1 - The Cytoskeleton
The cytoskeleton is composed of three major filaments:
● Actin filaments
○ Determine the shape of the cell’s surface, make the cytoskeleton dynamic,
and drive the pinching of a cell into two.
● Microtubules
○ Determine the positions of organelles, direct intracellular transport, and form
the mitotic spindle that segregates chromosomes in cell division.
● Intermediate filaments
○ Provide mechanical strength.
Actin
Actin is built from 2 rows of globular monomers (protofilaments). Actin provides the
building-blocks for dynamics, and is also an ATPase which hydrolyses ATP to ADP after
polymerization. A protofilament is polarized and consists of:
● Barbed end / + end: grows and shrinks fast → it takes less time
● Pointed end / - end: grows and shrinks slow → it takes more time
At steady state, the filaments grow as fast as they disassemble and polymerization stops
(equilibrium).
The growing rate (Kon) depends on concentration (more subunits → fast polymerization)
The shrinking rate (Koff) does not depend on concentration.
The critical concentration (Cc) is the concentration of subunits at equilibrium (equal for the
+ and - end when they are both in ATP-form (T), because the energy release is the same).
𝐾𝑜𝑓𝑓
𝐶𝑐 = 𝐾𝑜𝑛
= 𝑘𝑑
Actin polymerization occurs more rapidly in the presence of pre-formed filaments. The
making of the first oligomers out of monomers is called nucleation (lag phase).
Actin as a monomer in the cytoplasm is always in its ATP-form (T). After polymerization,
actin will hydrolyze ATP to ADP. Because the - end grows slow, hydrolysis can catch up with
polymerization, so the - end already is in ADP-form (D) before polymerization. This makes
the addition of T-form subunits to the - end even more difficult. The critical concentration for
the - end becomes high, making it grow only when a lot of subunits are present. The critical
concentration for the + end is high, making it grow with a few subunits. In the treadmilling
range, the subunit concentration is enough to make the + end grow, but it leads to shrinkage
of the - end (depolymerization). This looks as though the whole filament shifts.
Actin in T-form polymerizes spontaneously because of hydrophobic interactions with water
molecules (energetically favorable). ATP-hydrolysis is needed because depolymerization
requires energy, and depolymerization is needed for the dynamics of actin.
Drugs like latrunculin and cytochalasin B depolymerize actin filaments, phalloidin can
stabilize actin filaments.
,Microtubules
Microtubule filaments are thick and sparse, and also very dynamic. They are attached to
the nucleus and reach out to the periphery by polymerization (also spontaneously), or shrink
by depolymerization → dynamic instability.
Microtubules are built from tubulin dimers (globular proteins) and are GTP-enzymes
(β-tubulin hydrolyzes GTP after polymerization). They consist of 13 protofilaments, making
them a rigid hollow tube. A protofilament is polarized and consists of:
● + end: the end where β-tubulin sticks out (grows fast)
● - end: the end where ɑ-tubulin sticks out (grows slower)
As long as the microtubule β-tubulin tip is in its T-form (GTP-cap), the microtubules are held
together. After loss of the GTP-cap (catastrophe) or after GTP-hydrolyzation, the
protofilaments become less attached and split apart in individual protofilaments and then in
dimers (D-form), which can convert into T-form in the cytoplasm.
Drugs like nocodazole and colchicine can depolymerize microtubule filaments, taxol can
stabilize microtubule filaments.
Regulation of cytoskeletal organization
Actin filaments can be organized in:
● Lamellipodia: flat part of the cell with a flat branched network of actin
● Filopodia: protrusions of the cell with parallel bundles of actin
● Cell cortex: branched and unbranched network of actin
● Stress fibres: contractile parts of the cell with antiparallel bundles of actin
Microtubules can be organized in:
● Fibroblasts: radial array starting from one point (nucleus) reaching out to the
periphery
● Plant cells: layers of microtubules parallel to the plasma membrane
● Intestinal epithelial cells: vertical parallel bundles with the - end attached to the
surface and the + end on the inside
● Neurons: parallel bundles with the + end in the axons, and antiparallel in the
dendrites
Actin is way more abundant in the cell than the critical concentration. The cytoskeleton is
regulated by the availability of the monomers (subunit sequestration).
● Thymosin: prevent assembly by binding/sequestering actin subunits
● Profilin: prevent assembly at the - end and promotes assembly at the + end by
binding actin monomers and concentrating them at the + end
● Stathmin: prevent assembly by binding/sequestering tubulin dimers and keeping
them incompetent for polymerization
Nucleation is regulated by the nucleation-promoting factor + Arp2/3 and other proteins.
Actin nucleation is regulated by nucleation-promoting complex + Arp2/3 with other
proteins. This attaches to the minus-end to prevent shrinkage and promotes growth of the
plus-end. Arp2/3 also can make branches of actin. This only occurs very close to the plasma
membrane.
Microtubules are nucleated with γ-tubulin ring complex (very abundant in centrosomes),
which can also make microtubule branches with augmin.
