Dynein Motor Research Articles

Cryo‐EM reveals the complex architecture of dynactin's shoulder region and pointed end

Dynactin is a 1.1 MDa complex that activates the molecular motor dynein for ultra‐processive transport along microtubules. In order to do this, it forms a tripartite complex with dynein and a coiled‐coil adaptor. Dynactin consists of an actin‐related filament whose length is defined by its flexible shoulder domain. Despite previous cryo‐EM structures, the molecular architecture of the shoulder and pointed end of the filament is still poorly understood due to the lack of high‐resolution information in these regions. Here we combine multiple cryo‐EM datasets and define precise masking strategies for particle signal subtraction and 3D classification. This overcomes domain flexibility and results in high‐resolution maps into which we can build the shoulder and pointed end. The unique architecture of the shoulder securely houses the p150 subunit and positions the four identical p50 subunits in different conformations to bind dynactin’s filament. The pointed end map allows us to build the first structure of p62 and reveals the molecular basis for cargo adaptor binding to different sites at the pointed end.

Cellular and Viral Determinants of HSV-1 Entry and Intracellular Transport towards Nucleus of Infected Cells.

HSV-1 employs cellular motor proteins and modulates kinase pathways to facilitate intracellular virion capsid transport. Previously, we and others have shown that the Akt inhibitor miltefosine inhibited virus entry. Herein, we show that the protein kinase C inhibitors staurosporine (STS) and gouml inhibited HSV-1 entry into Vero cells, and that miltefosine prevents HSV-1 capsid transport toward the nucleus. We have reported that the HSV-1 UL37 tegument protein interacts with the dynein motor complex during virus entry and virion egress, while others have shown that the UL37/UL36 protein complex binds dynein and kinesin causing a saltatory movement of capsids in neuronal axons. Co-immoprecipitation experiments confirmed previous findings from our laboratory that the UL37 protein interacted with the dynein intermediate chain (DIC) at early times post infection. This UL37-DIC interaction was concurrent with DIC phosphorylation in infected, but not mock-infected cells. Miltefosine inhibited dynein phosphorylation when added before, but not after virus entry. Inhibition of motor accessory protein dynactins (DCTN2, DCTN3), the adaptor proteins EB1 and the Bicaudal D homolog 2 (BICD2) expression using lentiviruses expressing specific shRNAs, inhibited intracellular transport of virion capsids toward the nucleus of human neuroblastoma (SK-N-SH) cells. Co-immunoprecipitation experiments revealed that the major capsid protein Vp5 interacted with dynactins (DCTN1/p150 and DCTN4/p62) and the end-binding protein (EB1) at early times post infection. These results show that Akt and kinase C are involved in virus entry and intracellular transport of virion capsids, but not in dynein activation via phosphorylation. Importantly, both the UL37 and Vp5 viral proteins are involved in dynein-dependent transport of virion capsids to the nuclei of infected cells.Importance. Herpes simplex virus type-1 enter either via fusion at the plasma membranes or endocytosis depositing the virion capsids into the cytoplasm of infected cells. The viral capsids utilize the dynein motor complex to move toward the nuclei of infected cells using the microtubular network. This work shows that inhibitors of the Akt kinase and kinase C inhibit not only viral entry into cells but also virion capsid transport toward the nucleus. In addition, the work reveals that the virion protein ICP5 (VP5) interacts with the dynein cofactor dynactin, while the UL37 protein interacts with the dynein intermediate chain (DIC). Importantly, neither Akt nor Kinase C was found to be responsible for phosphorylation/activation of dynein indicating that other cellular or viral kinases may be involved.

To nucleate or not, that is the question in neurons

Microtubules are the structural center of neurons, stretching in overlapping arrays from the cell body to the far reaches of axons and dendrites. They also act as the tracks for long-range transport mediated by dynein and kinesin motors. Transcription and most translation take place in the cell body, and newly made cargoes must be shipped from this site of synthesis to sites of function in axons and dendrites. This constant demand for transport means that the microtubule array must be present without gaps throughout the cell over the lifetime of the animal. This task is made slightly easier in many animals by the relatively long, stable microtubules present in neurons. However, even stable neuronal microtubules have ends that are dynamic, and individual microtubules typically last on the order of hours, while the neurons around them last a lifetime. “Birth” of new microtubules is therefore required to maintain the neuronal microtubule array. In this review we discuss the nucleation of new microtubules in axons and dendrites, including how and where they are nucleated. In addition, it is becoming clear that neuronal microtubule nucleation is highly regulated, with unexpected machinery impinging on the decision of whether nucleation sites are active or inactive through space and time.

