Premed · Premed · Cell Biology
Lecture 19: Cell Motility and Motor Proteins
Cell Biology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the structure and mechanism of myosin, kinesin, and dynein motor proteins
- Explain the molecular basis of muscle contraction (sliding filament model)
- Describe the steps and mechanisms of cell crawling
- Explain how intracellular transport is mediated by motor proteins on microtubules
- Discuss the regulation and coordination of motor protein activity
Lecture Content
I. Overview of Molecular Motors
Motor proteins are remarkable molecular machines that convert the chemical energy of ATP hydrolysis into directed mechanical work. Three major superfamilies of cytoskeletal motor proteins exist. Myosins move along actin filaments, generally toward the plus (barbed) end. Kinesins move along microtubules, with most family members traveling toward the plus end. Dyneins move along microtubules toward the minus end.
All motor proteins share a common structural organization: a motor (head) domain that binds the cytoskeletal track and hydrolyzes ATP, a neck or lever arm that amplifies the conformational changes produced by the ATPase cycle, and a tail domain that binds cargo such as organelles, vesicles, or other filaments. Processivity -- the ability to take multiple consecutive steps without detaching from the track -- varies among motor types and is critical for long-distance transport. Each step in the ATPase cycle is tightly coupled to a specific conformational change that produces movement, a principle known as mechanochemical coupling.
II. Myosin Motors
The myosin superfamily encompasses over 35 classes. Myosin II (conventional myosin) is responsible for muscle contraction and cytokinesis. Each myosin II molecule has two heavy chains (each with a globular motor domain, a lever arm, and a coiled-coil tail) plus two essential light chains and two regulatory light chains that wrap around the neck region. The tail domains associate to form bipolar thick filaments in muscle. Non-muscle myosin II generates contractile forces in stress fibers, the contractile ring during cytokinesis, and the cell cortex, regulated by phosphorylation of the regulatory light chain by MLCK and ROCK.
Myosin V is a processive, two-headed motor that walks along actin using a hand-over-hand mechanism with a step size of approximately 36 nm, matching the actin helical repeat. It transports vesicles, organelles, and mRNA in the cytoplasm. In melanocytes, it is critical for melanosome transport, and mutations cause Griscelli syndrome (pigmentation defects and immunodeficiency). Myosin I is a single-headed, non-processive motor that links actin to membranes and participates in endocytosis and membrane tension regulation. Myosin VI is unique in that it moves toward the minus (pointed) end of actin and is involved in endocytosis and Golgi organization.
<image>Myosin motor protein structures and mechanisms. Panel A: Domain structure of myosin II — two heavy chains with globular head (motor domain containing actin-binding site and ATP-binding pocket), neck/lever arm region with essential and regulatory light chains, and alpha-helical coiled-coil tail. Panel B: Myosin V hand-over-hand processive walking along an actin filament — two heads alternately bind actin, with the trailing head swinging forward ~36 nm per step (matching the actin helical repeat). Cargo vesicle attached to the tail via adaptor proteins. Panel C: The myosin II ATPase cycle (cross-bridge cycle) — (1) rigor state (no nucleotide, head tightly bound to actin); (2) ATP binding causes head release; (3) ATP hydrolysis cocks the head (recovery stroke); (4) head rebinds actin (weak then strong binding, Pi release triggers power stroke); (5) ADP release returns to rigor state.</image>
III. Muscle Contraction: The Sliding Filament Model
The sarcomere is the fundamental contractile unit of striated muscle, bounded by Z-discs at each end. Thin filaments consist of actin decorated with tropomyosin and the troponin complex, with their plus ends anchored at the Z-disc by alpha-actinin. Thick filaments are bipolar assemblies of myosin II, with a central bare zone and myosin heads projecting outward. Titin, a giant elastic protein, connects the thick filaments to the Z-discs and acts as a molecular spring. The characteristic banding pattern reflects the overlapping arrangement: the A band (region of thick filaments, constant length during contraction), the I band (thin filaments only, shortens during contraction), the H zone (central thick filaments only, shortens during contraction), and the M line (center of the sarcomere, cross-links thick filaments).
