BC 465 Lecture 7

Introductory Context

• Speaker: Matt, a dedicated 5th-year Ph.D. student at Colorado State University (CSU), actively collaborating with Dr. Jake DeLuca's research group.

• Goal of lecture: To provide a comprehensive review of the fundamental principles governing microtubule motors, specifically kinesin and dynein, with a strong emphasis on their intricate molecular structure, precise mechanistic cycles, complex regulatory mechanisms, supporting experimental evidence, and significant biomedical relevance.

Microtubule Fundamentals (Quick Review)

• Microtubules (MTs) are dynamic filamentous polymers composed of repeating heterodimers of α-tubulin and β-tubulin. These heterodimers assemble head-to-tail to form protofilaments, and typically 13 protofilaments associate laterally to form a hollow tubular structure.

• Plus (+) end: Characterized by exposed β-tubulin, this end exhibits rapid growth (polymerization) and shrinkage (depolymerization), a phenomenon known as dynamic instability. It is often oriented towards the cell periphery.

• Minus (–) end: Typically composed of exposed α-tubulin, this end is generally less dynamic and is often anchored in microtubule-organizing centers (MTOCs) such as the centrosome in animal cells.

• MTs are inherently polar due to the asymmetric nature of the α/β-tubulin dimer and their head-to-tail assembly; this polarity is crucial as it dictates the directionality of motor protein movement.

  • Minus-end–directed motor ⇒ Cytoplasmic dynein is the primary motor moving towards the minus end of MTs, essential for retrograde transport.

  • Plus-end–directed motor ⇒ The vast majority of kinesin motor proteins move towards the plus end (anterograde transport), although a small subset of kinesins are known to be minus-end directed.

Motor-Family Overview

• Dynein:

  • Only one major cytoplasmic isoform (Dynein-1) exists in most eukaryotic cells, yet it is responsible for performing a remarkably diverse array of cellular tasks, highlighting its adaptability and reliance on cofactors.

  • Forms very large, multi-subunit complexes ranging from 24 to 48 distinct subunits, crucially interacting with essential cofactors such as dynactin, various adapter proteins, and regulatory factors that modulate its activity and cargo specificity.

• Kinesins:

  • Humans possess approximately 45 different kinesin genes (KIFs, or Kinesin Superfamily Proteins), each often specialized for distinct cellular functions, reflecting a higher degree of task specialization compared to dynein.

  • Naming conventions: Historically confusing, with classification by KIF number (e.g., KIF5) or kinesin family number (e.g., kinesin-1). Current understanding categorizes them into 14 families (Kinesin-1 through Kinesin-14) based on sequence homology and motor domain location.

  • Core architecture is highly conserved across the family, comprising a motor domain, neck linker, coiled-coil stalk, and tail domain. Key differences that dictate cargo specificity, processivity, and regulatory mechanisms primarily reside in the flexible stalk and diverse C-terminal tail domains.

Shared & Complementary Cellular Roles

• Organelle and vesicle trafficking: Kinesins typically mediate anterograde transport (away from the cell center, towards the plus end), while dynein handles retrograde transport (towards the cell center, towards the minus end), ensuring efficient bidirectional movement of cargo throughout the cell.

• Nuclear positioning & neuronal signaling: Both motors are crucial for precise positioning of the nucleus within various cell types, and in neurons, they facilitate long-distance signaling by transporting mRNA, proteins, and signaling endosomes.

• Endocytosis/exocytosis, Golgi/ER organization: Motors are vital for the continuous trafficking of vesicles involved in endocytosis and exocytosis, as well as maintaining the dynamic organization and intricate morphology of the Golgi apparatus and endoplasmic reticulum networks.

• Mitotic spindle assembly & chromosome congression: Kinesins (e.g., Kinesin-5, Kinesin-12) are critical for establishing and maintaining spindle pole separation and assembling the bipolar spindle, while dynein (e.g., kinetochore dynein) is involved in chromosome capture, congression, and movements during mitosis.

• Cytoskeletal remodeling: Beyond transport, specific kinesins and dynein isoforms actively modulate microtubule dynamics, including promoting polymerization, facilitating depolymerization, and cross-linking MTs to form bundles or networks.

