Conserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein.

Processive transport by the microtubule motor cytoplasmic dynein requires the regulated assembly of a dynein-dynactin-adapter complex. Interactions between dynein and dynactin were initially ascribed to the dynein intermediate chain N-terminus and the dynactin subunit p150Glued. However, recent cryo-EM structures have not resolved this interaction, questioning its importance. The intermediate chain also interacts with Nde1/Ndel1, which compete with p150Glued for binding. We reveal that the intermediate chain N-terminus is a critical evolutionarily conserved hub that interacts with dynactin and Ndel1, the latter of which recruits LIS1 to drive complex assembly. In additon to revealing that the intermediate chain N-terminus is likely bound to p150Glued in active transport complexes, our data support a model whereby Ndel1-LIS1 must dissociate prior to LIS1 being handed off to dynein in temporally discrete steps. Our work reveals previously unknown steps in the dynein activation pathway, and provide insight into the integrated activities of LIS1/Ndel1 and dynactin/cargo-adapters.

Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport.

The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to microtubule plus-ends). Although LIS1 binding is required for dynein activity, its unbinding prior to initiation of cargo transport is equally important, since preventing dissociation leads to dynein dysfunction. To understand whether and how dynein-LIS1 binding is modulated, we engineered dynein mutants locked in a microtubule-bound (MT-B) or microtubule-unbound (MT-U) state. Whereas the MT-B mutant exhibits low LIS1 affinity, the MT-U mutant binds LIS1 with high affinity, and as a consequence remains almost irreversibly associated with microtubule plus-ends. We find that a monomeric motor domain is sufficient to exhibit these opposing LIS1 affinities, and that this is evolutionarily conserved between yeast and humans. Three cryo-EM structures of human dynein with and without LIS1 reveal microtubule-binding induced conformational changes responsible for this regulation. Our work reveals key biochemical and structural insight into LIS1-mediated dynein activation.

Zn2+ decoration of microtubules arrests axonal transport and displaces tau, doublecortin, and MAP2C.

Intracellular Zn2+ concentrations increase via depolarization-mediated influx or intracellular release, but the immediate effects of Zn2+ signals on neuron function are not fully understood. By simultaneous recording of cytosolic Zn2+ and organelle motility, we find that elevated Zn2+ (IC50 ≈ 5-10 nM) reduces both lysosomal and mitochondrial motility in primary rat hippocampal neurons and HeLa cells. Using live-cell confocal microscopy and in vitro single-molecule TIRF imaging, we reveal that Zn2+ inhibits activity of motor proteins (kinesin and dynein) without disrupting their microtubule binding. Instead, Zn2+ directly binds to microtubules and selectively promotes detachment of tau, DCX, and MAP2C, but not MAP1B, MAP4, MAP7, MAP9, or p150glued. Bioinformatic predictions and structural modeling show that the Zn2+ binding sites on microtubules partially overlap with the microtubule binding sites of tau, DCX, dynein, and kinesin. Our results reveal that intraneuronal Zn2+ regulates axonal transport and microtubule-based processes by interacting with microtubules.

The role of kinetochore dynein in checkpoint silencing is restricted to disassembly of the corona.

During mitosis, kinetochore-microtubule attachments are monitored by a molecular surveillance system known as the spindle assembly checkpoint. The prevailing model posits that dynein evicts checkpoint proteins (e.g., Mad1, Mad2) from stably attached kinetochores by transporting them away from kinetochores, thus contributing to checkpoint silencing. However, the mechanism by which dynein performs this function, and its precise role in checkpoint silencing remain unresolved. Here, we find that dynein's role in checkpoint silencing is restricted to evicting checkpoint effectors from the fibrous corona, and not the outer kinetochore. Dynein evicts these molecules from the corona in a manner that does not require stable, end-on microtubule attachments. Thus, by disassembling the corona through indiscriminate microtubule encounters, dynein primes the checkpoint signaling apparatus so it can respond to stable end-on microtubule attachments and permit cells to progress through mitosis. Accordingly, we find that dynein function in checkpoint silencing becomes largely dispensable in cells in which checkpoint effectors are excluded from the corona.