The sliding filament mechanism operates through cyclic interactions between myosin heads (cross-bridges) and actin thin filaments. During the power stroke, myosin pulls the thin filaments toward the M line. As a result, thin filaments slide past thick filaments and the sarcomere shortens. Critically, neither the thin nor the thick filaments themselves change length.
Excitation-contraction coupling translates the electrical signal into mechanical contraction. An action potential at the neuromuscular junction triggers ACh release, which depolarizes the muscle fiber. This depolarization spreads via T-tubules into the fiber interior. The voltage sensor (DHPR/L-type Ca2+ channel) on the T-tubule activates the ryanodine receptor (RyR) on the sarcoplasmic reticulum, releasing Ca2+ into the cytoplasm. Ca2+ binds troponin C, inducing a conformational change in the troponin complex that shifts tropomyosin on the actin filament, exposing myosin-binding sites. Cross-bridge cycling begins. Relaxation occurs when SERCA pumps Ca2+ back into the SR, tropomyosin returns to its blocking position, and cross-bridge cycling ceases.
<image>Muscle sarcomere structure and the sliding filament model. Panel A: Sarcomere structure — Z-discs at each end, thin filaments (actin + tropomyosin + troponin) extending inward, thick filaments (myosin II bipolar assemblies) in the center, titin spanning from Z-disc to M-line. Bands labeled: A band (thick filament region), I band (thin filament only region), H zone (thick filament only in center), M line. Panel B: Sliding filament mechanism during contraction — sarcomere at rest (top) and contracted (bottom); I band and H zone shorten, A band stays constant, thin filaments slide inward past thick filaments. Panel C: Ca2+ regulation of contraction — in relaxed state, tropomyosin covers myosin-binding sites on actin; Ca2+ binds troponin C, shifting tropomyosin to expose the binding sites; myosin heads engage actin for cross-bridge cycling.</image>
IV. Kinesin and Dynein: Microtubule-Based Motors
Kinesin-1 (conventional kinesin) is a plus-end-directed motor that moves cargo toward the cell periphery in most cells. It consists of two heavy chains, each with an N-terminal motor domain, a neck linker, a coiled-coil stalk, and a tail that binds cargo via light chains and adaptors. Kinesin-1 is highly processive, taking approximately 100 steps before detaching, using a hand-over-hand mechanism with a step size of 8 nm (one tubulin dimer along the protofilament) at a speed of roughly 1 micrometer per second. It transports vesicles, mitochondria, mRNA, and signaling complexes. Other kinesin families include kinesin-5 (Eg5, which separates spindle poles during mitosis), kinesin-13 (MCAK, a microtubule depolymerizer), and kinesin-14 (which moves toward the minus end).
Cytoplasmic dynein is a minus-end-directed motor that moves cargo toward the cell center. It is a massive complex (approximately 1.2 MDa) with two heavy chains containing AAA+ ATPase domains arranged in a ring, plus intermediate, light intermediate, and light chains. Dynein requires the dynactin complex (featuring p150Glued and an Arp1 mini-filament) for processivity and cargo binding. The dynein-dynactin complex is activated by cargo-specific adaptors such as BICD2 and Hook3. Cytoplasmic dynein is responsible for retrograde transport of vesicles and organelles, Golgi positioning near the centrosome, mitotic spindle positioning and chromosome movement, and retrograde axonal transport in neurons.
