Molecular Biology · End-of-chapter questions below · Part 1 of 2 · 10 questions per part
Part 1 of 2
Actin Filaments, Myosin Motors, and Cell Movement
A crawling neutrophil that hunts bacteria, a contracting muscle fiber, a dividing cell cleaving in two — all depend on a dynamic internal scaffold of protein filaments. The cytoskeleton is not a rigid cage but a living, constantly remodeling architecture that converts chemical energy into mechanical force.
In Part 1, you will explore:
Actin filament structure, polarity (barbed/pointed ends), and critical concentration
Treadmilling and ATP hydrolysis by actin
Nucleation: Arp2/3 complex and formins
Myosin II motor mechanism and the cross-bridge cycle
Actin-based cell protrusions: lamellipodia and filopodia
16.1 Actin Filament Structure and Dynamics
Actin monomers (G-actin, ~42 kDa) polymerize head-to-tail into helical F-actin filaments with structural polarity: the barbed (plus) end elongates rapidly and the pointed (minus) end elongates slowly. Each monomer carries one ATP that is hydrolyzed to ADP shortly after incorporation. Because ADP-actin dissociates more readily from the pointed end, a steady-state treadmilling occurs: ATP-actin adds to the barbed end while ADP-actin falls off the pointed end, maintaining filament length while subunits flow through.
The critical concentration (Cc) is the monomer concentration at which on-rate equals off-rate. Below Cc, filaments depolymerize; above Cc, they grow. The barbed-end Cc (~0.1 µM) is much lower than the pointed-end Cc (~0.7 µM), driving net treadmilling when total actin is between these values. Profilin accelerates ADP→ATP exchange on monomers, replenishing the ATP-actin pool; thymosin-β4 sequesters ATP-actin monomers, buffering the available pool.
Key term
Treadmilling
The steady-state behavior of an actin (or microtubule) filament in which net addition at one end equals net loss at the other, allowing the filament to maintain constant length while individual subunits transit through it. Requires continuous ATP (actin) or GTP (tubulin) hydrolysis.
16.2 Actin Nucleation: Arp2/3 and Formins
Spontaneous nucleation (forming a 3-subunit seed) is kinetically unfavorable, so cells use dedicated nucleators. The Arp2/3 complex (7 subunits, including two actin-related proteins) is activated by WASP/N-WASP or WAVE/Scar nucleation-promoting factors, themselves activated by Rho-family GTPases (Cdc42 activates WASP; Rac1 activates WAVE). Arp2/3 binds the side or barbed end of an existing filament and nucleates a new filament at a 70° branch angle, creating the dendritic network in lamellipodia. Formins (e.g., mDia1) nucleate unbranched filaments and remain processively attached to the barbed end, enabling long filament growth for filopodia, stress fibers, and the cytokinetic ring.
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Pause & Recall
Why does cytochalasin D (which caps barbed ends) disrupt lamellipodia but latrunculin A (which sequesters G-actin) also collapses filaments?
Cytochalasin D blocks barbed-end addition, halting polymerization-driven protrusion and allowing pointed-end disassembly to shorten filaments. Latrunculin A binds G-actin monomers, lowering the free monomer concentration below Cc so both ends lose subunits and filaments depolymerize. Both drugs therefore collapse lamellipodia, but through opposite mechanisms: one prevents growth while the other starves the filament of building blocks.
16.3 Myosin II and the Cross-Bridge Cycle
Myosin II is a dimeric motor: two heavy chains form a coiled-coil tail and two globular heads, each with an actin-binding site and an ATPase. The cross-bridge cycle proceeds as follows: (1) ATP binding releases myosin from actin; (2) ATP hydrolysis to ADP+Pi "cocks" the head into a high-energy conformation; (3) the head binds actin weakly; (4) Pi release triggers the power stroke—the head swings ~10 nm, moving actin; (5) ADP release tightens the rigor bond. Multiple cross-bridges cycle asynchronously so force is continuous. Myosin II assembles into bipolar thick filaments via its tail domain, enabling it to slide antiparallel actin filaments past each other.
In skeletal muscle, actin (thin) and myosin II (thick) filaments are organized into sarcomeres—the repeating contractile unit flanked by Z-discs. Contraction is regulated by the troponin–tropomyosin complex: at rest, tropomyosin blocks myosin-binding sites on actin. Ca²⁺ released from the SR binds troponin C, shifting tropomyosin to expose binding sites and enable cross-bridge cycling (sliding filament model).
