Regulation Of Dynein Activity Research Articles

Self-sustaining oscillation of two axonemal microtubules based on a stochastic bonding model between microtubules and dynein.

The motility of cilia and flagella plays important physiological roles, and there has been a great deal of research on the mechanisms underlying the motility of molecular motors. Although recent molecular structural analyses have revealed the components of the ciliary axoneme, the mechanisms involved in the regulation of dynein activity are still unknown, and how multiple dyneins coordinate their movements remains unclear. In particular, the mode of binding for axonemal dynein has not been elucidated. In this study, we constructed a thermodynamic stochastic model of microtubule-dynein coupling and reproduced the experiments of Aoyama and Kamiya on the minimal component of axonemal microtubule-dynein. We then identified the binding mode of axonemal dynein and clarified the relationship between dynein activity distribution and axonemal movement. Based on our numerical results, the slip-bond mechanism agrees quantitatively with the experimental results in terms of amplitude, frequency, and propagation velocity, implying that axial microtubule-dynein coupling may follow a slip-bond mechanism. Moreover, the frequency and propagation velocity decayed in proportion to the fourth power of microtubule length, and the critical load of the trigger for the oscillation agreed well with Euler's critical load.

Nde1 and Ndel1: Outstanding Mysteries in Dynein-Mediated Transport.

Cytoplasmic dynein-1 (dynein) is the primary microtubule minus-end directed molecular motor in most eukaryotes. As such, dynein has a broad array of functions that range from driving retrograde-directed cargo trafficking to forming and focusing the mitotic spindle. Dynein does not function in isolation. Instead, a network of regulatory proteins mediate dynein’s interaction with cargo and modulate dynein’s ability to engage with and move on the microtubule track. A flurry of research over the past decade has revealed the function and mechanism of many of dynein’s regulators, including Lis1, dynactin, and a family of proteins called activating adaptors. However, the mechanistic details of two of dynein’s important binding partners, the paralogs Nde1 and Ndel1, have remained elusive. While genetic studies have firmly established Nde1/Ndel1 as players in the dynein transport pathway, the nature of how they regulate dynein activity is unknown. In this review, we will compare Ndel1 and Nde1 with a focus on discerning if the proteins are functionally redundant, outline the data that places Nde1/Ndel1 in the dynein transport pathway, and explore the literature supporting and opposing the predominant hypothesis about Nde1/Ndel1’s molecular effect on dynein activity.

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles

To build and maintain mitotic spindle architecture, molecular motors exert spatially regulated forces on microtubules (MT) minus-ends. This spatial regulation is required to allow proper chromosomes alignment through the organization of kinetochore fibers (k-fibers). NuMA was recently shown to target dynactin to MT minus-ends and thus to spatially regulate dynein activity. However, given that k-fibers are embedded in the spindle, our understanding of the machinery involved in the targeting of proteins to their minus-ends remains limited. Intraflagellar transport (IFT) proteins were primarily studied for their ciliary roles but they also emerged as key regulators of cell division. Taking advantage of MT laser ablation, we show here that IFT88 concentrates at k-fibers minus-ends and is required for their re-anchoring into spindles by controlling NuMA accumulation. Indeed, IFT88 interacts with NuMA and is required for its enrichment at newly generated k-fibers minus-ends. Combining nocodazole washout experiments and IFT88 depletion, we further show that IFT88 is required for the reorganization of k-fibers into spindles and thus for efficient chromosomes alignment in mitosis. Overall, we propose that IFT88 could serve as a mitotic MT minus-end adaptor to concentrate NuMA at minus-ends thus facilitating k-fibers incorporation into the main spindle.

DnaJB6 is a RanGTP-regulated protein required for microtubule organization during mitosis.

ABSTRACTBipolar spindle organization is essential for the faithful segregation of chromosomes during cell division. This organization relies on the collective activities of motor proteins. The minus-end-directed dynein motor complex generates spindle inward forces and plays a major role in spindle pole focusing. The dynactin complex regulates many dynein functions, increasing its processivity and force production. Here, we show that DnaJB6 is a novel RanGTP-regulated protein. It interacts with the dynactin subunit p150Glued (also known as DCTN1) in a RanGTP-dependent manner specifically in M-phase, and promotes spindle pole focusing and dynein force generation. Our data suggest a novel mechanism by which RanGTP regulates dynein activity during M-phase.

In vivo analyses of radial spoke transport, assembly, repair and maintenance.

