Structural basis for cytoplasmic dynein-1 regulation by Lis1.

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1 Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.

Rigid-body fitting to atomic force microscopy images for inferring probe shape and biomolecular structure.

Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhAC, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available.

Postulated Process of Axoneme Organization in the Male Gametogenesis of Malaria Parasite Plasmodium berghei.

The ultrastructural features of axoneme organization within the cytoplasm and exflagellation were investigated in detail in microgametes of a malaria parasite, Plasmodium berghei, by electron and fluorescence microscopy. The kinetosomes (basal bodies) of the microgamete were characterized by an electron dense mass in which singlet microtubules (MTs) were embedded. Around the kinetosomes, several singlet and doublet MTs were recognized in transverse sections. Incomplete doublets with growing B-tubule were also observed. As precursors of the axoneme, arrays of over three doublets showed a tendency to encircle the central pair MTs. Some of the doublet MTs were already equipped with inner and outer dynein arms. In the microgamete, which lacks an intraflagellar transport (IFT) system, self-assembly of microtubular and associated components appeared to proceed stepwise from singlet MTs through arrays of one to nine doublet MTs, surrounding the central pair, to form the complete axoneme in a quite short time. At exflagellation, some extra doublets were occasionally included between the axoneme and the flagellar membrane. At high magnification, the outer dynein arm of the Plasmodium microgamete had a pistol-like shape representing a three-headed dynein molecule like that of other Alveolata.

Collective motility of dynein linear arrays built on DNA nanotubes.

Dynein motor proteins usually work as a group in vesicle transport, mitosis, and ciliary/flagellar beating inside cells. Despite the obvious importance of the functions of dynein, the effect of inter-dynein interactions on collective motility remains poorly understood due to the difficulty in building large dynein ensembles with defined geometry. Here, we describe a method to build dynein ensembles to investigate the collective motility of dynein on microtubules. Using electron microscopy, we show that tens to hundreds of cytoplasmic dynein monomers were anchored along a 4- or 10-helix DNA nanotube with an average periodicity of 19 or 44 nm (a programmed periodicity of 14 or 28 nm, respectively). They drove the sliding movement of DNA nanotubes along microtubules at a velocity of 170-620 nm/s. Reducing the stiffness of DNA nanotubes made the nanotube movement discontinuous and considerably slower. Decreasing the spacing between motors simply slowed down the nanotube movement. This slowdown was independent of the number of motors involved but heavily dependent on motor-motor distance. This suggests that steric hindrance or mechanical coupling between dynein molecules was responsible for the slowdown. Furthermore, we observed cyclical buckling of DNA nanotubes on microtubules, reminiscent of ciliary/flagellar beating. These results highlight the importance of the geometric arrangement of dynein motors on their collective motility.

Structure of the dynein-2 complex and its assembly with intraflagellar transport trains.

Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery that is crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4-MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60-WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag conformation, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60-WDR34-light chain subcomplex renders dynein-2 monomeric and relieves autoinhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits controls dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into the interaction of dynein-2 with IFT trains and the origin of diverse functions in the dynein family.

Primary ciliary dyskinesia in Japan: systematic review and meta-analysis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare genetic disorder. Although the genetic tests and new diagnostic algorithms have recently been recommended, clinical signs and electron microscope (EM) findings have historically been the mainstays of diagnosis in Asia. To characterize PCD previously reported in Japan, we conducted a systematic review and meta-analysis. METHODS: A search using MEDLINE, EMBASE, and Japana Centra Revuo Medicina (in Japanese) databases was carried out to identify articles reporting PCD, Kartagener syndrome, or immotile cilia syndrome in Japanese patients and published between 1985 and 2015. RESULTS: After excluding duplication from 334 reports, we extracted 316 patients according to the criteria. Diagnosis was most frequently made in adulthood (148 patients [46.8%] ≥ 18 years old, 24 patients [7.6%] < 1 year old, 68 patients [21.5%] 1-17 years old and 76 patients [24.1%] lacking information). Of the 230 patients (72.8%) who received EM examination, there were patients with inner dynein arm (IDA) defects (n = 55; 23.9%), outer dynein arm (ODA) defects (14; 6.1%), both ODA and IDA defects (57; 24.8%), other structural abnormalities (25; 10.9%), no abnormalities (4; 1.7%), and no detailed conclusion or description (75; 32.6%). CONCLUSION: Delayed diagnosis of this congenital disease with high frequency of IDA defects and low frequency of ODA defects appear to be historical features of PCD reported in Japan, when EM was a main diagnostic tool. This review highlights problems experienced in this field, and provides basic information to establish a modernized PCD diagnosis and management system in the future.

Directionality of dynein is controlled by the angle and length of its stalk.

The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of dynein by engineering a Saccharomyces cerevisiae dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the dynein stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the minus end of the microtubule.

Nanometer-accuracy distance measurements between fluorophores at the single-molecule level.

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

The Motion of An Inv Nodal Cilium: a Realistic Model Revealing Dynein-Driven Ciliary Motion with Microtubule Mislocalization.