Selected Kinesin Subtypes & Functions

• Kinesin-1 (KIF5, canonical kinesin): The classic example of a processive plus-end directed motor, primarily responsible for bulk anterograde transport of large organelles (e.g., mitochondria), vesicles, and protein complexes over long distances, particularly in axons.

• Kinesin-3 (KIF1): Known for its rapid movement, this family is specialized for fast axonal vesicle transport, including synaptic vesicle precursors, and is particularly enriched in neurons.

• Kinesin-5 (Eg5 in vertebrates, BimC in fungi): A homotetrameric kinesin motor with two motor domains at each end. It functions as a plus-end directed motor that slides antiparallel microtubules past each other, crucial for separating spindle poles during bipolar spindle formation in mitosis.

• Kinesin-13 (MCAK - Mitotic Centromere-Associated Kinesin): Unique among kinesins, Kinesin-13 lacks transport ability. Instead, its motor domain is optimized for ATP-dependent microtubule depolymerase activity, inducing catastrophes (rapid shortening) at microtubule ends, playing a key role in spindle dynamics and chromosome segregation.

• Note: Despite using microtubules as tracks, many kinesins are also integral modulators of microtubule dynamics, demonstrating their dual roles in transport and cytoskeletal regulation.

Canonical Kinesin Architecture

• Kinesin-1, a homodimer, serves as the prototype for understanding kinesin architecture:

  • N-terminal Motor/Head domain: This highly conserved globular domain contains the ATP-binding site (ATPase activity) and the microtubule-binding site. It converts chemical energy from ATP hydrolysis into mechanical force.

  • Neck linker: A short, flexible polypeptide segment immediately C-terminal to the motor domain. It acts as a critical mechanical lever, undergoing a conformational change (docking) upon ATP binding in the front head, which propels the rear head forward and defines the direction of the step.

  • Coiled-coil stalk: A stiff, rod-like region formed by the intertwining of two alpha-helices from the two monomeric units. This domain is responsible for dimerization and contributes significantly to the structural diversity between different kinesin isoforms.

  • C-terminal Tail domain: Highly variable in sequence and structure across different kinesin families. This domain is primarily responsible for cargo binding, either directly or through interactions with specific adapter proteins or other motors.

Kinesin Mechanochemical Cycle (Hand-over-Hand)

Kinesin-1 moves processively along a microtubule by a "hand-over-hand" mechanism, ensuring one head is always bound to the MT:

1. Initial state: One head (the rear or trailing head) is bound to the microtubule with bound $ext{ADP}$, exhibiting weak microtubule affinity. The other head (the front or leading head) is usually ATP-bound and strongly coupled to the MT.

2. ATP binding and neck linker docking: The leading head, previously weakly bound, now strongly binds ATP. This ATP binding induces a conformational change in that head, causing its adjacent neck linker to dock tightly onto the motor domain. This docking event swings the trailing head forward by approximately $16\, ext{nm}$, which corresponds precisely to the distance of two tubulin dimers along a protofilament.

3. Hydrolysis and weakening: The newly advanced rear head (now the trailing head) quickly hydrolyzes its bound ATP to $ext{ADP}+ ext{P<em>i}$. The release of inorganic phosphate ($ext{P</em>i}$) renders this head weak-binding to the microtubule, priming it for detachment and subsequent forward movement.

4. ADP release and strong binding: Concurrently, the now leading head (which was previously the front head) releases its $ext{ADP}$. This nucleotide exchange makes the leading head strongly bound to the microtubule, completing the cycle and re-establishing the strong-binding/weak-binding state, ready for the next step.

• The coordination between the two heads is remarkably tight, with ATP binding to one head promoting the release of the other head, leading to very high processivity. Kinesin-1 can take hundreds of steps along a single microtubule without detaching, efficiently transporting cargo over long distances.

Kinesin Autoinhibition & Activation

• Kinesins are typically kept in an inactive or "autoinhibited" state in the absence of cargo, preventing futile ATP hydrolysis and uncontrolled movement.

• Folded conformation: In the autoinhibited state, the motor domains interact directly with the tail domain, forming a compact, folded conformation. This folding simultaneously blocks both the motor domains' ability to bind to microtubules and the tail domains' ability to bind to cargo.