V. Intracellular Transport
Neurons provide the most dramatic illustration of motor protein-dependent transport because their axons can extend up to a meter from the cell body. Anterograde transport (cell body to synapse) is carried out by kinesin along axonal microtubules, whose plus ends point distally. Kinesin carries vesicles, mitochondria, and synaptic precursors. Retrograde transport (synapse to cell body) is mediated by dynein, carrying signaling endosomes, autophagosomes, and neurotrophic factor signals. Fast axonal transport operates at 1 to 5 micrometers per second for vesicles and organelles, while slow axonal transport (0.2 to 10 mm per day for cytoskeletal proteins and enzymes) actually consists of short bursts of fast transport interspersed with pauses.
Organelle positioning throughout the cell depends on motor proteins. ER tubules are extended along microtubules by kinesin-1. The Golgi is maintained near the centrosome by dynein. Mitochondrial distribution involves both kinesin and dynein, with the Miro/Milton adaptor complex sensing local Ca2+ to arrest mitochondria at sites where energy demand is high.
Cargo selectivity is achieved through motor-cargo linkage via adaptor proteins. Rab GTPases on vesicle membranes recruit specific motor adaptors. Multiple motors can be simultaneously attached to the same cargo, with their activities coordinated through tug-of-war or cooperative mechanisms.
<image>Intracellular transport by motor proteins. Panel A: Neuron schematic — cell body (left) with nucleus, axon extending rightward, synapse (right). Microtubules with plus ends pointing distally. Kinesin-1 (blue) carrying vesicles anterograde (toward synapse); cytoplasmic dynein (red) carrying endosomes/signaling complexes retrograde (toward cell body). Panel B: Motor-cargo linkage — Rab GTPase on vesicle membrane recruiting a specific adaptor protein that connects to either kinesin or dynein-dynactin. Panel C: Organelle positioning — ER network extended by kinesin along microtubules; Golgi held near the centrosome by dynein; mitochondria distributed by both motors with Miro/Milton sensing local calcium to stop mitochondria where energy demand is high.</image>
VI. Cell Crawling and Amoeboid Movement
Cell crawling on a substrate, as performed by fibroblasts, epithelial cells, and immune cells, follows a coordinated four-step cycle. Protrusion of the leading edge is driven by Arp2/3-mediated branched actin polymerization pushing the membrane forward, with Rac and Cdc42 activated at the leading edge. Adhesion follows as new focal contacts form between integrins on the protruded membrane and ECM proteins, recruiting talin, vinculin, and paxillin to create focal complexes that mature into focal adhesions. Traction is generated by myosin II-driven contraction of actin stress fibers, activated through the RhoA/ROCK pathway. Retraction of the cell rear occurs as trailing adhesions disassemble (aided by calpain protease) and the cell tail detaches, allowing the cell body to move forward.
Chemotaxis, the directed migration toward a chemical attractant, involves sensing of extracellular gradients through surface receptors (GPCRs or RTKs). Internal amplification mechanisms establish front-rear polarity: PI3K produces PIP3 at the leading edge to recruit GEFs for Rac, while PTEN at the rear degrades PIP3 to maintain polarity. Positive feedback loops reinforce this polarization.
Amoeboid movement is a faster, more rounded mode of migration that does not require strong substrate adhesions. Used by leukocytes, Dictyostelium, and some cancer cells, it relies on cortical actin contraction and bleb-based protrusion. Because it is less dependent on integrins, amoeboid movement allows rapid navigation through three-dimensional tissue environments.
<image>Cell crawling mechanism. Panel A: Four-step cycle of cell crawling — (1) protrusion of the leading edge by actin polymerization (Arp2/3-driven branched network pushing the membrane forward), (2) new integrin-based adhesions forming at the front (focal complexes shown as green dots), (3) myosin II-mediated contraction of the cell body generating traction force (stress fibers shown with contractile arrows), (4) retraction of the trailing edge as rear adhesions disassemble. Panel B: Signaling polarity in a chemotaxing cell — gradient of chemoattractant (high concentration at top), PI3K and PIP3 enriched at the leading edge (blue), PTEN and myosin II enriched at the rear (red), Rac active at front, Rho active at rear.</image>
