# Lecture 17: The Cytoskeleton: Microtubules
## Cell Biology
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## Learning Objectives
By the end of this lecture, students will be able to:
1. Describe the structure, assembly, and polarity of microtubules
2. Explain dynamic instability and its regulation
3. Describe the organization of microtubules by the centrosome and MTOC
4. Explain the structure and function of cilia and flagella
5. Discuss the mechanism of action of microtubule-targeting drugs
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## Lecture Content
### I. Microtubule Structure
Microtubules are the largest cytoskeletal elements, forming hollow cylindrical polymers with an outer diameter of approximately 25 nm and an inner lumen of about 15 nm. They are composed of **tubulin heterodimers**, each consisting of one alpha-tubulin and one beta-tubulin subunit (each approximately 55 kDa). Both alpha- and beta-tubulin bind GTP, but only beta-tubulin hydrolyzes its GTP after the heterodimer is incorporated into the polymer. The heterodimers align head-to-tail to form linear **protofilaments**, and **13 protofilaments** arranged in parallel constitute the microtubule wall.
Like actin filaments, microtubules are **polar** structures. The **plus end**, where beta-tubulin is exposed, is the faster-growing end. The **minus end**, where alpha-tubulin is exposed, grows more slowly and is often anchored at a microtubule-organizing center (MTOC). Because all protofilaments are oriented identically, the entire microtubule has a consistent structural polarity.
Microtubules undergo several **post-translational modifications** that regulate their interactions with motor proteins and MAPs. These include acetylation of alpha-tubulin at Lys-40 (which marks stable, long-lived microtubules), tyrosination and detyrosination of the alpha-tubulin C-terminus, and polyglutamylation and polyglycylation. Together, these modifications create a "tubulin code" that fine-tunes microtubule function. **Gamma-tubulin** is found at the centrosome and plays a unique role in microtubule nucleation. The gamma-tubulin ring complex (gamma-TuRC), composed of 13 gamma-tubulins arranged in a ring, serves as a template for the 13-protofilament arrangement.
### II. Dynamic Instability
Microtubules exhibit a remarkable behavior called **dynamic instability**, in which individual microtubules stochastically switch between phases of growth and rapid shrinkage. This behavior is explained by the **GTP cap model**. GTP-tubulin dimers add to the plus end, and after incorporation, beta-tubulin hydrolyzes GTP to GDP with a characteristic delay. A growing microtubule therefore maintains a **GTP cap** at its plus end, which stabilizes the straight conformation of the protofilaments. If GTP hydrolysis catches up to the tip and the GTP cap is lost, the GDP-tubulin protofilaments, which have an inherently curved conformation that is strained in the straight lattice, peel outward. The result is rapid depolymerization, a transition called **catastrophe**.
The key parameters of dynamic instability include the **growth rate** (approximately 1 to 2 micrometers per minute), the much faster **shrinkage rate** (approximately 10 to 30 micrometers per minute), **catastrophe** (the transition from growth to shrinkage), **rescue** (the transition from shrinkage back to growth, when a new GTP cap is re-established), and **pause** (a state of neither growth nor shrinkage). Dynamic instability allows microtubules to rapidly explore the cell interior through a "search and capture" mechanism in which growing microtubules probe for targets such as kinetochores and the cell cortex. Unlike actin, which has different critical concentrations at its two ends, microtubules have a single critical concentration because dynamics are dominated by the plus end.
Microtubule dynamic instability. Panel A: Microtubule structure — 13 protofilaments forming a hollow tube, alpha/beta-tubulin heterodimers shown with GTP (green) on beta-tubulin at the plus end (GTP cap) and GDP (red) in the older lattice. Plus end (beta-tubulin exposed) and minus end (alpha-tubulin exposed) labeled. Panel B: Dynamic instability cycle — growing microtubule with GTP cap (straight protofilaments); loss of GTP cap triggers catastrophe (protofilaments curl outward, rapid depolymerization); rescue re-establishes GTP cap and resumes growth. Panel C: Graph showing length of a single microtubule over time with abrupt switches between growth (gradual increase) and shrinkage (rapid decrease), with catastrophe and rescue events labeled.
### III. Microtubule-Organizing Centers and the Centrosome
The **centrosome** is the primary MTOC in animal cells. Located near the nucleus, it organizes both the interphase microtubule array and the mitotic spindle. Each centrosome contains two **centrioles**, barrel-shaped structures composed of nine triplet microtubules in a 9+0 arrangement, oriented perpendicular to each other (mother and daughter centriole). The mother centriole bears subdistal and distal appendages. Surrounding the centrioles is the **pericentriolar material (PCM)**, an amorphous matrix containing gamma-TuRC complexes that nucleate microtubules, along with scaffolding proteins such as pericentrin and CDK5RAP2. Microtubule minus ends are anchored at the centrosome, with plus ends radiating outward. The centrosome duplicates once per cell cycle during S phase through centriole duplication.
Other MTOCs include **basal bodies** (modified centrioles at the base of cilia and flagella), the **spindle pole body** in yeast (embedded in the nuclear envelope), and distributed gamma-tubulin-dependent nucleation sites in plant cells (which lack centrosomes). The **Golgi apparatus** also serves as a secondary MTOC in many cell types, with proteins like CLASP and AKAP450 recruiting gamma-TuRC to Golgi membranes.