• Relief mechanisms (activation):

  • Cargo/adaptor binding: The primary activation mechanism involves the binding of specific cargo or associated adaptor proteins (e.g., JIP3, Sunday Driver) to the C-terminal tail domain. This binding event sterically disrupts the motor-tail interaction, unfolding the kinesin into an elongated, active state.

  • Post-translational modifications (PTMs): Phosphorylation, acetylation, or ubiquitination of specific residues, particularly on the neck or tail domains, can induce conformational changes that relieve autoinhibition or modulate activity. For example, phosphorylation of Kinesin-1's tail can activate it.

  • Allosteric regulation by scaffold proteins: Some kinesins can be activated by forming part of a larger protein scaffold, sometimes even involving dynein. For instance, HOOK3 can bind simultaneously to KIF1C (a kinesin) and dynein, promoting KIF1C activation and potentially coordinating bidirectional transport.

Cytoplasmic Dynein Architecture (Monomeric Heavy Chain ≈$1.2\, ext{MDa}$)

Cytoplasmic dynein-1 is a massive motor protein, typically forming a homodimer, with each monomer containing a single heavy chain and multiple light, intermediate, and light-intermediate chains:

• Heavy Chain Structure (per monomer):

  • Tail domain: Located at the N-terminus, this region is responsible for dimerization of the two heavy chains and serves as the primary attachment site for cargo, adaptor proteins, and the dynactin complex.

  • Linker domain: A long, flexible coiled-coil element located between the tail and the AAA+ ring. The linker undergoes significant conformational changes (bending and straightening) during the ATP hydrolysis cycle, acting as the primary force-generating element (power stroke).

  • AAA+ ring: The central enzymatic core of dynein, comprising six tandem AAA+ (ATPases Associated with various cellular Activities) domains arranged in a hexameric ring. Only the first AAA domain (AAA1) possesses potent ATPase activity essential for the motor cycle, while others modulate the overall function.

  • Coiled-coil stalk + Microtubule-binding domain (MTBD) at tip: Extending from the AAA+ ring (specifically from AAA4/5), this long, slender coiled-coil stalk (approximately $25\, ext{nm}$ from the ring) positions the critical MTBD at its very tip. The MTBD binds to microtubules in an ATP-sensitive manner; its affinity for MTs is modulated by conformational changes transmitted down the stalk from the AAA+ ring.

• Associated Chains: In addition to the heavy chains, cytoplasmic dynein contains several accessory chains that decorate the tail domain:

  • Intermediate chains (ICs): Play a role in targeting dynein to specific cellular locations and interacting with cargo activators.

  • Light intermediate chains (LICs): Implicated in cargo binding and regulatory interactions.

  • Light chains (LCs): Involved in specific cargo interactions and fine-tuning dynein's activity.

Dynein Power Stroke & Stochastic Walking

Dynein's stepping mechanism is distinct from kinesin, characterized by a more stochastic and less coordinated gait:

1. ATP binding to AAA1: When ATP binds to the AAA1 domain in the leading head, it triggers a significant conformational change within the AAA+ ring. This change is transmitted through the stalk to the MTBD, causing a decrease in its affinity for the microtubule and leading to detachment of this head.

2. Linker bending and biased random walk: Simultaneously with MTBD detachment, ATP binding causes the linker domain to bend and unbind from the AAA+ ring. The detached head, now freely diffusing in a somewhat biased manner (often forward, but also sideways or even backward), searches for a new binding site on the microtubule lattice.

3. Rebinding and $ext{P<em>i}$ release (power stroke): Upon rebinding to a new microtubule site, the AAA1 domain quickly hydrolyzes ATP and releases inorganic phosphate ($ext{P</em>i}$). This $ext{P_i}$ release triggers a conformational recovery within the AAA+ ring, causing the previously bent linker to straighten. This straightening motion acts as the power stroke, pulling the cargo forward by an average step size of approximately $8\, ext{nm}$. Importantly, due to its stochastic nature, the step size can be variable, and the motor frequently makes side and back steps.

4. ADP release and cycle reset: Finally, $ext{ADP}$ is released from AAA1, which typically strengthens the binding of the leading head to the microtubule and resets the system for the next ATP binding event.

