Microtubule Gliding Velocity Research Articles

A neuropathy-associated kinesin KIF1A mutation hyper-stabilizes the motor-neck interaction during the ATPase cycle.

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the β7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on β7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.

Photo-control of the mitotic kinesin Eg5 using a novel photochromic inhibitor composed of a spiropyran derivative.

In this study, we synthesized a novel photochromic inhibitor of the mitotic kinesin Eg5, which is composed of the photochromic compound spiropyran to photo-control the function of Eg5. The compound (S)-2, 3-dispiropyran propionic acid (DSPPA) exhibits reversible spiropyran-merocyanine photo-isomerization upon irradiation with visible or ultra-violet light. DSPPA induced reversible changes in the inhibitory effect on Eg5 ATPase and motor activities, which correlates with the spiropyran-merocyanine photo-isomerization. Microtubule-dependent ATPase activity was significantly more inhibited by the spiropyran isomer of DSPPA than by the merocyanine isomer. Additionally, an in vitro motility assay revealed that the microtubule gliding velocity was reduced more by the spiropyran isomer than by the merocyanine isomer. This indicates that the spiropyran derivative may be useful in regulating the function of the mitotic kinesin.

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors' anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted "membrane-anchored" gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

Effect of Active Kinesin Motor Density on Microtubules During Self-Assembly of Spools

Active self-assembly systems are energy driven and can organize into various structures. Microtubules and their associated motor proteins, such as kinesin, are widely used to study active self-assembly of higher order structures, such as linear bundles and spools. Microtubules are polymers composed of tubulin that are found in the cytoskeleton. Kinesin motors convert ATP into energy through hydrolysis and walk along microtubules. Microtubules functionalized with biotin and streptavidin bind together and form bundles and spools when gliding. The spools are able to maintain their shape and continue to rotate in the presence of ATP. We use gliding assays to investigate the effect of the active motor density on microtubules during spool formation. By tuning the velocity of gliding microtubules, we can effectively tune the kinesin density on microtubules. There was no significant change in average spool circumference and no reduction in spool density over a 10-fold reduction in microtubule gliding velocity. We find spool characteristics are robust against active kinesin density on microtubules.

Photocontrol of the mitotic kinesin Eg5 using a novel S-trityl-L-cysteine analogue as a photochromic inhibitor

Because the mitotic kinesin Eg5 is essential for the formation of bipolar spindles during eukaryotic cell division, it has been considered as a potential target for cancer treatment. A number of specific and potent inhibitors of Eg5 are known. S-trityl-L-cysteine is one of the inhibitors of Eg5 whose molecular mechanism of inhibition was well studied. The trityl group of S-trityl-L-cysteine was shown to be a key moiety required for potent inhibition. In this study, we synthesized a novel photochromic S-trityl-L-cysteine analogue, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4'- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (ACTAB), composed of a trityl group, azobenzene and N-acetyl-L-cysteine, which exhibits cis-trans photoisomerization in order to photocontrol the function of Eg5. ACTAB exhibited cis-trans photoisomerization upon alternating irradiation at two different wavelengths in the visible range, 400 and 480 nm. ACTAB induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with the cis-trans photoisomerization. Compared with cis-ACTAB, trans-ACTAB reduced ATPase activity and microtubule gliding velocity more significantly. These results suggest that ACTAB could be used as photochromic inhibitor of Eg5 to achieve photocontrol of living cells.

Cooperative Effects in Transport Systems Driven by Diffusively Anchored Motors

Intracellular trafficking of membrane-bound organelles is a fundamental process essential to many cellular functions including cell growth and signaling. A variety of such organelles are transported by motor proteins such as kinesins, dyneins and myosins, walking on cellular tracks made of microtubules and actin filaments. Since studying the mechanical and biochemical functioning of these transport systems inside the cell is extremely challenging, transport is often mimicked in-vitro, for example by gliding motility assays. There, cytoskeletal filaments are propelled by motors that are conventionally immobilized on a rigid substrate. In contrast, when transporting membrane-bound organelles inside a cell the motors are diffusively anchored - either directly or via adaptor molecules - to lipid bilayers. Such resulting ‘loose’ coupling may induce motor co-ordination and is likely to change the collective motor dynamics.In this study, we investigate the collective behavior of motor proteins anchored to lipid bilayers. Using truncated kinesin-1 motors with a streptavidin-binding-peptide tag we performed gliding motility assays on streptavidin-loaded biotinylated supported lipid bilayers. Our results suggest a dependence of the microtubule gliding velocity on both, the motor density as well as the microtubule length. Based on measurements of the diffusion constants and the velocity of motors and microtubules, a theoretical model is developed to determine (i) the number of microtubule-attached motors and (ii) the force produced by an ensemble of motors.