Conserved Roles for the Dynein Intermediate Chain and Ndel1 in Assembly and Activation of Dynein.

Cytoplasmic dynein, the primary retrograde microtubule transport motor within cells, must be activated for processive motility through the regulated assembly of a dynein-dynactin-adapter (DDA) complex. The interaction between dynein and dynactin was initially ascribed to the N-terminus of the dynein intermediate chain (IC) and a coiled-coil of the dynactin subunit p150 Glued . However, cryo-EM structures of DDA complexes have not resolve these regions of the IC and p150 Glued , raising questions about the importance of this interaction. The IC N-terminus (ICN) also interacts with the dynein regulators Nde1/Ndel1, which compete with p150 Glued for binding to ICN. Using a combination of approaches, we reveal that the ICN plays critical, evolutionarily conserved roles in DDA assembly by interacting with dynactin and Ndel1, the latter of which recruits the DDA assembly factor LIS1 to the dynein complex. In contrast to prior models, we find that LIS1 cannot simultaneously bind to Ndel1 and dynein, indicating that LIS1 must be handed off from Ndel1 to dynein in temporally discrete steps. Whereas exogenous Ndel1 or p150 Glued disrupts DDA complex assembly in vitro , neither perturbs preassembled DDA complexes, indicating that the IC is stably bound to p150 Glued within activated DDA complexes. Our study reveals previously unknown regulatory steps in the dynein activation pathway, and provides a more complete model for how the activities of LIS1/Ndel1 and dynactin/cargo-adapters are integrated to regulate dynein motor activity.

Using Budding Yeast as a Model to Understand Dynein-Mediated Cargo Transport.

Cytoplasmic dynein-1 is a minus end-directed microtubule motor that transports numerous cargoes in cell types throughout the evolutionary spectrum. Dynein is regulated by various motor-intrinsic and motor-extrinsic factors that enhance its processivity, recruit it to various cellular sites, or otherwise promote or restrict its activity. Studying dynein activity in higher eukaryotes is complicated by various factors, including the myriad functions in which this motor participates, and the consequential pleotropic effects associated with disrupting its activity. Budding yeast has long been a powerful model system for understanding this enormous motor protein complex, which is highly conserved between yeast and humans at the primary sequence and structural levels. Studies in budding yeast are simplified by the fact that dynein only performs one known function in this organism: to position the mitotic spindle at the site of cell division. Monitoring dynein-mediated spindle movements in budding yeast provides a powerful tool for the quantitative measurements of various motility parameters, and a system with which to assess the consequence of mutations in dynein or its regulators. Here, we provide detailed protocols to perform quantitative measurements of dynein activity in live cells using a combination of fluorescence microscopy and computational methods to track and quantitate dynein-mediated spindle movements. These methods are broadly applicable to anyone that wishes to perform fluorescence microscopy on budding yeast.

The microtubule-associated protein She1 coordinates directional spindle positioning by spatially restricting dynein activity.

Dynein motors move the mitotic spindle to the cell division plane in many cell types, including in budding yeast, in which dynein is assisted by numerous factors including the microtubule-associated protein (MAP) She1. Evidence suggests that She1 plays a role in polarizing dynein-mediated spindle movements toward the daughter cell; however, how She1 performs this function is unknown. We find that She1 assists dynein in maintaining the spindle in close proximity to the bud neck, such that, at anaphase onset, the chromosomes are segregated to mother and daughter cells. She1 does so by attenuating the initiation of dynein-mediated spindle movements within the mother cell, thus ensuring such movements are polarized toward the daughter cell. Our data indicate that this activity relies on She1 binding to the microtubule-bound conformation of the dynein microtubule-binding domain, and to astral microtubules within mother cells. Our findings reveal how an asymmetrically localized MAP directionally tunes dynein activity by attenuating motor activity in a spatially confined manner.

New insights into the mechanism of dynein motor regulation by lissencephaly-1.

