CB lec 9 & 10

What is the cytoskeleton?

• A network of protein polymers that gives cells shape and organization<br>• Supports vesicular transport<br>• Provides mechanical support<br>• Drives cell movement<br>• Helps separate genetic and cytoplasmic material during cell division<br>• Main types: intermediate filaments, microtubules, and actin filaments

What are the three major types of cytoskeletal polymers?

• Intermediate filaments<br>• Microtubules<br>• Actin filaments

Why do different cell types have different shapes?

• Different cell types have different cytoskeletal architectures<br>• The cytoskeleton creates shapes that support specialized cell functions<br>• Examples include nerve cells, epithelial cells, muscle cells, and red blood cells

How does the cytoskeleton support vesicular transport?

• Cytoskeletal polymers provide tracks for vesicles<br>• The tracks can provide directionality<br>• Motor proteins move vesicles along these tracks toward their target membranes

How does the cytoskeleton provide mechanical support?

• Different cytoskeletal polymers have different structural properties<br>• Intermediate filaments form strong, rope-like structures that resist tension<br>• These structures help cells withstand mechanical stress

Why is cytoskeletal polarity important for movement?

• Polarized filaments have distinct plus and minus ends<br>• Motor proteins recognize these directional differences<br>• This allows cargo and cellular structures to move in specific directions

What are intermediate filaments?

• Rope-like cytoskeletal polymers<br>• About 10 nm in diameter<br>• Provide mechanical strength and resistance to tension<br>• Help cells withstand physical stress

How are intermediate filaments assembled?

• Individual protein subunits associate to form dimers<br>• Dimers associate in an antiparallel arrangement<br>• Larger structures are formed from these associated subunits<br>• These structures pack together to form a strong, rope-like filament

Why are intermediate filaments rope-like and mechanically strong?

• Their subunits pack together into a strong, cable-like structure<br>• The rope-like organization allows forces to be distributed along the filament<br>• This gives intermediate filaments high tensile strength

Are intermediate filaments polarized?

• No<br>• Their two ends are structurally equivalent<br>• They do not have distinct plus and minus ends

Do intermediate filament subunits bind nucleotides?

• No<br>• Intermediate filament assembly does not require ATP or GTP binding or hydrolysis

What are cytoplasmic intermediate filaments?

• Intermediate filaments found throughout the cytoplasm<br>• Keratin is an important example<br>• They provide mechanical strength to cells and tissues

How does keratin help hold epithelial cells together?

• Keratin intermediate filaments extend through the cytoplasm<br>• They connect to cell-cell junctions such as desmosomes<br>• This mechanically links neighboring epithelial cells<br>• The resulting cell sheet can resist stretching and tension

What are desmosomes?

• Cell-cell junctions that mechanically connect neighboring epithelial cells<br>• Intermediate filaments such as keratin attach to the desmosomes<br>• They help distribute mechanical forces across the epithelial sheet

What are hemidesmosomes?

• Cell junctions that anchor intermediate filaments to the extracellular matrix<br>• They help attach epithelial cells to the underlying basement membrane

What happens if keratin intermediate filaments are defective?

• Epithelial cells become less resistant to mechanical stress<br>• Stretching or pressure can cause the epithelial sheet to rupture<br>• Certain keratin mutations can cause epithelial blistering

What are nuclear intermediate filaments in animal cells?

• Lamins<br>• They form the nuclear lamina underneath the inner nuclear membrane<br>• They provide mechanical support to the nucleus

What is the nuclear lamina?

• A network of lamin intermediate filaments underneath the inner nuclear membrane<br>• Provides structural support to the nucleus

What happens to the nuclear lamina during mitosis?

• Lamins are phosphorylated<br>• Phosphorylation causes the nuclear lamina to disassemble<br>• This contributes to breakdown of the nuclear envelope during mitosis

What can mutations in nuclear lamins cause?