• Dynein heads are weakly coordinated: Unlike kinesin, the two heads of a dynein dimer are only loosely coordinated. This allows for what is often described as a "drunken-sailor" stepping pattern, characterized by frequent side and even back steps. This less rigid coordination, however, confers an excellent ability to bypass cellular obstacles and navigate crowded actin or microtubule networks.

Dynein Processivity Requires Cofactors

While a single dynein dimer can take some steps, its high processivity and efficiency for long-distance transport in cells critically depend on its interaction with specific cofactors:

• Dynactin: A large, multi-protein complex (over 1 MDa) composed of approximately 11 subunits. It is built around a short actin-related protein 1 (ARP1) filament and a β-actin filament. Dynactin contains periodic "grooves" that are thought to precisely dock one or two dynein tails, effectively creating a stable platform for dynein.

• Cargo adapter proteins (e.g., BICD2, HOOK1-3, HAP1, Spindly): These are diverse coiled-coil proteins that act as molecular bridges. They possess distinct binding sites for both the cargo (or cargo-associated proteins) and the dynein tail, as well as specific binding sites for the dynactin complex. When these adapters simultaneously bind dynactin and one or more dynein dimers' tails, they form a highly stable and activated "Dynein–Dynactin–Adapter" (DDA) complex. This DDA assembly effectively aligns two dynein dimers (resulting in four total motor domains), leading to significantly faster movement, enhanced processivity, and more robust transport capabilities, though the exact mechanism for this speed increase is still under investigation.

• LIS1 (lissencephaly protein / PAFAH1B1): A non-motor dynein-associated protein critical for brain development. LIS1 stabilizes dynein in an open, high-affinity state, preventing its autoinhibition and enhancing its microtubule binding and stepping. Mutations in LIS1 lead to severe developmental disorders like lissencephaly (a smooth brain phenotype due to impaired neuronal migration).

Dynein Autoinhibition

Similar to kinesins, cytoplasmic dynein also has an autoinhibited state to regulate its activity:

• Φ-conformation (Phi-conformation): In its inactive form, dynein can adopt a compact, folded, and autoinhibited "Phi" state. In this conformation, the two heavy chain tails are crossed, and the motor domains are stacked, physically preventing both microtubule binding and productive interaction with adapter proteins.

• Transition to open state: The transition from the autoinhibited Φ-conformation to an open, active state is assisted by several factors, including the binding of LIS1, specific nucleotide states (e.g., nucleotide-free or ADP-bound states), and the initial binding of cargo-adaptor complexes.

• In vitro observations: Studies in vitro have shown that even during active transport, dynein occasionally re-folds into the autoinhibited Φ-conformation, an event likened to the "Star Wars AT-AT tripping" analogy. This re-folding can lead to premature self-termination of a transport run, potentially serving as a built-in regulatory mechanism.

Other Dyneins

Beyond cytoplasmic dynein-1, other dynein families perform specialized roles:

• Dynein-2 (IFT dynein, intraflagellar transport dynein): A distinct cytoplasmic dynein isoform primarily responsible for driving retrograde intraflagellar transport (IFT) within cilia and flagella. IFT is a crucial process for building and maintaining these organelles by transporting structural and signaling molecules from the tip back to the base.

• Axonemal dyneins: Specialized, multi-headed dynein motors found in the axonemes of cilia and flagella. These motors are responsible for powering the bending and beating motions of these organelles by sliding adjacent doublet microtubules past one another, essential for motility (e.g., sperm movement, mucociliary clearance).

Comparison of Kinesin vs Dynein

Experimental Approaches Highlighted

Biophysical and cellular techniques have been instrumental in deciphering motor protein function:

• Microtubule co-sedimentation assay: An in vitro biochemical assay used to determine the ability of a protein (e.g., a motor domain) to bind to assembled microtubules. Proteins are mixed with microtubules and centrifuged. If the protein binds to MTs, it pellets with the MTs; otherwise, it remains in the supernatant. The assay is often performed with and without ATP to reveal the nucleotide-dependent MT affinity of the motor.

