04 — Kinesin, Dynein, and Intracellular Transport

Prerequisite Concepts

• Microtubule structure, polarity, and dynamic instability (Topic 03)
• ATP hydrolysis and mechanochemical coupling (Topic 02 — myosin)
• Basic cell organization: ER, Golgi, endosomes, lysosomes

Core Explanation

Kinesin-1: The Prototypical Plus-End Motor

Kinesin-1 (conventional kinesin) is a tetramer of two heavy chains (motor domains + stalk + tail) and two light chains. Each heavy chain has a globular motor domain at the N-terminus that binds microtubules and ATP.

Mechanochemical Cycle (Hand-Over-Hand Walking)

Kinesin-1 is processive — it takes ~100 steps along a microtubule before dissociating, enabling single-motor transport. The cycle:

1. Leading head (ADP-bound) binds the microtubule → ADP release.
2. ADP release triggers the trailing head's neck linker to swing forward (the "zippering" of the neck linker onto the motor domain), throwing the trailing head ~16 nm forward — exactly two tubulin dimers.
3. The new leading head binds ATP; the cycle repeats.

Step size: 8 nm (one tubulin dimer) per motor domain cycle, but because the motor is dimeric and walks hand-over-hand, the center of mass moves 16 nm per ATP hydrolyzed (two 8-nm steps). Each step consumes one ATP.

Directionality: Kinesin vs Dynein

Most kinesins (kinesin-1 through kinesin-12) are plus-end-directed — they walk toward the cell periphery. However, the kinesin-14 family (including Ncd in Drosophila and HSET in humans) has a C-terminal motor domain and walks toward the minus end. Always say "most kinesins" when discussing directionality.

Cytoplasmic dynein (dynein-1) is the major minus-end-directed motor — it walks toward the centrosome/cell center. A second isoform, dynein-2, powers intraflagellar transport in cilia.

Cytoplasmic Dynein and the Dynactin Complex

Cytoplasmic dynein is a massive complex (~1.5 MDa):

• Heavy chains: Motor domain (AAA+ ATPase ring) with the microtubule-binding stalk.
• Intermediate chains, light intermediate chains, light chains: Structural and cargo-binding roles.

Dynein's mechanism differs fundamentally from kinesin's. The motor domain is an AAA+ ATPase ring; ATP hydrolysis in the ring drives a linker domain swing, which is amplified by the stalk into a ~8–32 nm step toward the minus end. Dynein can take variable step sizes — it is less stereotypic than kinesin.

Dynactin is an essential cofactor — a large, multi-subunit complex that:

• Increases dynein processivity (dynein alone is weakly processive).
• Links dynein to cargo via cargo adaptors (e.g., BICD2, Hook3, Spindly).
• Contains a short actin-like filament (Arp1) that participates in cargo binding.

Biological Roles of Microtubule Motors

Axonal Transport

Neurons face an extreme logistics challenge: axons can be >1 meter long, yet protein synthesis is largely restricted to the cell body. Anterograde transport (cell body → synapse) is driven by kinesins (kinesin-1, kinesin-3); retrograde transport (synapse → cell body) is driven by cytoplasmic dynein. Defects in axonal transport cause neurodegeneration — mutations in kinesin (KIF5A) and dynactin (p150^Glued^) are linked to ALS and hereditary spastic paraplegia.

Organelle Positioning

• ER: Kinesin-1 pulls ER tubules toward the periphery; dynein pulls them inward. This tug-of-war shapes the ER network.
• Golgi: Dynein pulls the Golgi toward the centrosome (perinuclear positioning). Blocking dynein disperses the Golgi throughout the cytoplasm.
• Mitochondria: Both kinesin-1 (via Miro/Milton adaptors) and dynein position mitochondria according to local energy demands.
• Endosomes/Lysosomes: Dynein drives late endosome and lysosome movement toward the perinuclear region for lysosomal degradation.

Mitotic Spindle Roles

• Kinesin-5 (Eg5): Bipolar, plus-end-directed — slides antiparallel microtubules apart at the spindle midzone, driving spindle pole separation. Eg5 is a cancer drug target (monastrol, ispinesib).
• Kinesin-13 (MCAK): A non-motile kinesin that depolymerizes microtubule ends (catastrophe factor) — regulates spindle microtubule dynamics.
• Kinesin-7 (CENP-E): Carries chromosomes to the metaphase plate.
• Cytoplasmic dynein: Anchors at the cell cortex and pulls on astral microtubules to position the spindle; also focuses spindle poles.

Cilia and Flagella

Motile Cilia: The 9+2 Axoneme

Motile cilia and flagella (the terms are structurally equivalent; flagella are longer) contain a core axoneme with the "9+2" arrangement:

• 9 outer doublet microtubules (A tubule: 13 protofilaments, complete; B tubule: 10 protofilaments, incomplete, fused to A).
• 2 central singlet microtubules with a central pair complex.

Axonemal dynein (distinct from cytoplasmic dynein) forms arms projecting from the A tubule of each doublet toward the B tubule of the adjacent doublet. ATP-driven dynein power strokes produce sliding between adjacent doublets. Because doublets are anchored by nexin links and radial spokes connect to the central pair, sliding is converted into bending.

Key components:

• Outer dynein arms: Increase beat frequency.
• Inner dynein arms: Control waveform (bend shape).
• Radial spokes: Transmit signals from the central pair to the dynein arms.
• Nexin links: Resist sliding, converting it to bending.

Primary Cilia: 9+0 and Sensory Signaling

Primary cilia have a "9+0" axoneme (9 outer doublets, no central pair, no dynein arms) and are non-motile (with rare exceptions — nodal cilia are 9+0 but motile via dynein). They function as sensory antennae:

• Enriched in receptors: Hedgehog, Wnt, PDGFRα, and GPCRs.
• Hedgehog signaling in vertebrates depends absolutely on primary cilia — Smoothened accumulates in the cilium upon pathway activation, and Gli transcription factors are processed within the cilium.
• Ciliopathies (polycystic kidney disease, Bardet-Biedl syndrome, Joubert syndrome) result from defects in ciliary assembly (intraflagellar transport) or signaling components.
• Intraflagellar transport (IFT): Kinesin-2 drives anterograde IFT toward the ciliary tip; dynein-2 drives retrograde IFT back to the base.

Disease Connections

• Hereditary spastic paraplegia: Mutations in kinesin (KIF5A) impair axonal transport in corticospinal tract neurons.
• ALS: Mutations in dynactin (p150^Glued^) disrupt retrograde transport.
• Primary ciliary dyskinesia (Kartagener syndrome): Dynein arm defects → immotile cilia → chronic respiratory infections, situs inversus (nodal cilia fail to establish left-right asymmetry).
• Polycystic kidney disease: Defective primary cilia signaling in renal tubule epithelia → cyst formation.
• Retinitis pigmentosa: IFT mutations cause photoreceptor degeneration (the connecting cilium is a modified primary cilium).

Common Misconceptions and Exam Traps

• Wrong: "All kinesins are plus-end-directed." Correct: The kinesin-14 family (Ncd, HSET) are minus-end-directed. Say "most kinesins."
• Wrong: "Dynein walks toward the plus end." Correct: Cytoplasmic dynein is minus-end-directed (retrograde transport).
• Wrong: "Primary cilia are just vestigial organelles." Correct: Primary cilia are active signaling hubs essential for Hedgehog, Wnt, and other developmental pathways.
• Wrong: "Cilia and flagella are different structures." Correct: They share the same core axoneme (9+2); cilia are shorter and more numerous; flagella are longer and typically singular or paired. The terms are descriptive, not structurally distinct.