• Mutations in nuclear intermediate filament proteins can cause human diseases<br>• These diseases are often called laminopathies<br>• One example associated with lamin mutations is progeria

What are microtubules?

• Hollow, tube-like cytoskeletal polymers<br>• About 25 nm in diameter<br>• Built from alpha-beta tubulin heterodimers<br>• Important for intracellular transport, cell organization, chromosome movement, and cilia/flagella

What is the basic building block of a microtubule?

• An alpha-beta tubulin heterodimer<br>• Contains one alpha-tubulin and one beta-tubulin

How are microtubules structured?

• They form hollow tubes<br>• The tube is made of 13 protofilaments<br>• Protofilaments are built from alpha-beta tubulin heterodimers

Are microtubules polarized?

• Yes<br>• They have a plus end and a minus end<br>• The two ends have different properties and different rates of polymerization

What nucleotide is associated with tubulin?

• GTP<br>• Both alpha- and beta-tubulin bind GTP<br>• GTP hydrolysis is important for regulating microtubule dynamics

What is the difference between GTP bound to alpha-tubulin and beta-tubulin?

• GTP on alpha-tubulin is tightly bound and mainly structural<br>• GTP on beta-tubulin can be hydrolyzed<br>• Beta-tubulin GTP hydrolysis helps regulate microtubule assembly and disassembly

What is the plus end of a microtubule?

• The end associated with beta-tubulin<br>• Generally grows and shrinks more rapidly<br>• GTP-tubulin is added more readily at this end

What is the minus end of a microtubule?

• The end associated with alpha-tubulin<br>• Generally grows and shrinks more slowly<br>• It is often anchored at a microtubule-organizing center

What happens when GTP-tubulin is added to a microtubule?

• GTP-tubulin is added to the growing end<br>• After incorporation, the GTP associated with beta-tubulin is eventually hydrolyzed to GDP<br>• GTP-tubulin at the end helps stabilize the microtubule

What is a GTP cap?

• A region of GTP-tubulin at the end of a growing microtubule<br>• Helps stabilize the microtubule and allows continued growth

What happens if microtubule growth is faster than GTP hydrolysis?

• A GTP cap is maintained at the growing end<br>• The microtubule remains relatively stable and continues to grow

What happens when microtubule growth slows and the GTP cap is lost?

• GDP-tubulin becomes exposed at the end<br>• The microtubule becomes unstable<br>• Protofilaments peel away<br>• Rapid depolymerization occurs

What is microtubule catastrophe?

• A rapid transition from microtubule growth to rapid depolymerization<br>• Usually occurs when the stabilizing GTP cap is lost

What is microtubule rescue?

• A transition from microtubule shrinkage back to growth<br>• The microtubule begins polymerizing again

What is treadmilling?

• Tubulin subunits are added preferentially at one end and removed from the other<br>• The filament can maintain a relatively similar length while subunits move through it<br>• For microtubules, GTP-tubulin can be added at the plus end while GDP-tubulin is lost at the minus end

What are the major stages of microtubule assembly?

• Nucleation: formation of an initial stable microtubule seed<br>• Elongation: addition of tubulin subunits to the existing microtubule

What is nucleation?

• The initial formation of a stable microtubule seed<br>• It is the difficult first step before rapid elongation can occur

What is elongation?

• Addition of new tubulin subunits to the ends of an existing microtubule<br>• It allows the microtubule to grow after nucleation

What is the core component of the microtubule nucleation complex?

• Gamma-tubulin<br>• Gamma-tubulin is part of the gamma-tubulin ring complex, or gamma-TuRC

What is the gamma-tubulin ring complex (gamma-TuRC)?

• A microtubule nucleation complex<br>• Contains gamma-tubulin<br>• Provides a template that promotes formation of a new microtubule

Which end of a microtubule is associated with the gamma-TuRC?

• The minus end<br>• The gamma-TuRC nucleates and anchors the minus end while the plus end extends outward

What is a microtubule-organizing center (MTOC)?