Radial spokes (RSs) are multiprotein complexes that regulate dynein activity. In the cell body, RS proteins (RSPs) are present in a 12S precursor, which enters the flagella and converts into the axoneme-bound 20S spokes consisting of a head and stalk. To study RS dynamics in vivo, we expressed fluorescent protein (FP)-tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pf1, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co-migrated by intraflagellar transport (IFT). The transport was elevated during flagellar assembly and IFT of RSP4-FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP-tagged RSPs were mated to untagged cells with, without, or with partial RSs. Tagged RSPs were incorporated in a spotted fashion along wild-type-derived flagella indicating an exchange of RSs. During the repair of pf1-derived axonemes, RSP4-FP is added onto the preexisting spoke stalks with little exchange of RSP3. Thus, RSP3 and RSP4 are transported together but appear to separate inside flagella during the repair of RSs. The 12S RS precursor encompassing both proteins could represent a transport form to ensure stoichiometric delivery of RSPs into flagella by IFT.

Microtubule cross-linking activity of She1 ensures spindle stability for spindle positioning.

Dynein mediates spindle positioning in budding yeast by pulling on astral microtubules (MTs) from the cell cortex. The MT-associated protein She1 regulates dynein activity along astral MTs and directs spindle movements toward the bud cell. In addition to localizing to astral MTs, She1 also targets to the spindle, but its role on the spindle remains unknown. Using function-separating alleles, live-cell spindle assays, and in vitro biochemical analyses, we show that She1 is required for the maintenance of metaphase spindle stability. She1 binds and cross-links MTs via a C-terminal MT-binding site. She1 can also self-assemble into ring-shaped oligomers. In cells, She1 stabilizes interpolar MTs, preventing spindle deformations during movement, and we show that this activity is regulated by Ipl1/Aurora B phosphorylation during cell cycle progression. Our data reveal how She1 ensures spindle integrity during spindle movement across the bud neck and suggest a potential link between regulation of spindle integrity and dynein pathway activity.

Two-headed outer- and inner-arm dyneins of Leishmania sp bear conserved IQ-like motifs

Dyneins are high molecular weight microtubule based motor proteins responsible for beating of the flagellum. The flagellum is important for the viability of trypanosomes like Leishmania. However, very little is known about dynein and its role in flagellar motility in such trypanosomatid species. Here, we have identified genes in five species of Leishmania that code for outer-arm dynein (OAD) heavy chains α and β, and inner-arm dynein (IAD) heavy chains 1α and 1β using BLAST and MSA. Our sequence analysis indicates that unlike the three-headed outer-arm dyneins of Chlamydomonas and Tetrahymena, the outer-arm dyneins of the genus Leishmania are two-headed, lacking the γ chain like that of metazoans. N-terminal sequence analysis revealed a conserved IQ-like calmodulin binding motif in the outer-arm α and inner-arm 1α dynein heavy chain in the five species of Leishmania similar to Chlamydomonas reinhardtii outer-arm γ. It was predicted that both motifs were incapable of binding calmodulin. Phosphorylation site prediction revealed conserved serine and threonine residues in outer-arm dynein α and inner-arm 1α as putative phosphorylation sites exclusive to Leishmania but not in Trypanosoma brucei suggesting that regulation of dynein activity might be via phosphorylation of these IQ-like motifs in Leishmania sp.

Efficient Endocytic Uptake and Maturation in Drosophila Oocytes Requires Dynamitin/p50.

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. In this study, we examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of Yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. We therefore propose that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor colocalized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation.

Stress-Induced CDK5 Activation Disrupts Axonal Transport via Lis1/Ndel1/Dynein.

Axonal transport is essential for neuronal function, and defects in transport are associated with multiple neurodegenerative diseases. Aberrant cyclin-dependent kinase 5 (CDK5) activity, driven by the stress-induced activator p25, also is observed in these diseases. Here we show that elevated CDK5 activity increases the frequency of nonprocessive events for a range of organelles, including lysosomes, autophagosomes, mitochondria, and signaling endosomes. Transport disruption induced by aberrant CDK5 activation depends on the Lis1/Ndel1 complex, which directly regulates dynein activity. CDK5 phosphorylation of Ndel1 favors a high affinity Lis1/Ndel/dynein complex that blocks the ATP-dependent release of dynein from microtubules, inhibiting processive motility of dynein-driven cargo. Similar transport defects observed in neurons from a mouse model of amyotrophic lateral sclerosis are rescued by CDK5 inhibition. Together, these studies identify CDK5 asa Lis1/Ndel1-dependent regulator of transport in stressed neurons, and suggest that dysregulated CDK5 activity contributes to the transport deficits observed during neurodegeneration.