Lissencephaly ('smooth brain') is a severe brain disease associated with numerous symptoms, including cognitive impairment, and shortened lifespan. The main causative gene of this disease - lissencephaly-1 (LIS1) - has been a focus of intense scrutiny since its first identification almost 30 years ago. LIS1 is a critical regulator of the microtubule motor cytoplasmic dynein, which transports numerous cargoes throughout the cell, and is a key effector of nuclear and neuronal transport during brain development. Here, we review the role of LIS1 in cellular dynein function and discuss recent key findings that have revealed a new mechanism by which this molecule influences dynein-mediated transport. In addition to reconciling prior observations with this new model for LIS1 function, we also discuss phylogenetic data that suggest that LIS1 may have coevolved with an autoinhibitory mode of cytoplasmic dynein regulation.

Pac1/LIS1 stabilizes an uninhibited conformation of dynein to coordinate its localization and activity.

Dynein is a microtubule motor that transports many different cargos in various cell types and contexts. How dynein is regulated to perform these activities with spatial and temporal precision remains unclear. Human dynein is regulated by autoinhibition, whereby intermolecular contacts limit motor activity. Whether this mechanism is conserved throughout evolution, whether it can be affected by extrinsic factors, and its role in regulating dynein function remain unclear. Here, we use a combination of negative stain electron microscopy, single-molecule assays, genetic, and cell biological techniques to show that autoinhibition is conserved in budding yeast, and plays a key role in coordinating in vivo dynein function. Moreover, we find that the Lissencephaly-related protein, LIS1 (Pac1 in yeast), plays an important role in regulating dynein autoinhibition. Our studies demonstrate that, rather than inhibiting dynein motility, Pac1/LIS1 promotes dynein activity by stabilizing the uninhibited conformation, which ensures appropriate dynein localization and activity in cells.

Molecular basis for dyneinopathies reveals insight into dynein regulation and dysfunction.

Cytoplasmic dynein plays critical roles within the developing and mature nervous systems, including effecting nuclear migration, and retrograde transport of various cargos. Unsurprisingly, mutations in dynein are causative of various developmental neuropathies and motor neuron diseases. These 'dyneinopathies' define a broad spectrum of diseases with no known correlation between mutation identity and disease state. To circumvent complications associated with dynein studies in human cells, we employed budding yeast as a screening platform to characterize the motility properties of seventeen disease-correlated dynein mutants. Using this system, we determined the molecular basis for several classes of etiologically related diseases. Moreover, by engineering compensatory mutations, we alleviated the mutant phenotypes in two of these cases, one of which we confirmed with recombinant human dynein. In addition to revealing molecular insight into dynein regulation, our data provide additional evidence that the type of disease may in fact be dictated by the degree of dynein dysfunction.

Effectors of the spindle assembly checkpoint are confined within the nucleus of Saccharomyces cerevisiae.

The spindle assembly checkpoint (SAC) prevents erroneous chromosome segregation by delaying mitotic progression when chromosomes are incorrectly attached to the mitotic spindle. This delay is mediated by mitotic checkpoint complexes (MCCs), which assemble at unattached kinetochores and repress the activity of the anaphase promoting complex/cyclosome (APC/C). The cellular localizations of MCCs are likely critical for proper SAC function, yet remain poorly defined. We recently demonstrated that in mammalian cells, in which the nuclear envelope disassembles during mitosis, MCCs diffuse throughout the spindle region and cytoplasm. Here, we employed an approach using binucleate yeast zygotes to examine the localization dynamics of SAC effectors required for MCC assembly and function in budding yeast, in which the nuclear envelope remains intact throughout mitosis. Our findings indicate that in yeast, MCCs are confined to the nuclear compartment and excluded from the cytoplasm during mitosis.

She1 affects dynein through direct interactions with the microtubule and the dynein microtubule-binding domain.

Cytoplasmic dynein is an enormous minus end-directed microtubule motor. Rather than existing as bare tracks, microtubules are bound by numerous microtubule-associated proteins (MAPs) that have the capacity to affect various cellular functions, including motor-mediated transport. One such MAP is She1, a dynein effector that polarizes dynein-mediated spindle movements in budding yeast. Here, we characterize the molecular basis by which She1 affects dynein, providing the first such insight into which a MAP can modulate motor motility. We find that She1 affects the ATPase rate, microtubule-binding affinity, and stepping behavior of dynein, and that microtubule binding by She1 is required for its effects on dynein motility. Moreover, we find that She1 directly contacts the microtubule-binding domain of dynein, and that their interaction is sensitive to the nucleotide-bound state of the motor. Our data support a model in which simultaneous interactions between the microtubule and dynein enables She1 to directly affect dynein motility.

