “D form” (Koff is high, Kon is low, Koff/Kon=Cc (D) is high)
• Functional consequence: actin networks can flow or translocate by subunit turnover without the monomers themselves moving long
distances.
• Steady state: equilibrium phase with actin filament with subunits coming on and off (no change in % actin subunits in filaments if time after
polymerization initiation becomes longer)
Microtubule dynamics: dynamic instability
• Microtubule dynamic instability is driven by GTP hydrolysis,
which induces conformational changes in protofilaments
• Order: Minus-end – (GTP)-α-tubulin – GDP-β-tubulin
– GTP-β-tubulin – Plus-end
• MTs show dynamic instability: single filaments stochastically
switch between phases of growth and rapid shrinkage
(catastrophe) and occasionally resume growth (rescue).
• GTP cap model: growing plus end is capped by GTP-β-tubulin; hydrolysis to GDP weakens lateral/longitudinal contacts and, upon loss of
the GTP cap, protofilaments splay and rapid depolymerization (catastrophe) occurs. Regaining a GTP cap allows rescue and regrowth.
• Dynamic instability underlies rapid reorganization of MT arrays (e.g., mitotic spindle assembly)
Energetics & role of nucleotide hydrolysis:
• Polymer assembly is driven mainly by favourable non-covalent interactions (hydrophobic/electrostatic) → polymerization itself is
spontaneous when [subunit] > CC
• ATP/GTP hydrolysis is not required to polymerize, but hydrolysis changes the conformation and binding affinity of incorporated subunits
→ creates kinetic asymmetry and enables dynamic behaviours (treadmilling, catastrophe/rescue).
• In other words: hydrolysis converts a thermodynamically stable polymer into a kinetically dynamic structure.
Cytoskeleton-active drugs:
• Actin-targeting (source: sponges and fungi):
o Latrunculin: depolymerizes filaments; binds actin subunits
o Cytochalasin B: depolymerizes filaments; caps filament plus ends
o Phalloidin: stabilizes filaments; binds along filaments
• Microtubules-targeting (source: plants and synthetic):
o Taxol (paclitaxel): stabilizes filaments; binds along filaments
o Nocodazole: depolymerizes filaments; binds tubulin subunits
o Colchicine: depolymerizes filaments; caps both filament ends
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, Summary:
• Cytoskeletons are dynamic because they are built of subunits, which can polymerize and depolymerize
• Polymerization of actin (ATPase) and tubulin (GTPase) does not require energy of ATP/GTP hydrolysis
• Nucleotide hydrolysis makes actin and microtubules dynamic
• The two ends of microtubules and actin filaments are structurally and kinetically different
• Nucleation is a rate-limiting step of cytoskeletal filament assembly
• Different drugs can stabilize or destabilize cytoskeletal filaments
Overview: Regulation of cytoskeletal organization & dynamics
Cytoskeletal behaviour in cells is steered by many regulatory factors that control where filaments form, how fast they grow/shrink, how they are
organized into higher-order structures, and when they are severed or stabilized.
Different cell types build distinct actin and microtubule architectures (stress fibers, lamellipodia, axonal bundles, radial centrosomal arrays, etc.)
by combining the same basic building blocks with different regulatory proteins.
Typical cellular architectures:
• Actin arrays in a migrating fibroblast:
o Lamellipodium: branched network (Arp2/3-dependent) that pushes the membrane.
o Filopodia: tight, parallel bundles that probe the environment.
o Cortex: network beneath the plasma membrane (branched/unbranched) providing stiffness.
o Stress fibers: contractile bundles (α-actinin–spaced) engaged with myosin for contractility.
• Microtubule arrays: radial centrosomal rays in fibroblasts, long sheets in muscle cells, long parallel bundles in axons/dendrites, and
specialized axonemes in cilia/flagella.