• A cellular site where microtubules are nucleated and organized<br>• In animal cells, the centrosome is a major MTOC

Why does microtubule polymerization happen faster at an MTOC?

• The MTOC contains microtubule nucleation complexes such as gamma-TuRCs<br>• These complexes provide a template for microtubule nucleation<br>• This bypasses the slow nucleation step that would otherwise occur in the cytoplasm

Why do many microtubules radiate from the centrosome?

• The centrosome acts as a major MTOC<br>• It nucleates and anchors microtubule minus ends<br>• Plus ends extend outward into the cytoplasm<br>• This creates a polarized microtubule array

What is the centrosome?

• A major microtubule-organizing center in animal cells<br>• Nucleates microtubules<br>• Anchors their minus ends<br>• Allows plus ends to extend into the cytoplasm

What is the effect of taxol on microtubules?

• Taxol stabilizes microtubules<br>• It prevents normal microtubule depolymerization<br>• This can cause excessive microtubule polymerization and disrupt normal microtubule dynamics

Why can microtubule drugs such as taxol be used as anticancer drugs?

• Cell division requires dynamic microtubules<br>• Drugs that disrupt microtubule dynamics can interfere with chromosome movement and cell division<br>• Rapidly dividing cancer cells can therefore be affected<br>• Normal rapidly dividing cells can also be affected

What are kinesin and dynein?

• Microtubule-based motor proteins<br>• Use ATP hydrolysis to generate directional movement along microtubules<br>• Transport cargo such as vesicles and organelles

What are the two basic domains of a microtubule motor protein?

• Head/motor domain: binds the microtubule and uses ATP<br>• Tail domain: interacts with cargo or cargo-binding proteins

What does the head domain of kinesin or dynein do?

• Binds to the microtubule<br>• Uses ATP hydrolysis to generate movement

What does the tail domain of kinesin or dynein do?

• Interacts with cargo<br>• Helps transport vesicles, organelles, or other cellular materials

What is the energy source for kinesin and dynein movement?

• ATP hydrolysis<br>• ATP provides the energy needed for directional movement along microtubules

Which direction does kinesin I move on a microtubule?

• Toward the plus end of the microtubule

Which direction does dynein move on a microtubule?

• Toward the minus end of the microtubule

How do microtubules support vesicle transport?

• Microtubules provide polarized tracks<br>• Kinesin generally moves cargo toward the plus end<br>• Dynein generally moves cargo toward the minus end<br>• This allows directional transport through the cell

How do dynein motors generate the beating motion of cilia and flagella?

• Dynein motors attach to one microtubule doublet<br>• Dynein walks toward the minus end of an adjacent microtubule doublet<br>• This creates a sliding force between neighboring microtubules<br>• Linking proteins restrict the sliding<br>• The restricted sliding is converted into bending

What is the energy source for cilia and flagella movement?

• ATP hydrolysis by dynein motors

How is dynein movement related to flagellum or cilium bending?

• Dynein attempts to slide neighboring microtubule doublets relative to one another<br>• Linking proteins restrict the sliding<br>• The restricted sliding produces bending of the flagellum or cilium

What is nexin?

• A linking protein that connects neighboring microtubule doublets in cilia and flagella<br>• Helps convert microtubule sliding into bending

What happens if nexin is removed from a flagellum or cilium?

• Microtubule doublets can slide past one another instead of producing normal bending<br>• Normal coordinated bending is disrupted

Why can dysfunctional ciliary dynein cause chronic lung infections?

• Ciliated cells in the respiratory tract use cilia to move mucus and trapped particles<br>• Dysfunctional dynein prevents normal ciliary movement<br>• Mucus and inhaled particles are not cleared effectively<br>• This can increase the risk of chronic respiratory infections

What are actin filaments?

• Thin, flexible cytoskeletal polymers<br>• About 6–7 nm in diameter<br>• Built from actin monomers<br>• Important for cell shape, movement, contraction, and cell division

What is the basic building block of an actin filament?