Motor Regulation Results in Distal Forces that Bend Partially Disintegrated Chlamydomonas Axonemes into Circular Arcs

The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella.

Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.

Axonemal radial spokes

The radial spoke (RS) is a complex of at least 23 proteins that works as a mechanochemical transducer between the central-pair apparatus and the peripheral microtubule doublets in eukaryotic flagella and motile cilia. The RS contributes to the regulation of the activity of dynein motors, and thus to flagellar motility. Despite numerous biochemical, physiological and structural studies, the mechanism of the function of the radial spoke remains unclear. Detailed knowledge of the 3D structure of the RS protein complex is needed in order to understand how RS regulates dynein activity. Here we review the most important findings on the structure of the RS, including results of our recent cryo-electron tomographic analysis of the RS protein complex.

Neurodegenerative Mutation in Cytoplasmic Dynein Alters Its Organization and Dynein-Dynactin and Dynein-Kinesin Interactions

A single amino acid change, F580Y (Legs at odd angles (Loa), Dync1h1Loa), in the highly conserved and overlapping homodimerization, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin. We found that the Loa mutation in the heavy chain leads to increased affinity of this subunit of cytoplasmic dynein to light intermediate and a population of intermediate chains and a suppressed association of dynactin to dynein. These data suggest that the Loa mutation drives the assembly of cytoplasmic dynein toward a complex with lower affinity to dynactin and thus impairing transport of cargos that tether to the complex via dynactin. In addition, we detected up-regulation of kinesin light chain 1 (KLC1) and its increased association with dynein but reduced microtubule-associated KLC1 in the Loa samples. We provide a model describing how up-regulation of KLC1 and its interaction with cytoplasmic dynein in Loa could play a regulatory role in restoring the retrograde and anterograde transport in the Loa neurons.

Regulation of dynein-driven microtubule sliding by the axonemal protein kinase CK1 in Chlamydomonas flagella

Experimental analysis of isolated ciliary/flagellar axonemes has implicated the protein kinase casein kinase I (CK1) in regulation of dynein. To test this hypothesis, we developed a novel in vitro reconstitution approach using purified recombinant Chlamydomonas reinhardtii CK1, together with CK1-depleted axonemes from the paralyzed flagellar mutant pf17, which is defective in radial spokes and impaired in dynein-driven microtubule sliding. The CK1 inhibitors (DRB and CK1-7) and solubilization of CK1 restored microtubule sliding in pf17 axonemes, which is consistent with an inhibitory role for CK1. The phosphatase inhibitor microcystin-LR blocked rescue of microtubule sliding, indicating that the axonemal phosphatases, required for rescue, were retained in the CK1-depleted axonemes. Reconstitution of depleted axonemes with purified, recombinant CK1 restored inhibition of microtubule sliding in a DRB- and CK1-7-sensitive manner. In contrast, a purified "kinase-dead" CK1 failed to restore inhibition. These results firmly establish that an axonemal CK1 regulates dynein activity and flagellar motility.

Dynein-Driven Mitotic Spindle Positioning Restricted to Anaphase by She1p Inhibition of Dynactin Recruitment

Dynein is a minus-end-directed microtubule motor important for mitotic spindle positioning. In budding yeast, dynein activity is restricted to anaphase when the nucleus enters the bud neck, yet the nature of the underlying regulatory mechanism is not known. Here, the microtubule-associated protein She1p is identified as a novel regulator of dynein activity. In she1 Delta cells, dynein is activated throughout the cell cycle, resulting in aberrant spindle movements that misposition the spindle. We also found that dynactin, a cofactor essential for dynein motor function, is a dynamic complex whose recruitment to astral microtubules (aMTs) increases dramatically during anaphase. Interestingly, loss of She1p eliminates the cell-cycle regulation of dynactin recruitment and permits enhanced dynactin accumulation on aMTs throughout the cell cycle. Furthermore, localization of the dynactin complex to aMTs requires dynein, suggesting that dynactin is recruited to aMTs via interaction with dynein and not the microtubule itself. Lastly, we present evidence supporting the existence of an incomplete dynactin subcomplex localized at the SPB, and a complete complex that is loaded onto aMTs from the cytoplasm. We propose that She1p restricts dynein-dependent spindle positioning to anaphase by inhibiting the association of dynein with the complete dynactin complex.