, The centrosome is built around centrioles:
● Attached two each other by a linker
● Built from 9 triplets of microtubules (1 complete with 13 protofilaments, 2 incomplete
with 10 protofilaments)
● SAS6 has the capacity to make this 9-fold symmetry
A lot of microtubules also nucleate non-centrosomal: the Golgi-apparatus, nuclear envelope,
and other microtubules all have γ-tubulin.
Actin polymerization regulating factors:
● Formin: + end bound factor that accelerates actin growth at the + end, by interacting
with profilin, which are bound to actin-monomers
● Capping proteins: prevent actin polymerization and depolymerization at the + ends
Microtubule polymerization regulating factors:
● Plus-end tracking proteins (+TIPs): can be polymerizing or depolymerizing
○ XMAP215: accelerate growth of the growing + end (like formin in actin)
○ Kinesin-13: induce catastrophes by breaking down the GTP-cap
There are actin binding proteins that can control stability, bundling, packing, orientation and
membrane attachment of the filaments after they have grown:
● Tropomyosin (muscle): makes actin more accessible to myosin motors
● Fimbrin: makes very tight actin bundles
● ɑ-actinin: makes antiparallel loose bundles
● Filamin: stabilizes branched actin bundles
● Spectrin/ERM: attach actin to the plasma membrane
Not all cytoskeletal structures are dynamic (such as stable microvilli on intestinal epithelial
cells):
Microtubule-associated proteins (MAP’s) stabilize and bundle microtubules or link them to
other structures:
● MAP2: link to dendrites
● Plectrin: link intermediate filaments
● Tau: link to axons
Primary cilia are stable microtubuli components, grown from a centriole, and are important
for organizing sensory and signaling functions. All cilia (primary and motile) have a
microtubule core (axoneme) made out of 9 doublets. Axonemal dyneins are attached to a
doublet and can move to the other doublet, which moves the two doublets apart. In cilia the
doublets are connected so this movement results in a wave of bending (sperm).
Actin-destabilizing and severing proteins:
● Cofilin: binds (ADP-)actin filaments, increases their twist and destabilizes them
● Gelsolin: severs actin filaments and caps their + ends
Microtubule-severing enzymes:
● Katanin: binds to the surface and uses energy of ATP-hydrolysis to extract tubulin
subunits from the microtubules, causing their breakage. Two things can happen:
○ Microtubule breakdown
○ Microtubule multiplication (when it’s not too fast), because of growth
amplification.
The cytoskeleton is composed of three major filaments:
● Actin filaments
○ Determine the shape of the cell’s surface, make the cytoskeleton dynamic,
and drive the pinching of a cell into two.
● Microtubules
○ Determine the positions of organelles, direct intracellular transport, and form
the mitotic spindle that segregates chromosomes in cell division.
● Intermediate filaments
○ Provide mechanical strength.
Actin
Actin is built from 2 rows of globular monomers (protofilaments). Actin provides the
building-blocks for dynamics, and is also an ATPase which hydrolyses ATP to ADP after
polymerization. A protofilament is polarized and consists of:
● Barbed end / + end: grows and shrinks fast → it takes less time
● Pointed end / - end: grows and shrinks slow → it takes more time
At steady state, the filaments grow as fast as they disassemble and polymerization stops
(equilibrium).
The growing rate (Kon) depends on concentration (more subunits → fast polymerization)
The shrinking rate (Koff) does not depend on concentration.
The critical concentration (Cc) is the concentration of subunits at equilibrium (equal for the
+ and - end when they are both in ATP-form (T), because the energy release is the same).
𝐾𝑜𝑓𝑓
𝐶𝑐 = 𝐾𝑜𝑛
= 𝑘𝑑
Actin polymerization occurs more rapidly in the presence of pre-formed filaments. The
making of the first oligomers out of monomers is called nucleation (lag phase).
Actin as a monomer in the cytoplasm is always in its ATP-form (T). After polymerization,
actin will hydrolyze ATP to ADP. Because the - end grows slow, hydrolysis can catch up with
polymerization, so the - end already is in ADP-form (D) before polymerization. This makes
the addition of T-form subunits to the - end even more difficult. The critical concentration for
the - end becomes high, making it grow only when a lot of subunits are present. The critical
concentration for the + end is high, making it grow with a few subunits. In the treadmilling
range, the subunit concentration is enough to make the + end grow, but it leads to shrinkage
of the - end (depolymerization). This looks as though the whole filament shifts.
Actin in T-form polymerizes spontaneously because of hydrophobic interactions with water
molecules (energetically favorable). ATP-hydrolysis is needed because depolymerization
requires energy, and depolymerization is needed for the dynamics of actin.
Drugs like latrunculin and cytochalasin B depolymerize actin filaments, phalloidin can
stabilize actin filaments.
,Microtubules
Microtubule filaments are thick and sparse, and also very dynamic. They are attached to
the nucleus and reach out to the periphery by polymerization (also spontaneously), or shrink
by depolymerization → dynamic instability.