### IV. Microtubule-Associated Proteins (MAPs)
**Stabilizing MAPs** bind along microtubules and reduce dynamic instability. **MAP2** is specific to dendrites, where it cross-links and stabilizes microtubules. **Tau** is specific to axons and promotes microtubule assembly and stability. Hyperphosphorylated tau detaches from microtubules and forms neurofibrillary tangles, a pathological hallmark of Alzheimer's disease and other tauopathies including frontotemporal dementia and progressive supranuclear palsy. **MAP4** is the ubiquitous non-neuronal stabilizing MAP.
**Plus-end tracking proteins (+TIPs)** specifically associate with growing microtubule plus ends. **EB1** autonomously tracks growing plus ends and serves as a platform for recruiting other +TIPs. **CLIP-170** links plus ends to other cellular structures. **CLASP** regulates microtubule dynamics at the cell cortex. **APC**, in addition to its role in Wnt signaling, tracks microtubule plus ends. These +TIPs collectively regulate microtubule behavior at the cell cortex, kinetochore capture during mitosis, and cell migration.
**Destabilizing factors** promote microtubule disassembly. **Stathmin/Op18** sequesters free tubulin dimers and promotes catastrophe. **Katanin** is an AAA-ATPase that severs microtubules, releasing them from the centrosome and generating new plus ends. **Kinesin-13 (MCAK)** is unusual among kinesins in that it does not walk along microtubules but instead uses ATP hydrolysis to peel protofilaments from microtubule ends, promoting depolymerization.
### V. Cilia and Flagella
Cilia and flagella are microtubule-based cellular projections that serve motile and sensory functions. **Motile cilia and flagella** share a core structure called the **axoneme**, organized in a 9+2 arrangement: nine outer doublet microtubules (each consisting of a complete 13-protofilament A-tubule and an incomplete 10-protofilament B-tubule) surround two central singlet microtubules (the central pair). **Dynein arms** (outer and inner) on the A-tubule generate the sliding force that bends the cilium. **Nexin links** between adjacent doublets convert this sliding motion into bending. **Radial spokes** connect the outer doublets to the central pair and help regulate dynein activity. Each cilium or flagellum is anchored by a **basal body** with a 9+0 triplet microtubule arrangement identical to a centriole. Cilia beat in coordinated wave-like patterns, while flagella produce sinusoidal waves.
**Primary ciliary dyskinesia (Kartagener syndrome)** results from mutations in dynein arm genes (such as DNAH5 and DNAI1), producing immotile cilia. Patients suffer from chronic sinusitis, bronchiectasis, and infertility. Approximately half have situs inversus (reversal of left-right body asymmetry) because nodal cilia, which normally establish left-right patterning during embryonic development, are also affected.
**Primary (non-motile) cilia** have a 9+0 axoneme (no central pair, no dynein arms) and are present as a single cilium on nearly all mammalian cells. They function as sensory antennae, concentrating signaling receptors on their surface. Notably, **Hedgehog signaling** occurs at the primary cilium, with Smoothened, Patched, and Gli proteins all localizing there. **Intraflagellar transport (IFT)** is essential for building and maintaining cilia. The IFT-B complex and kinesin-2 mediate anterograde transport (base to tip), while the IFT-A complex and cytoplasmic dynein 2 drive retrograde transport (tip to base). Defects in cilia cause **ciliopathies** including polycystic kidney disease (involving the PC1/PC2 mechanosensory complex on the primary cilium), Bardet-Biedl syndrome (involving BBSome proteins that serve as IFT cargo adaptors), and Joubert syndrome (cerebellar vermis hypoplasia).
Structure of motile cilia and primary cilia. Panel A: Cross-section of a motile cilium showing the 9+2 axoneme — 9 outer doublet microtubules (A and B tubules labeled), 2 central singlet microtubules, outer and inner dynein arms on A-tubule, nexin links between doublets, and radial spokes connecting to the central pair. Basal body (9+0 triplet) shown at the base. Panel B: Diagram of dynein-driven ciliary bending — dynein on one doublet walks toward the minus end of the adjacent doublet, but nexin links constrain sliding so the force is converted to bending. Panel C: Primary cilium (9+0) on a cell surface with IFT particles — kinesin-2 carrying IFT-B (anterograde, green arrows) and cytoplasmic dynein 2 carrying IFT-A (retrograde, red arrows). Hedgehog signaling components (Patched, Smoothened) shown localized to the ciliary membrane.
### VI. Microtubule-Targeting Drugs
Microtubule-targeting drugs represent a major class of anticancer agents, exploiting the absolute dependence of mitotic cell division on microtubule dynamics. **Microtubule-stabilizing agents** include **taxol (paclitaxel)**, which binds beta-tubulin on the inner surface of the microtubule, preventing depolymerization. By blocking spindle dynamics, taxol causes mitotic arrest and ultimately apoptosis. Originally isolated from the bark of the Pacific yew tree (*Taxus brevifolia*), it is used for breast, ovarian, and lung cancer. **Epothilones** share a similar mechanism and are effective against taxol-resistant tumors.
**Microtubule-destabilizing agents** include **colchicine**, which binds free tubulin dimers and prevents polymerization (used clinically for gout to reduce neutrophil migration, but too toxic for anticancer use at required doses); **vinca alkaloids** (vincristine, vinblastine), which bind at the interface between tubulin dimers to prevent polymerization and promote depolymerization (used for leukemias, lymphomas, and solid tumors); and **nocodazole**, a reversible depolymerizing agent widely used in experimental settings.
All of these drugs arrest cells in mitosis because they prevent proper spindle dynamics. The spindle assembly checkpoint detects the resulting unattached kinetochores and enforces a mitotic arrest, which eventually triggers apoptosis.
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