• Squid giant axon fractionation (1980s): Pioneering experiments using the easily accessible and large squid giant axon cytosolic extract led to the discovery of kinesin. Differential centrifugation and biochemical purification revealed a motor activity (the "squid translocator") responsible for moving organelles along microtubules.

• Bead motility assays: In these in vitro assays, purified motor proteins are attached to microscopic beads. The beads are then observed walking along surface-bound microtubules. Optical tweezers can be used to trap the beads and precisely quantify the force generated by individual motors (typically a few piconewtons, pN).

• Gliding assays: The inverse of bead motility assays. In a gliding assay, motor proteins are adsorbed onto a coverslip surface, and fluorescently labeled microtubules are added. The motors, acting in unison, transport the MTs across the surface like a "crowd-surfing" phenomenon, allowing for measurement of bulk motor velocity and processivity.

• Single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy: A powerful technique that illuminates only a thin layer near the coverslip surface, dramatically reducing background noise. This allows for the visualization and tracking of individual, fluorescently tagged motors (e.g., with GFP or bright organic dyes) in real-time, providing nanometer-resolution insights into their stepping, force generation, and interactions.

• Rapalog inducible dimerization in cells: A chemogenetic approach where engineered components (e.g., a motor domain and a vesicle-targeting domain) can be acutely linked within living cells by the addition of a small molecule drug (rapalog). This allows researchers to precisely control and trace the anterograde versus retrograde flux of specific organelles or vesicles in a live cellular context.

• Cryo-electron microscopy (Cryo-EM) & negative-stain EM: High-resolution structural techniques allowing for the visualization of macromolecules in their near-native state. Cryo-EM, in particular, has achieved sub-$\&lt;4\,\text{Å}$ reconstructions of complex assemblies like dynein, dynactin, and adapter complexes, providing unprecedented detail on their interaction interfaces. Electron tomography can further visualize multi-dynein teams in situ.

Physiological & Pathological Relevance

Motor proteins are critical for maintaining cellular function and their dysregulation is implicated in numerous diseases:

• Axonal transport: Neurons have extremely long axons (ranging from micrometers to meters in length, e.g., in giraffes). Kinesin and dynein are responsible for transporting essential cargoes over these vast distances. Kinesin typically transports dynein itself anterogradely, while dynein returns used kinesin and other retrograde cargo. A precise bidirectional balance is crucial; imbalances lead to axonal degeneration.

• Neural migration & cortical layering: During brain development, radial glial cells undergo repeated division and migration, guided by motor proteins. Even mild mutations in genes encoding dynein, kinesin, or tubulin can severely disrupt these processes, leading to developmental brain disorders such as lissencephaly (characterized by a smooth cerebral cortex due to a lack of gyri and sulci) or polymicrogyria (excessive small folds).

• Neurodegeneration & motor neuron disease: Impaired dynein/kinesin balance and defects in axonal transport are hallmarks of many neurodegenerative disorders. These include the accumulation of vesicle jams and protein aggregates (e.g., Tau tangles in Alzheimer’s disease, α-synuclein in Parkinson's, and SOD1/TDP-43 aggregates in Amyotrophic Lateral Sclerosis - ALS), which disrupt neuronal function and lead to cell death.

• Nuclear positioning: In many cell types, including budding yeast, cortical dynein plays a vital role. In yeast, it actively pulls the nucleus into the burgeoning daughter cell during cell division. Similar mechanisms involving kinesins and dyneins are employed in filamentous fungi and various animal cells to ensure proper nuclear migration and positioning within the cytoplasm.

Quantitative Nuggets & Useful Numbers

• MT diameter: Approximately $25\, ext{nm}$, a consistent and crucial track dimension for motors.

• Kinesin/dynein step: Each ATP hydrolysis event typically propels the motor forward by $8 ext{–}16\, ext{nm}$, representing a remarkable conversion of chemical to mechanical energy at the nanoscale.

• Max velocity (axonal transport): Motor proteins can achieve maximum velocities of approximately $1\, rac{ ext{µm}}{ ext{s}}$, which, over the course of hours or days, allows for transport over meters of axonal length.

• Force per motor: A single kinesin motor can generate approximately $5\, ext{pN}$ of force, while a single head of dynein generates about $1 ext{–}2\, ext{pN}$. However, dynein often works in teams facilitated by dynactin/adaptors to generate significant total force.