Microtubules are built from tubulin dimers (globular proteins) and are GTP-enzymes
(β-tubulin hydrolyzes GTP after polymerization). They consist of 13 protofilaments, making
them a rigid hollow tube. A protofilament is polarized and consists of:
● + end: the end where β-tubulin sticks out (grows fast)
● - end: the end where ɑ-tubulin sticks out (grows slower)
As long as the microtubule β-tubulin tip is in its T-form (GTP-cap), the microtubules are held
together. After loss of the GTP-cap (catastrophe) or after GTP-hydrolyzation, the
protofilaments become less attached and split apart in individual protofilaments and then in
dimers (D-form), which can convert into T-form in the cytoplasm.
Drugs like nocodazole and colchicine can depolymerize microtubule filaments, taxol can
stabilize microtubule filaments.
Regulation of cytoskeletal organization
Actin filaments can be organized in:
● Lamellipodia: flat part of the cell with a flat branched network of actin
● Filopodia: protrusions of the cell with parallel bundles of actin
● Cell cortex: branched and unbranched network of actin
● Stress fibres: contractile parts of the cell with antiparallel bundles of actin
Microtubules can be organized in:
● Fibroblasts: radial array starting from one point (nucleus) reaching out to the
periphery
● Plant cells: layers of microtubules parallel to the plasma membrane
● Intestinal epithelial cells: vertical parallel bundles with the - end attached to the
surface and the + end on the inside
● Neurons: parallel bundles with the + end in the axons, and antiparallel in the
dendrites
Actin is way more abundant in the cell than the critical concentration. The cytoskeleton is
regulated by the availability of the monomers (subunit sequestration).
● Thymosin: prevent assembly by binding/sequestering actin subunits
● Profilin: prevent assembly at the - end and promotes assembly at the + end by
binding actin monomers and concentrating them at the + end
● Stathmin: prevent assembly by binding/sequestering tubulin dimers and keeping
them incompetent for polymerization
Nucleation is regulated by the nucleation-promoting factor + Arp2/3 and other proteins.
Actin nucleation is regulated by nucleation-promoting complex + Arp2/3 with other
proteins. This attaches to the minus-end to prevent shrinkage and promotes growth of the
plus-end. Arp2/3 also can make branches of actin. This only occurs very close to the plasma
membrane.
Microtubules are nucleated with γ-tubulin ring complex (very abundant in centrosomes),
which can also make microtubule branches with augmin.
, The centrosome is built around centrioles:
● Attached two each other by a linker
● Built from 9 triplets of microtubules (1 complete with 13 protofilaments, 2 incomplete
with 10 protofilaments)
● SAS6 has the capacity to make this 9-fold symmetry
A lot of microtubules also nucleate non-centrosomal: the Golgi-apparatus, nuclear envelope,
and other microtubules all have γ-tubulin.
Actin polymerization regulating factors:
● Formin: + end bound factor that accelerates actin growth at the + end, by interacting
with profilin, which are bound to actin-monomers
● Capping proteins: prevent actin polymerization and depolymerization at the + ends
Microtubule polymerization regulating factors:
● Plus-end tracking proteins (+TIPs): can be polymerizing or depolymerizing
○ XMAP215: accelerate growth of the growing + end (like formin in actin)
○ Kinesin-13: induce catastrophes by breaking down the GTP-cap
There are actin binding proteins that can control stability, bundling, packing, orientation and
membrane attachment of the filaments after they have grown:
● Tropomyosin (muscle): makes actin more accessible to myosin motors
● Fimbrin: makes very tight actin bundles
● ɑ-actinin: makes antiparallel loose bundles
● Filamin: stabilizes branched actin bundles
● Spectrin/ERM: attach actin to the plasma membrane
Not all cytoskeletal structures are dynamic (such as stable microvilli on intestinal epithelial
cells):
Microtubule-associated proteins (MAP’s) stabilize and bundle microtubules or link them to
other structures:
● MAP2: link to dendrites
● Plectrin: link intermediate filaments
● Tau: link to axons
Primary cilia are stable microtubuli components, grown from a centriole, and are important
for organizing sensory and signaling functions. All cilia (primary and motile) have a
microtubule core (axoneme) made out of 9 doublets. Axonemal dyneins are attached to a
doublet and can move to the other doublet, which moves the two doublets apart. In cilia the
doublets are connected so this movement results in a wave of bending (sperm).
Actin-destabilizing and severing proteins:
● Cofilin: binds (ADP-)actin filaments, increases their twist and destabilizes them
● Gelsolin: severs actin filaments and caps their + ends
Microtubule-severing enzymes:
● Katanin: binds to the surface and uses energy of ATP-hydrolysis to extract tubulin
subunits from the microtubules, causing their breakage. Two things can happen:
○ Microtubule breakdown
○ Microtubule multiplication (when it’s not too fast), because of growth
amplification.