• An actin monomer<br>• Actin monomers are globular proteins

What is the structure of an actin monomer?

• A globular protein<br>• Contains an ATP-binding cleft<br>• Actin monomers polymerize to form actin filaments

How is an actin filament structured?

• It is a helical polymer<br>• It consists of two actin strands twisted around each other<br>• It is about 6–7 nm in diameter<br>• It has a repeat of about 37 nm

Are actin filaments polarized?

• Yes<br>• The actin monomers have the same orientation within the filament<br>• This creates distinct plus and minus ends

What is the plus end of an actin filament?

• The end where actin subunits are added more easily<br>• It generally grows faster than the minus end<br>• It is more likely to contain ATP-actin

What is the minus end of an actin filament?

• The end where actin subunits are removed more readily<br>• It generally grows more slowly<br>• It is more likely to contain ADP-actin

What nucleotide associates with actin?

• ATP<br>• Actin monomers exist mainly in the ATP-bound form in the cytosol<br>• ATP is hydrolyzed after incorporation into the filament

What happens to ATP-actin after it is incorporated into an actin filament?

• ATP is gradually hydrolyzed to ADP<br>• ADP-actin is less favorable for filament stability<br>• The minus end therefore tends to contain more ADP-actin

What is actin treadmilling?

• Actin subunits are added at the plus end<br>• Actin subunits are removed from the minus end<br>• ATP-actin is added at the plus end<br>• ADP-actin is commonly lost from the minus end

What are the major stages of actin filament assembly?

• Nucleation: formation of a stable actin seed<br>• Elongation: addition of actin subunits to the filament<br>• Treadmilling: addition at one end and loss at the other

What is an actin filament capper?

• A protein that binds to one end of an actin filament<br>• Controls the addition and removal of actin subunits at that end

What is CapZ?

• An actin filament capping protein<br>• Caps the plus end of actin filaments<br>• Prevents actin subunits from being added to or removed from that end

What is tropomodulin?

• An actin filament capping protein<br>• Caps the minus end of actin filaments<br>• Helps regulate actin filament length

Why do CapZ and tropomodulin have different effects on actin polymerization?

• They bind to different ends of the actin filament<br>• CapZ binds the plus end<br>• Tropomodulin binds the minus end<br>• Each capper controls subunit addition and removal at its specific end

What is the Arp2/3 complex?

• An actin nucleation complex<br>• Promotes formation of new actin filaments<br>• Creates branched actin networks

What type of actin structure does Arp2/3 organize?

• Branched actin filament networks<br>• These networks are especially important near the plasma membrane

How can an extracellular signal cause localized actin polymerization?

• A signal molecule binds to a cell-surface receptor<br>• The receptor activates a signaling pathway<br>• The pathway activates proteins that regulate actin assembly<br>• Arp2/3 promotes local actin polymerization<br>• The resulting actin network can change cell shape and direction of movement

How does localized actin polymerization help a cell move?

• Actin polymerization can push the plasma membrane outward<br>• This creates structures such as leading edges or protrusions<br>• Localized polymerization helps establish the direction of cell movement

How can a GPCR signal lead to localized actin polymerization?

• An extracellular signal activates a GPCR<br>• The GPCR activates a trimeric G protein<br>• The signaling pathway activates a small GTPase<br>• The small GTPase activates the Arp2/3 complex<br>• Arp2/3 promotes local actin polymerization<br>• The localized actin network changes cell shape and movement

What are myosins?

• Actin-based motor proteins<br>• Use ATP hydrolysis to generate movement along actin filaments<br>• Different myosins perform different cellular functions

What is myosin II?

• An actin-based motor protein<br>• Important for muscle contraction and other forms of cellular contraction<br>• Uses ATP to move toward the plus end of actin filaments

What are the basic parts of myosin II?