Key term
Sarcomere
The fundamental contractile unit of striated muscle, bounded by Z-discs. The A-band contains thick filaments (myosin II); the I-band contains thin filaments (actin) alone; the H-zone is where thick and thin filaments do not overlap. During contraction, the I-band and H-zone narrow while A-band length is constant.
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Pause & Recall
How does rigor mortis illustrate the biochemistry of the cross-bridge cycle?
After death, ATP production ceases. Without ATP, myosin heads cannot detach from actin (step 1 of the cross-bridge cycle requires ATP binding). All myosin heads lock in the "rigor" conformation tightly bound to actin, making muscles rigid. Rigor mortis resolves 24–48 hours post-mortem as protease activity degrades the thin and thick filament proteins.
Practice questions — Part 1Score: 0 / 10
1. At which end of an actin filament is the critical concentration lowest, favoring net addition?
The barbed (plus) end has a critical concentration of ~0.1 µM, far lower than the pointed end (~0.7 µM). When free actin is between these values, net addition occurs at the barbed end and net loss at the pointed end — the hallmark of treadmilling.
2. Which nucleotide state of actin is found preferentially at the pointed end during treadmilling?
ATP is hydrolyzed to ADP shortly after actin incorporation. The pointed end contains older, ADP-bound subunits that dissociate readily. ADP-actin's higher Cc drives net depolymerization at the pointed end, while ATP-actin addition at the barbed end drives polymerization.
3. The Arp2/3 complex nucleates actin branches at what angle to the mother filament?
Arp2/3 creates a ~70° branch angle, generating a dendritic network of filaments in lamellipodia. This branched architecture converts barbed-end polymerization into protrusive force against the plasma membrane. The angle is set by the geometry of Arp2/3 binding to the mother filament.
4. Which Rho-family GTPase activates WAVE/Scar to stimulate Arp2/3 in lamellipodia?
Rac1 activates the WAVE/Scar complex, which in turn activates Arp2/3, driving lamellipodium formation. Cdc42 activates WASP/N-WASP to promote filopodia. RhoA activates formins and myosin II for stress fibers and contractility. Ran is a GTPase involved in nuclear transport, not cytoskeletal regulation.
5. During the myosin cross-bridge cycle, what event constitutes the "power stroke"?
Pi release after actin binding triggers the conformational change in the myosin neck region (power stroke), swinging the lever arm ~10 nm and displacing the actin filament. ATP binding causes detachment; ADP+Pi hydrolysis cocks the head; Pi release drives movement; ADP release locks the rigor bond.
6. Which protein sequesters ATP-actin monomers to buffer the free monomer pool?
Thymosin-β4 binds ATP-actin monomers and prevents their spontaneous polymerization, maintaining a large sequestered pool. When polymerization is stimulated, profilin exchanges monomers from thymosin-β4 and delivers them to formin-capped barbed ends. Cofilin severs ADP-actin filaments to create new ends; tropomyosin stabilizes filaments against cofilin.
7. In skeletal muscle, Ca²⁺ binding to troponin C shifts tropomyosin to expose myosin-binding sites on actin. Which troponin subunit anchors the complex to actin?
Troponin T (TnT) tethers the complex to tropomyosin along the actin filament. Troponin I (TnI) inhibits myosin ATPase activity in the absence of Ca²⁺. Troponin C (TnC) binds Ca²⁺ and relieves TnI inhibition. Plasma TnT and TnI are cardiac biomarkers used clinically to diagnose myocardial infarction.
8. Formins remain processively at the barbed end during elongation. Which property makes them useful for building filopodia rather than the branched networks of lamellipodia?
Formins use their FH1 domain (profilin-binding) and FH2 domain (barbed-end association) to thread actin subunits onto a growing unbranched filament. This generates the long, parallel actin bundles characteristic of filopodia, stress fibers, and the cytokinetic contractile ring. Arp2/3 generates branched networks; cofilin severs; capping protein blocks barbed ends.
9. Cofilin/ADF severs and depolymerizes actin filaments. What nucleotide state does cofilin preferentially target?
Cofilin/ADF binds with high affinity to ADP-actin — the older, more weakly associated subunits near the pointed end — and severs the filament and promotes pointed-end depolymerization. This releases ADP-actin monomers that profilin recharges with ATP, replenishing the monomer pool for barbed-end assembly.