α-Synuclein Oligomers Impair Neuronal Microtubule-Kinesin Interplay

Early α-synuclein (α-Syn)-induced alterations are neurite pathologies resulting in Lewy neurites. α-Syn oligomers are a toxic species in synucleinopathies and are suspected to cause neuritic pathology. To investigate how α-Syn oligomers may be linked to aberrant neurite pathology, we modeled different stages of α-Syn aggregation in vitro and investigated the interplay of α-Syn aggregates with proteins involved in axonal transport. The interaction of wild type α-Syn (WTS) and α-Syn variants (E57K, A30P, and aSyn(30-110)) with kinesin, tubulin, and the microtubule (MT)-associated proteins, MAP2 and Tau, is stronger for multimers than for monomers. WTS seeds but not α-Syn oligomers significantly and dose-dependently reduced Tau-promoted MT assembly in vitro. In contrast, MT gliding velocity across kinesin-coated surfaces was significantly decreased in the presence of α-Syn oligomers but not WTS seeds or fibrils (aSyn(30-110) multimers). In a human dopaminergic neuronal cell line, mild overexpression of the oligomerizing E57K α-Syn variant significantly impaired neurite network morphology without causing profound cell death. In accordance with these findings, MT stability, neuritic kinesin, and neuritic kinesin-dependent cargoes were significantly reduced by the presence of α-Syn oligomers. In summary, different α-Syn species act divergently on the axonal transport machinery. These findings provide new insights into α-Syn oligomer-driven neuritic pathology as one of the earliest events in synucleinopathies.

Neck Rotation and Neck Mimic Docking in the Noncatalytic Kar3-associated Protein Vik1

Kar3Vik1 is a heterodimeric kinesin with one catalytic subunit (Kar3) and one noncatalytic subunit (Vik1). Vik1 experiences conformational changes in regions analogous to the force-producing elements in catalytic kinesins. A molecular mechanism by which Kar3 could trigger Vik1's release from microtubules was revealed. These findings will serve as the prototype for understanding the motile mechanism of kinesin-14 motors in general. It is widely accepted that movement of kinesin motor proteins is accomplished by coupling ATP binding, hydrolysis, and product release to conformational changes in the microtubule-binding and force-generating elements of their motor domain. Therefore, understanding how the Saccharomyces cerevisiae proteins Cik1 and Vik1 are able to function as direct participants in movement of Kar3Cik1 and Kar3Vik1 kinesin complexes presents an interesting challenge given that their motor homology domain (MHD) cannot bind ATP. Our crystal structures of the Vik1 ortholog from Candida glabrata may provide insight into this mechanism by showing that its neck and neck mimic-like element can adopt several different conformations reminiscent of those observed in catalytic kinesins. We found that when the neck is α-helical and interacting with the MHD core, the C terminus of CgVik1 docks onto the central β-sheet similarly to the ATP-bound form of Ncd. Alternatively, when neck-core interactions are broken, the C terminus is disordered. Mutations designed to impair neck rotation, or some of the neck-MHD interactions, decreased microtubule gliding velocity and steady state ATPase rate of CgKar3Vik1 complexes significantly. These results strongly suggest that neck rotation and neck mimic docking in Vik1 and Cik1 may be a structural mechanism for communication with Kar3.