Regulating the pool of available subunits: sequestration
Cells keep total concentrations of actin and tubulin high, but regulate the available pool via binding proteins:
• Actin:
o Thymosin β4 (thymosin) sequesters free G-actin monomers preventing polymerization; binds subunits, prevents assembly
o Profilin binds actin monomers and concentrates them at sites of filament assembly; restricts actin polymerization to the plus end
• Tubulin:
o Stathmin binds tubulin dimers (subunits) and lowers their availability for MT assembly
Nucleation: defining network geometry
• Actin branching (Arp2/3 complex):
o Arp2/3 is activated by nucleation-promoting factors (NPFs) near membranes.
o Activated Arp2/3 nucleates a new filament from the side of an existing filament, producing a branch at ~70° that grows toward the
membrane → characteristic lamellipodial web.
• Microtubule nucleation (γ-TuRC): γ-tubulin ring complex templates 13 protofilaments and seeds MT growth (centrosome is rich in γ-TuRC).
• Branching MT nucleation (augmin): recruits γ-TuRC to sides of existing MTs (important in mitotic spindle amplification and in plants).
Centrosomes, centrioles and non-centrosomal nucleation:
• Centrioles: stable microtubule structures within the centrosome; cylindrical structures with 9-fold symmetry (protein: Sas-6) formed by
nine microtubule triplets (one complete + two partials); centrioles are embedded in pericentriolar material that contains γ-TuRCs.
• Non-centrosomal MT nucleation occurs at sites such as the Golgi, nuclear envelope (in muscle), and in many differentiated cells (e.g.,
neurons), enabling diverse MT architectures.
Proteins that act at filament ends:
• Actin end regulators:
o Formins: plus-end bound actin nucleation and elongation factors; interacts with profilin, accelerates actin growth at the plus end
o Capping proteins: prevent actin polymerization and depolymerization at actin plus ends (capped → growth at minus only)
• Microtubule plus end-tracking proteins (+TIPs) can accelerate growth or induce catastrophes
o EB proteins (e.g., EB3) bind the GTP-cap and mark growing MT ends.
o XMAP215 (polymerase) binds plus ends/tubulin dimers and speeds growth.
o Kinesin-13 (catastrophe factor) binds ends and promotes protofilament peeling → increases catastrophe frequency.
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The eukaryotic cytoskeleton is a set of dynamic filamentous networks whose assembly/disassembly controls cell shape, migration, intracellular
transport and cell division. Two major dynamic systems covered here are actin filaments (F-actin) and microtubules (MTs). Both are built from
globular subunits that bind and hydrolyze nucleotides (ATP for actin, GTP for tubulin). Nucleotide hydrolysis is not required to make the polymer
but it is what makes the polymers dynamic (treadmilling / dynamic instability).
• Actin: major driver of membrane protrusions, cell migration, contractility (with myosin), and cortical stiffness. Rapid turnover allows fast
cellular shape changes.
• Microtubules: provide long-range intracellular tracks for motors (kinesin/dynein), organize the mitotic spindle during cell division, and
determine major cell polarity axes by dynamic probing of space.
• The different kinetics and regulatory proteins (nucleation factors, capping proteins, severing proteins, stabilizers) tune filament behaviour
to cellular tasks.
Structure & polarity:
• Actin:
o Monomer = G-actin (binds ATP). Polymer = two long, staggered
protofilaments forming a right-handed helix (F-actin).
o Filament is polar: barbed (plus) end grows/shrinks faster; pointed
(minus) end grows/shrinks slower.
o ATP is bound in monomers and hydrolysed after incorporation
→ ATP-actin (T form) versus ADP-actin (D form).
o Polarity can be visualized experimentally (e.g., “decoration” with myosin
S1 fragments).
• Microtubules:
o Building block = α/β tubulin heterodimer (both bind GTP; GTP on β-
tubulin is hydrolysable after polymerization; α-tubulin’s GTP is structural).
o Microtubules are hollow cylinders typically made of 13 protofilaments
(in most eukaryotic cells).
o MTs are polar: plus end (β-tubulin exposed) is the rapidly growing end;
minus end (α-tubulin exposed) is the slower end and often anchored
(e.g., at centrosome).