10. What happens to the sarcomere I-band and H-zone width during muscle contraction?
The sliding filament model predicts that thin filaments slide toward the center of the sarcomere. The I-band (thin filaments only) and H-zone (thick filaments only) both narrow as overlap increases. The A-band (full length of thick filament) remains constant because myosin filaments do not change length — they are simply invaded by actin.
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Part 1 complete. Continue to Part 2 below.
Part 1 → Part 2
Actin and myosin power contraction and migration. Now we turn to microtubules — the longer, stiffer polymers that organize cell division, long-range transport, and cilia — and to intermediate filaments, which provide tensile strength to the cell.
Part 2 of 2
Microtubules, Motors, Cilia, and Intermediate Filaments
16.4 Microtubule Structure and Dynamic Instability
Microtubules (MTs) are hollow tubes (~25 nm diameter) made of αβ-tubulin heterodimers assembled into 13 protofilaments arranged in parallel. Like actin, MTs are polar: the plus end (β-tubulin exposed) is dynamic; the minus end (α-tubulin exposed) is typically anchored at the MTOC. Each αβ dimer carries one GTP on β-tubulin, hydrolyzed to GDP after assembly. The GTP-cap at the plus end stabilizes the MT; when GTP hydrolysis outpaces addition, the cap is lost and the MT undergoes rapid depolymerization — catastrophe. Re-growth from the shrinking end is called rescue. This dynamic instability allows MTs to explore cellular space, a key feature exploited during chromosome capture at kinetochores.
Key term
Dynamic Instability
The stochastic switching of individual microtubule plus ends between phases of slow growth (polymerization) and rapid shortening (catastrophe), driven by GTP hydrolysis. Drugs that prevent this — taxol (stabilizes) or colchicine/vincristine (depolymerize) — block cell division by freezing the mitotic spindle.
16.5 MTOCs, Centrosomes, and Spindle Formation
The microtubule-organizing center (MTOC) in animal cells is the centrosome, composed of a pair of centrioles surrounded by pericentriolar material (PCM). PCM contains γ-tubulin ring complexes (γ-TuRCs) that nucleate minus ends and anchor them; plus ends grow outward. Before mitosis, the centrosome duplicates (once per cell cycle, S phase) and the two centrosomes separate to form the poles of the bipolar mitotic spindle. Kinetochore MTs attach kinetochores on sister chromatids; polar MTs from opposite poles interdigitate; astral MTs contact the cell cortex to position the spindle.
16.6 Kinesin and Dynein: Microtubule-Based Motors
Most kinesins (kinesin-1 through kinesin-14) move toward the MT plus end (anterograde transport in axons: away from the soma), carrying organelles, vesicles, and mRNA-protein complexes. The kinesin-1 head binds MT, hydrolyzes ATP, and "walks" hand-over-hand in ~8 nm steps per ATP. Cytoplasmic dynein moves toward the minus end (retrograde in axons: toward soma). Dynein requires the multi-subunit dynactin complex and cargo adaptors (BICD2, Hook proteins) for activation and processivity. Dynein also powers ciliary/flagellar beating as axonemal dynein.
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Pause & Recall
How does taxol (paclitaxel) kill rapidly dividing cancer cells while also causing peripheral neuropathy as a side effect?
Taxol binds polymerized tubulin in the inner face of the MT lumen and prevents dynamic instability. In cancer cells, this freezes the mitotic spindle, preventing chromosome segregation and triggering apoptosis at the spindle assembly checkpoint. In post-mitotic neurons — which rely on axonal transport via kinesin and dynein along stable MTs — taxol disrupts the normal MT dynamics needed for transport, causing "dying-back" neuropathy in long sensory and motor axons. The peripheral nervous system is particularly vulnerable because long axons depend heavily on MT dynamics for cargo delivery.
16.7 Cilia and Flagella
Motile cilia and flagella share the 9+2 axoneme: nine outer doublets of A- and B-tubules surrounding a central pair of singlets, all connected by radial spokes and nexin links. Outer dynein arms walk along adjacent B-tubules, converting ATP hydrolysis to sliding. Because doublets are cross-linked, sliding is converted to bending. Primary (non-motile) cilia lack the central pair (9+0) and dynein arms; they function as sensory antennae (Hedgehog signaling, olfaction, photoreception). Intraflagellar transport (IFT) uses kinesin-2 (anterograde, outward) and dynein-2 (retrograde, inward) to deliver and retrieve ciliary components along the axoneme. Defects in IFT or axonemal dynein cause primary ciliary dyskinesia (PCD), with chronic respiratory infections, situs inversus, and infertility.