Mechanism of flagellar oscillation–bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm

Oscillatory movement of eukaryotic flagella is caused by dynein-driven microtubule sliding in the axoneme. The mechanical feedback from the bending itself is involved in the regulation of dynein activity, the main mechanism of which is thought to be switching of the activity of dynein between the two sides of the central pair microtubules. To test this, we developed an experimental system using elastase-treated axonemes of sperm flagella, which have a large Ca(2+)-induced principal bend (P-bend) at the base. On photoreleasing ATP from caged ATP, they slid apart into two bundles of doublets. When the distal overlap region of the slid bundles was bent in the direction opposite to the basal P-bend, backward sliding of the thinner bundle was induced along the flagellum including the bent region. The velocity of the backward sliding was significantly lower than that of the forward sliding, supporting the idea that the dynein activity alternated between the two sides of the central pair on bending. Our results show that the combination of the direction of bending and the conformational state of dynein-microtubule interaction induce the switching of the dynein activity in flagella, thus providing the basis for flagellar oscillation.

Keeping an eye on I1: I1 dynein as a model for flagellar dynein assembly and regulation

Among the major challenges in understanding ciliary and flagellar motility is to determine how the dynein motors are assembled and localized and how dynein-driven outer doublet microtubule sliding is controlled. Diverse studies, particularly in Chlamydomonas, have determined that the inner arm dynein I1 is targeted to a unique structural position and is critical for regulating the microtubule sliding required for normal ciliary/flagellar bending. As described in this review, I1 dynein offers additional opportunities to determine the principles of assembly and targeting of dyneins to cellular locations and for studying the mechanisms that regulate dynein activity and control of motility by phosphorylation.

NudEL targets dynein to microtubule ends through LIS1

Dynein is a minus-end-directed microtubule motor with critical roles in mitosis, membrane transport and intracellular transport. Several proteins regulate dynein activity, including dynactin, LIS1 (refs 2, 3) and NudEL (NudE-like). Here, we identify a NUDEL homologue in budding yeast and name it Ndl1. The ndl1delta null mutant shows decreased targeting of dynein to microtubule plus ends, an essential element of the model for dynein function. We find that Ndl1 regulates dynein targeting through LIS1, with which it interacts biochemically, but not through CLIP170, another plus-end protein involved in dynein targeting. Ndl1 is found at far fewer microtubule ends than are LIS1 and dynein. However, when Ndl1 is present at a plus end, the molar amount of Ndl1 approaches that of LIS1 and dynein. We propose a model in which Ndl1 binds transiently to the plus end to promote targeting of LIS1, which cooperatively recruits dynein.

Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning.

Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), we found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning.

Regulation of flagellar dynein by the axonemal central apparatus

Numerous studies indicate that the central apparatus, radial spokes, and dynein regulatory complex form a signaling pathway that regulates dynein activity in eukaryotic flagella. This regulation involves the action of several kinases and phosphatases anchored to the axoneme. To further investigate the role of the central apparatus in this signaling pathway, we have taken advantage of a microtubule-sliding assay to assess dynein activity in central apparatus defective mutants of Chlamydomonas. Axonemes isolated from both pf18 and pf15 (lacking the entire central apparatus) and from pf16 (lacking the C1 central microtubule) have reduced microtubule-sliding velocity compared with wild-type axonemes. Based on functional analyses of axonemes isolated from radial spokeless mutants, we hypothesized that inhibitors of casein kinase 1 (CK1) and cAMP dependent protein kinase (PKA) would rescue dynein activity and increase microtubule-sliding velocity in central pairless mutants. Treatment of axonemes isolated from both pf18 and pf16 with DRB, a CK1 inhibitor, but not with PKI, a PKA inhibitor, restored dynein activity to wild-type levels. The DRB-induced increase in dynein-driven microtubule sliding was inhibited if axonemes were first incubated with the phosphatase inhibitor, microcystin. Inhibiting CK1 in pf15 axonemes, which lack the central pair as well as PP2A [Yang et al., 2000: J. Cell Sci. 113:91-102], did not increase microtubule-sliding velocity. These data are consistent with a model in which the central apparatus, and specifically the C1 microtubule, regulate dynein through interactions with the radial spokes that ultimately alter the activity of CK1 and PP2A. These data are also consistent with localization of axonemal CK1 and PP2A near the dynein arms.