Kinetics: on/off rates and the critical concentration:
• Kinetic constants:
o kon (M-1 s-1) = rate constant for subunit addition
o koff (s-1) = rate constant for subunit loss
• If the plus and the minus ends are both in (“T”) form:
o Plus-end: koff is high, kon is high
o Minus-end: koff is low, kon is low
• At equilibrium (steady state): kon [subunit] = koff
• Critcal concentration (CC) for an end: CC = koff / kon
o If [subunit] > CC → net growth at that end
o If [subunit] < CC → net shrinkage at that end
• Because kon and koff differ between barbed/pointed (or plus/minus) ends, each end has different kinetic behaviour even though the
thermodynamic free-energy change per incorporation is the same.
1|Page
,Actin dynamics: nucleation, elongation, treadmilling:
• Nucleation (formation of the first oligomer) is rate-limiting for polymerization. Adding pre-formed
filament “seeds” shortens the lag and speeds up reaching steady state. Actin polymerization
occurs more rapidly in the presence of pre-formed filaments.
• Treadmilling: when [G-actin] is between the critical concentrations for ATP- and ADP-states, you
observe net addition at the barbed (+) end and net loss at the pointed (–) end.
o Mechanism: newly added subunits are ATP-bound (T form) and hydrolysis to
ADP lags behind incorporation → barbed end stays T-rich and stable, pointed
end tends to be D-rich and unstable.
o Condition for classic treadmilling: CC (T) < [actin] < CC (D)
o Plus-end: Actin-ATP – “T form” (Koff is low, Kon is high, Koff/Kon=Cc (T) is low)
o Minus-end: Actin-ADP – “D form” (Koff is high, Kon is low, Koff/Kon=Cc (D) is high)
• Functional consequence: actin networks can flow or translocate by subunit turnover without the monomers themselves moving long
distances.
• Steady state: equilibrium phase with actin filament with subunits coming on and off (no change in % actin subunits in filaments if time after
polymerization initiation becomes longer)
Microtubule dynamics: dynamic instability
• Microtubule dynamic instability is driven by GTP hydrolysis,
which induces conformational changes in protofilaments
• Order: Minus-end – (GTP)-α-tubulin – GDP-β-tubulin
– GTP-β-tubulin – Plus-end
• MTs show dynamic instability: single filaments stochastically
switch between phases of growth and rapid shrinkage
(catastrophe) and occasionally resume growth (rescue).
• GTP cap model: growing plus end is capped by GTP-β-tubulin; hydrolysis to GDP weakens lateral/longitudinal contacts and, upon loss of
the GTP cap, protofilaments splay and rapid depolymerization (catastrophe) occurs. Regaining a GTP cap allows rescue and regrowth.
• Dynamic instability underlies rapid reorganization of MT arrays (e.g., mitotic spindle assembly)
Energetics & role of nucleotide hydrolysis:
• Polymer assembly is driven mainly by favourable non-covalent interactions (hydrophobic/electrostatic) → polymerization itself is
spontaneous when [subunit] > CC
• ATP/GTP hydrolysis is not required to polymerize, but hydrolysis changes the conformation and binding affinity of incorporated subunits
→ creates kinetic asymmetry and enables dynamic behaviours (treadmilling, catastrophe/rescue).
• In other words: hydrolysis converts a thermodynamically stable polymer into a kinetically dynamic structure.