16.8 Intermediate Filaments and Nuclear Lamins
Intermediate filaments (IFs) (~10 nm diameter, between actin and MTs) are made of tissue-specific proteins: keratins in epithelia, vimentin in mesenchymal cells, neurofilaments in neurons, desmin in muscle, and GFAP in astrocytes. IFs are non-polar, do not use motor proteins, and are not dynamic in the same way as actin or MTs — they provide tensile strength and resistance to mechanical stress. IF monomers coil into dimers, then antiparallel tetramers, then unit-length filaments that anneal end-to-end.
Nuclear lamins (A/C and B types) are IF proteins that line the inner nuclear envelope, forming the nuclear lamina. The lamina provides mechanical support to the nucleus and organizes heterochromatin at the nuclear periphery. Mutations in LMNA (lamin A/C) cause laminopathies, including Emery–Dreifuss muscular dystrophy and Hutchinson–Gilford progeria syndrome (accelerated aging). Lamin B is phosphorylated by Cdk1 at mitosis onset, causing lamina depolymerization and nuclear envelope breakdown.
Key term
Nuclear Lamina
A meshwork of lamin A/C and lamin B filaments lining the inner surface of the nuclear envelope. It provides structural rigidity to the nucleus, anchors chromatin, and disassembles at mitosis onset via Cdk1-mediated phosphorylation of lamins. Reassembles after chromosome segregation when Cdk1 is inactivated.
Practice questions — Part 2Score: 0 / 10
1. What nucleotide is carried on β-tubulin in a newly assembled microtubule, and what happens when it is hydrolyzed?
β-tubulin carries GTP when incorporated. GTP hydrolysis to GDP occurs after assembly, weakening lateral contacts between protofilaments. The GTP-cap (a few terminal GTP-tubulin subunits) prevents the curved GDP-tubulin protofilaments from splaying outward. Loss of the cap triggers rapid depolymerization (catastrophe).
2. How many protofilaments make up a typical cytoplasmic microtubule?
A microtubule typically consists of 13 protofilaments of αβ-tubulin heterodimers arranged head-to-tail in parallel, forming a hollow tube ~25 nm in outer diameter. This 13-protofilament arrangement creates a seam where the lattice is slightly mismatched and which may serve as a site for MT regulatory proteins.
3. γ-Tubulin ring complexes (γ-TuRCs) in the centrosome primarily nucleate which end of microtubules?
γ-TuRCs template the minus end of microtubules, anchoring it in the pericentriolar material. The plus end then grows outward into the cytoplasm. This gives the MT array a uniform polarity with all plus ends facing outward and all minus ends anchored at the centrosome — essential for directed vesicle transport and spindle formation.
4. In which direction does conventional kinesin-1 move along a microtubule?
Kinesin-1 moves from the minus end toward the plus end — anterograde transport in neurons (soma to axon terminal). Cytoplasmic dynein moves in the opposite direction (plus to minus end) — retrograde transport (axon terminal to soma). This opposing directionality allows bidirectional cargo transport on the same MT track.
5. The "9+2" axoneme of motile cilia contains outer dynein arms that slide adjacent doublets. Why does this sliding produce bending rather than doublet separation?
Nexin links (dynein regulatory complex) cross-link adjacent outer doublets and limit the extent of sliding. Because the doublets are tethered at the basal body and constrained by nexin, dynein-generated sliding forces are converted into bending of the entire axoneme. This is analogous to bending a hinged ruler — restricted sliding of parallel elements produces curvature.
6. Which intermediate filament protein is specific to astrocytes in the central nervous system?
GFAP (glial fibrillary acidic protein) is the signature IF of astrocytes and is upregulated after CNS injury (gliosis). Vimentin is found in mesenchymal/fibroblastic cells; desmin in muscle; neurofilaments in neurons. GFAP immunostaining is a standard tool in neuropathology to identify astrocytomas and reactive gliosis.
7. Mutations in the LMNA gene (encoding lamin A/C) cause Hutchinson–Gilford progeria. What is the molecular mechanism of the "progerin" mutant lamin A?