• Motor/head domain: binds actin and hydrolyzes ATP<br>• Lever arm: changes position during the power cycle<br>• Tail: helps myosin molecules interact with one another and form filaments

How do myosin II molecules form a bipolar filament?

• Myosin II molecules associate through their tails<br>• Their motor heads point outward from the center<br>• Motor heads are therefore present at both ends of the thick filament<br>• This creates a bipolar filament

What is the energy source for myosin II movement?

• ATP hydrolysis

Which direction does myosin II move on actin?

• Toward the plus end of the actin filament

What happens during the myosin II power cycle?

• Myosin begins with ADP and phosphate bound<br>• Myosin binds to actin<br>• Phosphate release strengthens actin binding and triggers the power stroke<br>• ADP is released<br>• ATP binds, causing myosin to detach from actin<br>• ATP is hydrolyzed, recocking the lever arm<br>• The cycle repeats

What is the rigor state of myosin?

• A state in which myosin is tightly bound to actin when ATP is absent<br>• This contributes to rigor mortis after death

Why does rigor mortis occur?

• After death, cells stop producing ATP<br>• Without ATP, myosin remains tightly bound to actin<br>• This cross-links the filaments and makes muscle tissue rigid<br>• Later, cellular proteases break down proteins and the rigidity resolves

What is the neuromuscular junction?

• The specialized synapse between a motor neuron and a muscle cell<br>• It allows a motor neuron to signal the muscle cell to contract

How is a signal transmitted from a motor neuron to a muscle cell?

• An action potential reaches the motor neuron terminal<br>• Voltage-gated Ca2+ channels open<br>• Ca2+ enters the nerve terminal<br>• Neurotransmitter is released<br>• Neurotransmitter binds receptors on the muscle cell membrane<br>• Ion channels open and Na+ enters<br>• The muscle membrane depolarizes and generates an action potential

What happens when an action potential reaches the motor neuron terminal?

• Voltage-gated Ca2+ channels open<br>• Ca2+ enters the nerve terminal<br>• Ca2+ triggers neurotransmitter release into the neuromuscular junction

What happens when neurotransmitter binds receptors on the muscle cell?

• Ion channels in the muscle membrane open<br>• Na+ enters the muscle cell<br>• The muscle membrane depolarizes<br>• An action potential is generated and spreads across the muscle cell

How does Prialt cause paralysis?

• Prialt is a cone snail toxin<br>• It blocks voltage-gated Ca2+ channels in nerve terminals<br>• Ca2+ cannot enter the motor neuron normally<br>• Neurotransmitter release is reduced or blocked<br>• The muscle cell is not properly activated<br>• This can cause paralysis

What are T-tubules?

• Deep invaginations of the muscle cell plasma membrane<br>• Extend deep into the muscle cell<br>• Carry action potentials from the cell surface into the interior of the cell

What is the sarcoplasmic reticulum (SR)?

• A specialized form of ER in muscle cells<br>• Stores Ca2+<br>• Releases Ca2+ in response to muscle excitation

How are T-tubules and the SR arranged in skeletal muscle?

• T-tubules extend deep into the muscle cell<br>• The SR closely associates with the T-tubules<br>• Together they surround the myofibrils<br>• This allows excitation at the cell surface to rapidly trigger Ca2+ release throughout the muscle cell

How does an action potential cause Ca2+ to increase throughout a muscle cell?

• The action potential spreads along the plasma membrane<br>• It travels deep into the cell through T-tubules<br>• Voltage-sensitive proteins in the T-tubule membrane respond to the action potential<br>• These activate Ca2+ release channels in the adjacent SR<br>• The SR releases Ca2+ throughout the cytosol<br>• This rapidly increases cytoplasmic Ca2+ concentration

Why are T-tubules important for muscle contraction?

• They carry the action potential deep into the muscle cell<br>• This allows the signal to reach the SR throughout the cell<br>• The SR can then rapidly release Ca2+ near the contractile machinery