Metaphors & Analogies Shared

• “Little boots” cartoon from 1985: An early, intuitive visualization from a seminal paper depicting motor proteins as tiny walking shoes that attach to cargo and step along a filament.

• Dynein’s ‘drunken sailor’ gait vs kinesin’s ‘marching soldier’: This analogy vividly captures the difference in stepping coordination and path straightness between the two motor types.

• Dynein–dynactin–adapter complex ≈ four-horse team pulling a wagon: Illustrates the concerted, powerful effort generated by the assembly of multiple dyneins with their cofactors to efficiently pull large cargo.

• Φ-conformation likened to Star Wars AT-AT tripping: This analogy describes how an autoinhibited dynein occasionally re-folds during a run, causing it to prematurely disengage from the microtubule, similar to the AT-AT walker falling during an attack.

Key Concept Check (Mirrors Speaker’s Review Questions)

• Cargo coupling: Achieved primarily via the highly variable C-terminal tail domains of both motor types. Dynein typically requires dedicated coiled-coil adapter proteins (e.g., BICD2, HOOKs) and the dynactin complex to link to specific cargo, whereas many kinesins can bind cargo directly, but also utilize adapters.

• Force generation: The mechanical work is generated by ATP hydrolysis within the motor domain. This hydrolysis drives conformational changes, specifically the swinging of the neck linker in kinesin and the bending/straightening of the linker domain in dynein, which translates into linear movement along the microtubule.

• Coordination: Kinesin heads exhibit strict coordination, with nucleotide binding in one head directly influencing the attachment and detachment of the other, ensuring high processivity. In contrast, dynein heads are largely independent (stochastic); their weak coordination allows for more flexibility and obstacle bypass.

• Processivity: Kinesin-1 is inherently very highly processive as a dimer, capable of taking hundreds of steps due to its tight coordination. Dynein, however, achieves high processivity only when it forms the larger Dynein–Dynactin–Adapter (DDA) team, which aligns multiple dynein dimers and stabilizes their interaction with the microtubule.

• Regulation: Motor activity is tightly controlled through multiple mechanisms, including autoinhibitory folding (motor-tail interaction in kinesin, Φ-conformation in dynein), activation by specific cargo or adapter protein binding, various post-translational modifications (PTMs like phosphorylation), and even regulation by mechanical load.

Connections to Prior & Future Lectures

• Builds on prior microtubule dynamics lecture: Reinforces understanding of microtubule structure, their intrinsic polarity (plus/minus ends), the roles of Microtubule-Associated Proteins (MAPs), and the concepts of catastrophe and rescue in microtubule growth.

• Links forward to mitosis lecture: Provides essential groundwork for understanding mitotic processes, particularly the role of Eg5 in driving bipolar spindle formation and the function of kinetochore dynein in chromosome alignment and movement during cell division.

• Reinforces broader cell-biology themes: Emphasizes fundamental principles such as cellular polarity, energy transduction, the intricate relationship between molecular structure and biological function, and the critical roles of these motors in various disease mechanisms.

Ethical, Philosophical & Practical Implications

• Therapeutic angles: A deep understanding of motor protein regulation offers promising therapeutic targets for a range of human diseases. This includes designing interventions for neurodegenerative disorders (by enhancing axonal transport), addressing developmental disorders (by correcting neuronal migration defects), and developing novel anti-cancer drugs (e.g., Eg5 inhibitors that block mitotic progression).

• Bridging molecular to organismal physiology: The study of single-molecule biophysics, exemplified by experiments on kinesin and dynein, provides a powerful framework for connecting the detailed molecular mechanisms of proteins to their profound impact on cellular and even organismal physiology (e.g., how extremely long-distance axonal transport is adapted in large animals like giraffes).

Summary Cheat-Sheet

• Remember: “One dynein, many adapters; many kinesins, diverse tails.” This highlights the singular identity and reliance on cofactors for dynein versus the broad diversity and functional specialization of kinesins.

• Direction memory: A helpful mnemonic: Dynein = DYve into minus (–) end; KINesin = head to (+) KINetic front.

• For exams: Be prepared to accurately sketch the mechanochemical cycles of both kinesin and dy