"Wait anaphase" signals are not confined to the mitotic spindle.

The spindle assembly checkpoint ensures the faithful inheritance of chromosomes by arresting mitotic progression in the presence of kinetochores that are not attached to spindle microtubules. This is achieved through inhibition of the anaphase-promoting complex/cyclosome by a kinetochore-derived "wait anaphase" signal known as the mitotic checkpoint complex. It remains unclear whether the localization and activity of these inhibitory complexes are restricted to the mitotic spindle compartment or are diffusible throughout the cytoplasm. Here we report that "wait anaphase" signals are indeed able to diffuse outside the confines of the mitotic spindle compartment. Using a cell fusion approach to generate multinucleate cells, we investigate the effects of checkpoint signals derived from one spindle compartment on a neighboring spindle compartment. We find that spindle compartments in close proximity wait for one another to align all chromosomes before entering anaphase synchronously. Synchrony is disrupted in cells with increased interspindle distances and cellular constrictions between spindle compartments. In addition, when mitotic cells are fused with interphase cells, "wait anaphase" signals are diluted, resulting in premature mitotic exit. Overall our studies reveal that anaphase inhibitors are diffusible and active outside the confines of the mitotic spindle from which they are derived.

The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition.

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from "off" at the plus ends to "on" at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein-dynactin-Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end-directed dynein-dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched "on" by Num1, which induces Pac1/LIS1 removal.

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.

The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast owing to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Because of problems associated with fusing genes encoding fluorescent proteins (FPs) to the native α-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome-integrated FP-tubulin fusions negatively affect microtubule functions. Thus, a simple, economical and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP-Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of 30 FP-Tub1 plasmids that use various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function.

She1-mediated inhibition of dynein motility along astral microtubules promotes polarized spindle movements.

BACKGROUND: Cytoplasmic dynein motility along microtubules is critical for diverse cellular processes ranging from vesicular transport to nuclear envelope breakdown to mitotic spindle alignment. In yeast, we have proposed a regulated-offloading model to explain how dynein motility drives microtubule sliding along the cortex, powering transport of the nucleus into the mother-bud neck [1, 2]: the dynein regulator She1 limits dynein offloading by gating the recruitment of dynactin to the astral microtubule plus end, a prerequisite for offloading to the cortex. However, whether She1 subsequently affects cortically anchored dynein activity during microtubule sliding is unclear. RESULTS: Using single-molecule motility assays, we show that She1 strongly inhibits dynein movement along microtubules, acting directly on the motor domain in a manner independent of dynactin. She1 has no effect on the motility of either Kip2, a kinesin that utilizes the same microtubule track as dynein, or human kinesin-1, demonstrating the specificity of She1 for the dynein motor. At single-molecule resolution, She1 binds tightly to and exhibits diffusional behavior along microtubules. Diffusive She1 collides with and pauses motile dynein motors, prolonging their attachment to the microtubule. Furthermore, Aurora B/Ipl1 directly phosphorylates She1, and this modification appears to enhance the diffusive behavior of She1 along microtubules and its potency against dynein. In cells, She1 dampens productive microtubule-cortex interactions specifically in the mother compartment, polarizing spindle movements toward the bud cell. CONCLUSIONS: Our data reveal how inhibitory microtubule-associated proteins selectively regulate motor activity to achieve unidirectional nuclear transport and demonstrate a direct link between cell-cycle machinery and dynein pathway activity.

Astral microtubule asymmetry provides directional cues for spindle positioning in budding yeast.