Functional Dissection of LIS1 and NDEL1 Towards Understanding the Molecular Mechanisms of Cytoplasmic Dynein Regulation

LIS1 and NDEL1 are known to be essential for the activity of cytoplasmic dynein in living cells. We previously reported that LIS1 and NDEL1 directly regulated the motility of cytoplasmic dynein in an in vitro motility assay. LIS1 suppressed dynein motility and inhibited the translocation of microtubules (MTs), while NDEL1 dissociated dynein from MTs and restored dynein motility following suppression by LIS1. However, the molecular mechanisms and detailed interactions of dynein, LIS1, and NDEL1 remain unknown. In this study, we dissected the regulatory effects of LIS1 and NDEL1 on dynein motility using full-length or truncated recombinant fragments of LIS1 or NDEL1. The C-terminal fragment of NDEL1 dissociated dynein from MTs, whereas its N-terminal fragment restored dynein motility following suppression by LIS1, demonstrating that the two functions of NDEL1 localize to different parts of the NDEL1 molecule, and that restoration from LIS1 suppression is caused by the binding of NDEL1 to LIS1, rather than to dynein. The truncated monomeric form of LIS1 had little effect on dynein motility, but an artificial dimer of truncated LIS1 suppressed dynein motility, which was restored by the N-terminal fragment of NDEL1. This suggests that LIS1 dimerization is essential for its regulatory function. These results shed light on the molecular interactions between dynein, LIS1, and NDEL1, and the mechanisms of cytoplasmic dynein regulation.

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport. These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available. Building on the classic inverted motility assay, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface. The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA, quantum dots or a wide variety of antigens via biotinylated antibodies.

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport. These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available. Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface. The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

“Popoffs” Under a Transverse Force Reveal the Number and Location of Active Kinesin Motors During Motility Assays

Determination of the number of active motors pulling a single MT or bead during motility assays has proven difficult. Traditional protein concentration assays, such as Bradford, cannot distinguish between active and inactive motors. We attach a superparamagnetic bead to the (+) end of a microtubule. When placed in a magnet with uniform magnetic field gradient, the bead pulls on the MT with a controllable 0-10 pN force. If the force is perpendicular to the gliding direction of the MT, a short section of the MT “pops off” the surface every 2 to 5 s, as shown in the diagram. This detachment is characterized by rapid motion of the superparamagnetic bead in the direction of higher magnetic field gradient followed by normal microtubule gliding velocity when the MT is pulled taut. The length of the short section between “popoffs” is the distance between active kinesins along a microtubule.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Kinesin-Calmodulin fusion protein as a molecular shuttle

In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca(2+)-dependent reversible binding of K560-CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560-CaM in the absence of Ca(2+), while both dimeric and tetrameric configurations in the presence of Ca(2+). Further, Ca(2+)-dependent change in the configuration of K560-CaM was monitored by size-exclusion-HPLC and analytical ultracentrifugation. Finally, by total internal reflection fluorescence microscopy, we successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca(2+).

Ratchetlike Properties of In Vitro Microtubule Translocation by a Chlamydomonas Inner-Arm Dynein Species c in the Presence of Flow

To investigate the force generation properties of Chlamydomonas axonemal inner-arm dyneins in response to external force, we analyzed microtubule gliding on dynein-coated surfaces under shear flow. When inner-arm dynein c was used, microtubule translocation in the downstream direction accelerated with increasing flow speed in a manner that depended on the dynein density and ATP concentration. In contrast, the microtubule translocation velocity in the upstream direction was unaffected by the flow speed. The number of microtubules on the glass surface was almost constant with and without flow, suggesting that gliding acceleration was not simply caused by weakened dynein-microtubule binding. With other inner-arm dynein species, the microtubule gliding velocity was unaffected by the flow regardless of the flow direction or nucleotide concentration. The flow-generated force acting on a single dynein was estimated to be as small as ∼0.03 pN/dynein. These results indicate that dynein c possesses a ratchetlike property that allows acceleration only in one direction by a very small external force. This property should be important for slow- and fast-moving dyneins to function simultaneously within the axoneme.