BACKGROUND/AIMS: Nodal cilia that rotate in the ventral node play an important role in establishing left-right asymmetry during embryogenesis; however, inv mutant cilia present abnormal movement and induce laterality defects. The mechanism of their motility, which is regulated by dynein activation and microtubule arrangement, has not been fully understood. This study analyzed the dynein-triggered ciliary motion in the abnormal ultrastructure of the inv mutant, aiming to quantitatively evaluate the influence of microtubule mislocalization on the movement of the cilium. METHODS: We established a realistic 3-D model of an inv mutant cilium with an ultrastructure based on tomographic datasets generated by ultra-high voltage electron microscopy. The time-variant activation of the axonemal dynein force was simulated by pairs of point loads and embedded at dynein-mounted positions between adjacent microtubule doublets in this mathematical model. Utilizing the finite element method and deformable grid, the motility of the mutant cilium that is induced by various dynein activation hypotheses was investigated and compared to experimental observation. RESULTS: The results indicate that for the inv mutant, simulations of the ciliary movement with the engagement of dyneins based on the distance-controlled pattern in the partially activation scenario are broadly consistent with the observation; the shortening of the microtubules induces smaller movement amplitudes, while the angles of the mislocalized microtubules affect the pattern of the ciliary movement, and during the ciliary movement, the microtubules swing and twist in the mutant ciliary body. CONCLUSION: More generally, this study implies that dynein engagement is sensitive to subtle geometric changes in the axoneme, and thus, this geometry greatly influences the integrity of a well-formed ciliary rotation.

Motor Proteins.

Myosin motors power movements on actin filaments, whereas dynein and kinesin motors power movements on microtubules. The mechanisms of these motor proteins differ, but, in all cases, ATP hydrolysis and subsequent release of the hydrolysis products drives a cycle of interactions with the track (either an actin filament or a microtubule), resulting in force generation and directed movement.

Mutations in C11orf70 Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry Due to Defects of Outer and Inner Dynein Arms.

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling.

Asymmetric distribution and spatial switching of dynein activity generates ciliary motility.

Motile cilia and flagella are essential, highly conserved organelles, and their motility is driven by the coordinated activities of multiple dynein isoforms. The prevailing "switch-point" hypothesis posits that dyneins are asymmetrically activated to drive flagellar bending. To test this model, we applied cryo-electron tomography to visualize activity states of individual dyneins relative to their locations along beating flagella of sea urchin sperm cells. As predicted, bending was generated by the asymmetric distribution of dynein activity on opposite sides of the flagellum. However, contrary to predictions, most dyneins were in their active state, and the smaller population of conformationally inactive dyneins switched flagellar sides relative to the bending direction. Thus, our data suggest a "switch-inhibition" mechanism in which force imbalance is generated by inhibiting, rather than activating, dyneins on alternating sides of the flagellum.

Cryo-EM shows how dynactin recruits two dyneins for faster movement.

Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a dynein tail-dynactin-BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein-dynactin transport machine.

Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements.

Cilia are multifunctional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multimotor assemblies with kinesin-II. Combining these data with insights from single-particle EM, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2's mechanical 'linker' and 'stalk' domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2's recycling activity during IFT.

Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes.

Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms offers a route to increase the signal-to-noise of repetitive biological structures, such improving the information content and interpretability of tomograms. We discuss the potential for sub-volume averaging in highlighting and investigating specific processes in situ, focusing on microtubule structure and viral infection. We show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features, (ii) the in situ determination of the structure of individual viruses is possible as they infect a cell, and (iii) novel, transient processes can be imaged with high levels of detail.

A simple and fast approach for missing-wedge invariant classification of subtomograms extracted from filamentous structures.

Unsupervised classification of subtomograms extracted from cryo-electron tomograms is often challenging due to the presence of a missing wedge in tomographic data. Here, we propose a simple new approach to classify subtomograms extracted from cryo-electron tomograms of filamentous objects. This unsupervised classification approach uses the 1D projections of the subtomograms for classification and works independently of the orientations of the missing wedge. We applied this approach to subtomograms from eukaryotic cilia and successfully detected heterogeneity including structural polymorphism of dynein molecules.

TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization.

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

Application of scanning cytometry and confocal-microscopy-based image analysis for investigation the role of cytoskeletal elements during equine herpesvirus type 1 (EHV-1) infection of primary murine neurons.

Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the ß-tubulin fibres within the neurites of infected cells. Alterations in ß-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.

DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Diagnosis of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disorder of ciliary structure or function. It results in mucus accumulation and bacterial colonization of the respiratory tract which leads to chronic upper and lower airway infections, organ laterality defects, and fertility problems. We review the respiratory signs and symptoms of PCD, as well as the screening tests for and diagnostic investigation of the disease, together with details related to ciliary function, ciliary ultrastructure, and genetic studies. In addition, we describe the difficulties in diagnosing PCD by means of transmission electron microscopy, as well as describing patient follow-up procedures.