The G608G point mutation in LMNA activates a cryptic splice site that deletes 50 amino acids near the C-terminus, including the ZMPSTE24 cleavage site. Normal lamin A is farnesylated and then the farnesyl group is removed; progerin cannot be cleaved and remains permanently farnesylated, trapping it at the inner nuclear membrane. This disrupts nuclear architecture, chromatin organization, and DNA repair, causing premature aging.
8. Intraflagellar transport (IFT) in primary cilia uses kinesin-2 for anterograde transport. In which direction does anterograde IFT move?
Kinesin-2 (heterotrimeric KIF3A/KIF3B/KAP) powers anterograde IFT from the basal body outward to the ciliary tip, delivering axonemal components. Dynein-2 (IFT dynein) powers retrograde IFT from the ciliary tip back to the basal body, recycling IFT complexes and turnover products. Mutations in IFT genes cause a spectrum of ciliopathies (Bardet–Biedl, Joubert, Meckel syndromes).
9. During mitotic entry, Cdk1 phosphorylates nuclear lamins. What is the consequence for the nuclear envelope?
Cdk1 (with cyclin B) phosphorylates specific serine residues in lamin A/C and lamin B at mitosis onset, disrupting head-to-tail polymerization and causing the lamina to depolymerize. This triggers nuclear envelope breakdown, allowing spindle MTs to access chromosomes. After anaphase, Cdk1 is inactivated and protein phosphatase 2A dephosphorylates lamins, allowing re-polymerization and nuclear envelope reassembly around decondensing chromosomes.
10. Patients with primary ciliary dyskinesia (PCD) often have situs inversus (reversed organ positioning). Why?
During early embryogenesis, rotating motile cilia at the embryonic node (a transient structure) generate a leftward directional fluid flow that concentrates Nodal (a TGF-β family morphogen) on the left side. Nodal then activates downstream left-specific gene expression (Lefty, Pitx2). When node cilia are immotile (as in PCD), flow is absent, and organ laterality is determined randomly — giving ~50% situs inversus and ~50% normal, explaining why only half of PCD patients have situs inversus.
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Part 2 complete. Review the summary, then tackle the end-of-chapter questions.
Chapter 16 at a glance
Actin treadmilling is driven by ATP hydrolysis: barbed-end (plus) assembly uses ATP-actin; pointed-end (minus) loss releases ADP-actin, which profilin recharges.
Arp2/3 (activated by WAVE/WASP) creates 70° branched networks in lamellipodia; formins nucleate long unbranched filaments for filopodia and stress fibers.
Myosin II power stroke is triggered by Pi release after the ADP+Pi-myosin head binds actin; ATP binding causes head detachment (rigor mortis = ATP-free locked heads).
Microtubule dynamic instability is controlled by the GTP cap; taxol stabilizes, colchicine/vincristine depolymerize — both block mitosis.
Kinesin-1 moves toward the plus end (anterograde); cytoplasmic dynein moves toward the minus end (retrograde); axonemal dynein powers ciliary bending.
Nuclear lamins (A/C, B) form the lamina; Cdk1 phosphorylation causes NEBD at mitosis; LMNA mutations cause laminopathies including progeria.
End-of-Chapter Questions
Work through all sections. Section B tests recall, Section C asks for analysis, and Section D gives immediate feedback on key terms.
Section B — Recall Questions (10)
B1
Explain actin filament polarity and why treadmilling occurs at steady state.
Sample answer: Actin filaments are polar: the barbed (plus) end has a lower critical concentration (~0.1 µM) than the pointed (minus) end (~0.7 µM). When free actin concentration lies between these values, net addition occurs at the barbed end and net loss at the pointed end simultaneously — this is treadmilling. ATP hydrolysis by incorporated actin subunits creates an ADP-actin "older" filament body that favors disassembly at the pointed end.
B2
Describe how Rac1 activation leads to lamellipodium formation through the Arp2/3 complex.
Sample answer: Active Rac1-GTP binds and activates the WAVE/Scar complex at the plasma membrane. WAVE then activates Arp2/3, which binds the side of an existing actin filament and nucleates a new filament at a 70° branch angle. Successive branching events generate a dendritic network of short actin filaments whose barbed ends push against the plasma membrane, extending the lamellipodium.
B3
Describe the four key steps of the myosin II cross-bridge cycle.
Sample answer: (1) ATP binding to myosin causes detachment from actin. (2) ATP hydrolysis (→ ADP+Pi) cocks the myosin head into a high-energy conformation. (3) The primed head binds actin weakly. (4) Pi release triggers the power stroke — the neck region swings ~10 nm, displacing the actin filament. ADP then releases, tightening the rigor bond until another ATP binds.