Cortical force generators play a central role in the orientation and positioning of the mitotic spindle. In higher eukaryotes, asymmetrically localized cortical polarity determinants recruit or activate such force generators, which, through interactions with astral microtubules, position the mitotic spindle at the future site of cytokinesis. Recent studies in budding yeast have shown that, rather than the cell cortex, the astral microtubules themselves may provide polarity cues that are needed for asymmetric pulling on the mitotic spindle. Such asymmetry has been shown to be required for proper spindle positioning, and consequently faithful and accurate chromosome segregation. In this review, we highlight results that have shed light on spindle orientation in this classical model of asymmetric cell division, and review findings that may shed light on similar processes in higher eukaryotes.

Regulated offloading of cytoplasmic dynein from microtubule plus ends to the cortex.

Cytoplasmic dynein mediates spindle orientation from the cell cortex through interactions with astral microtubules, but neither the mechanism governing its cortical targeting nor the regulation thereof is well understood. Here we show that yeast dynein offloads from microtubule plus ends to the daughter cell cortex. Mutants with an engineered peptide inserted between the tail domain and the motor head retain wild-type motor activity but exhibit enhanced offloading and cortical targeting. Conversely, shortening the "neck" sequence between the tail and motor domains precludes offloading from the microtubule plus ends. Furthermore, chimeric mutants with mammalian dynein "neck" sequences rescue targeting and function. These findings provide direct support for an active microtubule-mediated delivery process that appears to be regulated by a conserved masking/unmasking mechanism.

Quantitative analysis of Pac1/LIS1-mediated dynein targeting: Implications for regulation of dynein activity in budding yeast.

LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik1 recruits approximately equal amounts of Pac1 and Dyn1. Overexpression of Pac1 enhanced plus end targeting of Dyn1 and vice versa, while affinity-purification of Dyn1 revealed that it exists in a complex with Pac1 in the absence of Bik1, leading us to conclude that the Pac1-Dyn1 complex preassembles in the cytoplasm prior to loading onto Bik1-decorated plus ends. Strikingly, we found that Pac1-overexpression augments cortical dynein activity through a mechanism distinct from loss of She1, a negative regulator of dynein-dynactin association. While Pac1-overexpression enhances the frequency of cortical targeting for dynein and dynactin, the stoichiometry of these complexes remains relatively unchanged at the plus ends compared to that in wild-type cells (∼3 dynein to 1 dynactin). Loss of She1, however, enhances dynein-dynactin association at the plus ends and the cell cortex, resulting in an apparent 1:1 stoichiometry. Our results reveal differential regulation of cortical dynein activity by She1 and Pac1, and provide a potentially new regulatory step in the off-loading model for dynein function.

Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation.

During animal development, microtubules (MTs) play a major role in directing cellular and subcellular patterning, impacting cell polarization and subcellular organization, thereby affecting cell fate determination and tissue architecture. In particular, when progenitor cells divide asymmetrically along an anterior-posterior or apical-basal axis, MTs must coordinate the position of the mitotic spindle with the site of cell division to ensure normal distribution of cell fate determinants and equal sequestration of genetic material into the two daughter cells. Emerging data from diverse model systems have led to the prevailing view that, during mitotic spindle positioning, polarity cues at the cell cortex signal for the recruitment of NuMA and the minus-end directed MT motor cytoplasmic dynein.(1) The NuMA/dynein complex is believed to connect, in turn, to the mitotic spindle via astral MTs, thus aligning and tethering the spindle, but how this connection is achieved faithfully is unclear. Do astral MTs need to search for and then capture cortical NuMA/dynein? How does dynein capture the astral MTs emanating from the correct spindle pole? Recently, using the classical model of asymmetric cell division-budding yeast S. cerevisiae-we successfully demonstrated that astral MTs assume an active role in cortical dynein targeting, in that astral MTs utilize their distal plus ends to deliver dynein to the daughter cell cortex, the site where dynein activity is needed to perform its spindle alignment function. This observation introduced the novel idea that, during mitotic spindle orientation processes, polarity cues at the cell cortex may actually signal to prime the cortical receptors for MT-dependent dynein delivery. This model is consistent with the observation that dynein/dynactin accumulate prominently at the astral MT plus ends during metaphase in a wide range of cultured mammalian cells.