Cytoskeleton-active drugs:
• Actin-targeting (source: sponges and fungi):
o Latrunculin: depolymerizes filaments; binds actin subunits
o Cytochalasin B: depolymerizes filaments; caps filament plus ends
o Phalloidin: stabilizes filaments; binds along filaments
• Microtubules-targeting (source: plants and synthetic):
o Taxol (paclitaxel): stabilizes filaments; binds along filaments
o Nocodazole: depolymerizes filaments; binds tubulin subunits
o Colchicine: depolymerizes filaments; caps both filament ends
2|Page
, Summary:
• Cytoskeletons are dynamic because they are built of subunits, which can polymerize and depolymerize
• Polymerization of actin (ATPase) and tubulin (GTPase) does not require energy of ATP/GTP hydrolysis
• Nucleotide hydrolysis makes actin and microtubules dynamic
• The two ends of microtubules and actin filaments are structurally and kinetically different
• Nucleation is a rate-limiting step of cytoskeletal filament assembly
• Different drugs can stabilize or destabilize cytoskeletal filaments
Overview: Regulation of cytoskeletal organization & dynamics
Cytoskeletal behaviour in cells is steered by many regulatory factors that control where filaments form, how fast they grow/shrink, how they are
organized into higher-order structures, and when they are severed or stabilized.
Different cell types build distinct actin and microtubule architectures (stress fibers, lamellipodia, axonal bundles, radial centrosomal arrays, etc.)
by combining the same basic building blocks with different regulatory proteins.
Typical cellular architectures:
• Actin arrays in a migrating fibroblast:
o Lamellipodium: branched network (Arp2/3-dependent) that pushes the membrane.
o Filopodia: tight, parallel bundles that probe the environment.
o Cortex: network beneath the plasma membrane (branched/unbranched) providing stiffness.
o Stress fibers: contractile bundles (α-actinin–spaced) engaged with myosin for contractility.
• Microtubule arrays: radial centrosomal rays in fibroblasts, long sheets in muscle cells, long parallel bundles in axons/dendrites, and
specialized axonemes in cilia/flagella.
Regulating the pool of available subunits: sequestration
Cells keep total concentrations of actin and tubulin high, but regulate the available pool via binding proteins:
• Actin:
o Thymosin β4 (thymosin) sequesters free G-actin monomers preventing polymerization; binds subunits, prevents assembly
o Profilin binds actin monomers and concentrates them at sites of filament assembly; restricts actin polymerization to the plus end
• Tubulin:
o Stathmin binds tubulin dimers (subunits) and lowers their availability for MT assembly
Nucleation: defining network geometry
• Actin branching (Arp2/3 complex):
o Arp2/3 is activated by nucleation-promoting factors (NPFs) near membranes.
o Activated Arp2/3 nucleates a new filament from the side of an existing filament, producing a branch at ~70° that grows toward the
membrane → characteristic lamellipodial web.
• Microtubule nucleation (γ-TuRC): γ-tubulin ring complex templates 13 protofilaments and seeds MT growth (centrosome is rich in γ-TuRC).
• Branching MT nucleation (augmin): recruits γ-TuRC to sides of existing MTs (important in mitotic spindle amplification and in plants).
Centrosomes, centrioles and non-centrosomal nucleation:
• Centrioles: stable microtubule structures within the centrosome; cylindrical structures with 9-fold symmetry (protein: Sas-6) formed by
nine microtubule triplets (one complete + two partials); centrioles are embedded in pericentriolar material that contains γ-TuRCs.
• Non-centrosomal MT nucleation occurs at sites such as the Golgi, nuclear envelope (in muscle), and in many differentiated cells (e.g.,
neurons), enabling diverse MT architectures.
Proteins that act at filament ends:
• Actin end regulators:
o Formins: plus-end bound actin nucleation and elongation factors; interacts with profilin, accelerates actin growth at the plus end
o Capping proteins: prevent actin polymerization and depolymerization at actin plus ends (capped → growth at minus only)
• Microtubule plus end-tracking proteins (+TIPs) can accelerate growth or induce catastrophes
o EB proteins (e.g., EB3) bind the GTP-cap and mark growing MT ends.
o XMAP215 (polymerase) binds plus ends/tubulin dimers and speeds growth.
o Kinesin-13 (catastrophe factor) binds ends and promotes protofilament peeling → increases catastrophe frequency.
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