B4
Explain how calcium regulates skeletal muscle contraction via the troponin–tropomyosin system.
Sample answer: At rest, tropomyosin lies over myosin-binding sites on actin, blocking cross-bridge formation. Calcium released from the sarcoplasmic reticulum binds troponin C (TnC), causing a conformational change that displaces troponin I from actin and shifts tropomyosin to expose the myosin-binding sites. Myosin can now cycle, and the thin and thick filaments slide past each other (sliding filament model), shortening the sarcomere.
B5
What is dynamic instability and how does the GTP cap regulate it?
Sample answer: Dynamic instability is the stochastic switching of individual microtubule plus ends between slow polymerization and rapid catastrophic depolymerization. β-tubulin carries GTP when incorporated; hydrolysis to GDP weakens lateral contacts. As long as GTP-tubulin subunits cap the plus end, GDP-tubulin protofilaments are prevented from splaying. When hydrolysis outpaces addition, the GTP cap is lost, GDP-tubulin protofilaments curl outward, and catastrophe ensues. Rescue (return to growth) occurs when new GTP-tubulin is incorporated.
B6
Describe the role of the centrosome and γ-TuRC in organizing cellular microtubules.
Sample answer: The centrosome (MTOC in animal cells) consists of two centrioles surrounded by pericentriolar material (PCM). PCM contains γ-tubulin ring complexes (γ-TuRCs), which template the minus ends of microtubules and anchor them. Plus ends grow outward into the cytoplasm, creating a radially symmetric MT array. Before mitosis, the centrosome duplicates so that two centrosomes can form the poles of a bipolar mitotic spindle.
B7
Compare the directionality and cellular roles of kinesin-1 and cytoplasmic dynein in neurons.
Sample answer: Kinesin-1 moves toward the MT plus end (anterograde: soma → axon terminal), transporting synaptic vesicle precursors, mitochondria, and mRNA. Cytoplasmic dynein moves toward the minus end (retrograde: axon terminal → soma), carrying signaling endosomes (containing NGF-TrkA complexes), damaged organelles, and recycled membrane. Dynein requires dynactin and cargo adaptors (Hook, BICD2) for processivity. Loss of kinesin-1 or dynein causes axonal degeneration diseases.
B8
Describe the 9+2 axoneme structure of motile cilia and how dynein-driven sliding produces ciliary beating.
Sample answer: The motile cilium axoneme has nine outer doublets (A- and B-tubules) surrounding a central pair (9+2). Outer dynein arms extend from each A-tubule and walk along the B-tubule of the adjacent doublet. Because doublets are constrained at the base and cross-linked by nexin links, sliding is converted into coordinated bending. Cyclic ATP-driven dynein activity produces the whip-like beat that propels mucus, eggs, and (in sperm flagella) the sperm cell itself.
B9
How do nuclear lamins contribute to nuclear architecture, and what happens to them at mitotic entry?
Sample answer: Lamin A/C and lamin B form the nuclear lamina — a meshwork beneath the inner nuclear envelope that provides mechanical rigidity, organizes chromatin, and tethers nuclear pore complexes. At mitosis onset, Cdk1/cyclin B phosphorylates specific serine residues in lamin tails, disrupting head-to-tail polymerization. The lamina depolymerizes, causing nuclear envelope breakdown (NEBD), which allows spindle MTs access to kinetochores. After anaphase, Cdk1 inactivation permits dephosphorylation and lamina reassembly around the daughter nuclei.
B10
How do intermediate filaments differ from actin filaments and microtubules in structure, polarity, and function?
Sample answer: Intermediate filaments (~10 nm) are non-polar (subunits are antiparallel), unlike polar actin (6 nm) and microtubules (25 nm). IFs do not use nucleotide hydrolysis for dynamics and do not support motor protein movement. They are highly tissue-specific (keratins in epithelia, vimentin in fibroblasts, GFAP in astrocytes, desmin in muscle, neurofilaments in neurons). Their primary function is mechanical — they resist tensile stress and anchor cell junctions. Mutations in keratin genes cause skin fragility (epidermolysis bullosa); LMNA mutations cause laminopathies.
Section C — Critical Thinking (5)
C1
Cofilin severs ADP-actin filaments near the pointed end, generating new barbed ends. Explain how cofilin activity paradoxically accelerates cell migration rather than simply depolymerizing the actin network.