Velocity-dependence of Cargo Loading onto Molecular Shuttles Demonstrates the Glue-like Character of Biotin/Streptavidin

Molecular shuttles based on biomolecular motors and their associated filaments are being developed to function as conveyor belts in the molecular factories of the future. An essential design element in these active nanoscale transport systems is cargo loading onto the shuttles. We demonstrate that molecular shuttle velocity has to be optimized to facilitate cargo attachment of nanospheres via biotin-streptavidin linkages. The biotin-streptavidin bond gains its ultimate strength on a timescale of milliseconds due to existence of metastable binding states. As a consequence of the glue-like character of this widely used intermolecular bond, the velocity of molecular shuttles has to be optimized to permit efficient attachment of cargo via biotin-streptavidin linkages.In our experiments, kinesin motor proteins adsorbed to a casein precoated surface were used to propel biotinylated microtubules which were coated with streptavidin at saturating dosages. The microtubule gliding velocity was varied between 50 nm/s and 450 nm/s by changing the kinesin substrate ATP concentration. Finally, biotinylated fluorescein-labeled nanospheres were added in concentrations ranging from 25 pM to 100 pM. Nanospheres attached to the surface and were loaded onto microtubules only as a result of collisions between gliding microtubules and nanospheres. Nanosphere attachment showed an unexpected optimum at an intermediate shuttle velocity.The attachment and detachment processes were modeled by combining rigorous mechanical engineering analysis with detailed physico-chemical models. This contribution will present both, the experimental details of our velocity dependent loading experiments and the theoretical model which explains the optimum on the basis of the complex binding energy landscape of the biotin streptavidin linkages.

Phosphorylation Relieves Autoinhibition of the Kinetochore Motor Cenp-E

During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.

Engineering Cooperativity in Biomotor-Protein Assemblies

A biosynthetic approach was developed to control and probe cooperativity in multiunit biomotor assemblies by linking molecular motors to artificial protein scaffolds. This approach provides precise control over spatial and elastic coupling between motors. Cooperative interactions between monomeric kinesin-1 motors attached to protein scaffolds enhance hydrolysis activity and microtubule gliding velocity. However, these interactions are not influenced by changes in the elastic properties of the scaffold, distinguishing multimotor transport from that powered by unorganized monomeric motors. These results highlight the role of supramolecular architecture in determining mechanisms of collective transport.

Interaction of metal salts with cytoskeletal motor protein systems

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 μM PbCl 2, 0.05 μM Pb(OAc) 2 and 0.01 μM HgCl 2. The in vitro results obtained for PbCl 2 correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 μM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295–298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 μM. Inhibition of tubulin assembly by mercury started at 2 μM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 μM Pb(NO 3) 2 and 0.1 μM HgCl 2 in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility.

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.

Chapter 63 Assaying Protein Phosphatases in Sperm and Flagella

Sperm and flagellar motility mechanism depends on protein phosphorylation. Dynein has been indicated as a potential site of convergence for regulation through phosphorylation/dephosphorylation by protein kinases and phosphatases. Phosphorylation of dynein has been correlated with increased ATPase activity and an increase in the velocity of dynein-mediated gliding of microtubules in vitro. An important role for protein phosphatases in motility regulation has been suggested by adding protein phosphatases or their inhibitors to flagellar models. A significant decrease in the gliding velocity of microtubules in vitro by dynein has been observed by preincubation of dynein with serinekhreonine protein phosphatase, SPP, recently purified from sea urchin sperm. The enzyme specifically dephosphorylates a single dynein-associated subunit phosphorylated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase. This chapter describes the assay for serine/threonine protein phosphatases using the dephosphorylation of type II regulatory subunit (RII) peptide phosphorylated by cAMP-dependent protein kinase as substrate and the considerations when designing experiments to examine the effect of protein phosphatases and kinases on the gliding velocity of microtubules in vitro by dynein. The methods presented are purification of the catalytic subunit of cAMP-dependent protein kinase, phosphorylation and purification of RII substrate, and protein phosphatase assay. The ability to assay dynein function by examining the propulsion of taxol-stabilized microtubules on glass surfaces offers a tool for examining the role of protein phosphorylation. All experiments examining effects on gliding of manipulation in solution are conducted in parallel with incubations using dynein that has been treated with [ 32 P]ATP and analyzed by sodium dodecyl phosphate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, to confirm changes in protein phosphorylation that are associated with changes in gliding velocity. A peptide substrate radiolabeled to a high specific activity is essential for the high sensitivity and reproducibility of the protein phosphatase activity.