Sample answer: Cofilin severs older ADP-actin filaments, generating new free barbed ends that are immediately available for polymerization. Because the barbed end has a much lower critical concentration than free monomer levels, rapid polymerization occurs at these new ends, pushing the membrane forward. Simultaneously, monomer recycled from cofilin-mediated depolymerization is recharged to ATP-actin by profilin and fed back to the growing barbed ends. Cofilin thereby amplifies the number of productive growing ends, accelerating protrusion-driven migration rather than simply collapsing the network.
C2
Taxol stabilizes microtubules while vincristine promotes depolymerization — yet both drugs block cell division. Explain the shared mechanism and why MT dynamics are essential for mitosis.
Sample answer: Both drugs prevent dynamic instability — the rapid growth/catastrophe switching that allows spindle MTs to search for and capture kinetochores. Taxol freezes MTs in the polymerized state; vincristine promotes depolymerization. In both cases, kinetochore MTs cannot generate the tension needed for satisfying the spindle assembly checkpoint (SAC). The SAC (via Mad2/BubR1/MCC) keeps APC/C inactive, arresting cells in mitosis and triggering apoptosis. Cancer cells, which divide frequently, are disproportionately affected; post-mitotic neurons are secondarily harmed because they require axonal MT dynamics for transport.
C3
Loss-of-function mutations in cytoplasmic dynein or dynactin cause motor neuron disease in humans. Propose two mechanisms by which impaired retrograde axonal transport leads to neurodegeneration.
Sample answer: (1) Trophic signal deprivation: neurons depend on retrograde transport of NGF/BDNF-receptor signaling endosomes from the axon terminal to the soma to promote survival gene expression. Impaired dynein blocks this signal, and neurons fail to receive survival cues, triggering apoptosis by a "dying-back" mechanism. (2) Proteotoxic stress: damaged organelles (mitochondria, protein aggregates) normally are transported retrogradely to the soma for proteasomal or lysosomal degradation. When dynein is impaired, misfolded proteins and damaged organelles accumulate in the axon, causing local proteotoxic stress, MT disruption, and eventual axonal degeneration.
C4
Hutchinson–Gilford progeria is caused by a single point mutation activating a cryptic splice site in LMNA. Explain why this "toxic gain-of-function" causes premature aging rather than simply losing lamin A.
Sample answer: Normal lamin A is transiently farnesylated to facilitate nuclear import; ZMPSTE24 then cleaves the farnesyl group for mature lamin A. The G608G mutation removes 50 amino acids including the ZMPSTE24 cleavage site, producing "progerin" — permanently farnesylated lamin A that is anchored in the inner nuclear membrane and cannot be released into the lamina network properly. Progerin disrupts lamina architecture, impairs heterochromatin organization (loss of H3K27me3), accumulates DNA damage, and activates premature senescence. This is a dominant toxic gain-of-function: one progerin allele damages nuclei even when the other allele produces normal lamin A — explaining why simple loss of lamin A (LMNA deletion) has different, milder phenotypes (e.g., dilated cardiomyopathy without accelerated aging).
C5
Only ~50% of patients with primary ciliary dyskinesia have situs inversus. Explain this statistic in terms of the developmental role of node cilia in left–right axis establishment.
Sample answer: Node cilia normally rotate clockwise, generating a leftward fluid flow that directs Nodal protein to the left side of the embryo, where it activates Lefty and Pitx2 — specifying left identity. In PCD, node cilia are immotile so no directional flow is generated, and Nodal distribution becomes random (stochastic). Approximately 50% of PCD embryos will randomly place organs on the left (normal = situs solitus) and ~50% on the right (situs inversus). This 50:50 distribution is the expected outcome of random chance, not a penetrant inverted phenotype — which is why approximately half of PCD patients have situs inversus rather than all of them.
Section D — Interactive Questions (5)
D1
What multi-subunit complex nucleates branched actin filaments in lamellipodia? (two-word answer)
D2
What term describes the rapid depolymerization phase of a microtubule after loss of its GTP cap? (one word)
D3
Which motor protein powers retrograde axonal transport (axon terminal → soma)? (one word)
D4
Which calcium-binding troponin subunit directly senses Ca²⁺ to initiate muscle contraction? (two words)
D5
What intermediate filament protein is characteristically expressed in astrocytes? (abbreviation, 4 letters)