Roles of the multivalent dynein adaptors BicD2 and RILP in neurons

Cytoplasmic dynein is responsible for all forms of retrograde transport in neurons and other cells. Work over several years has led to the identification of a class of coiled-coil domain containing “adaptor” proteins that are responsible for expanding dynein’s range of cargo interactions, as well as regulating dynein motor behavior. This brief review focuses first on the BicD family of adaptor proteins, which clearly serve to expand the number of dynein cargo interactions. RILP, another adaptor protein, also interacts with multiple proteins. Surprisingly, this is to mediate a series of steps within a common pathway, higher eukaryotic autophagy. These distinct features have important implications for understanding the full range of dynein adaptor functions

Ciliary motors locked closed by Shulin

Molecular Motors Motile cilia and flagella are vital cellular organelles with functions that include setting up the left-right body axis, clearing airways of mucus, and driving single-cell movements. Cilia beating is powered by arrays of dynein motors, the key force generators being the outer dynein arm (ODA) complexes. Using the protozoan Tetrahymena , Mali et al. identified a factor, which they name Shulin, that binds newly synthesized ODAs. Cryo–electron microscopy revealed how Shulin locks the dynein motors together by shutting off motor activity and facilitating delivery of ODAs from the cytoplasm to their final position in the cilia. Science , this issue p. [910][1] [1]: /lookup/doi/10.1126/science.abe0526

No hype in hyperspace

Targeted transport of intracellular vesicular cargos is mediated by myosin, dynein, and kinesin molecular motors operating on actin and microtubule cytoskeletal filaments (1–3). In this issue of Biophysical Journal, a new and powerful method is introduced to dynamically resolve the three-dimensional position and orientation of vesicles to help quantify and categorize these motions (4). The dynamic organization of the cytoskeleton is complex, with actin filaments and microtubules interacting throughout the cytoplasm.

Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking

ABSTRACT It is now clear that the intercellular transport on microtubules by dynein and kinesin-1 motors has an important role in the replication and spread of many viruses. Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded RNA virus of the Coronavirus family, which can infect swine of all ages and cause severe economic losses in the swine industry. Elucidating the molecular mechanisms of the intercellular transport of PEDV through microtubule, dynein and kinesin-1 will be crucial for understanding its pathogenesis. Here, we demonstrate that microtubule, dynein, and kinesin-1 are involved in PEDV infection and can influence PEDV fusion and accumulation in the perinuclear region but cannot affect PEDV attachment or internalization. Furthermore, we adopted a single-virus tracking technique to dynamically observe PEDV intracellular transport with five different types: unidirectional movement toward microtubule plus ends; unidirectional movement toward microtubule minus ends; bidirectional movement along the same microtubule; bidirectional movement along different microtubules and motionless state. Among these types, the functions of dynein and kinesin-1 in PEDV intercellular transport were further analyzed by single-virus tracking and found that dynein and kinesin-1 mainly transport PEDV to the minus and plus ends of the microtubules, respectively; meanwhile, they also can transport PEDV to the opposite ends of the microtubules different from their conventional transport directions and also coordinate the bidirectional movement of PEDV along the same or different microtubules through their cooperation. These results provided deep insights and references to understand the pathogenesis of PEDV as well as to develop vaccines and treatments.

Regulation of lysosomal positioning via TMEM55B phosphorylation.

Lysosomes are dynamic organelles that are transported along microtubules bidirectionally via kinesin and dynein motor proteins. Lysosomal positioning, which is determined by the balance of the bidirectional lysosomal movement, changes under various conditions and affects lysosomal functions such as autophagy and antigen presentation. A recent study by Takemasu et al. (Phosphorylation of TMEM55B by Erk/MAPK regulates lysosomal positioning. J. Biochem. 2019; 166:175-185) has shown that phosphorylation of the transmembrane protein TMEM55B is involved in the retrograde lysosomal trafficking towards the perinuclear region. They found that TMEM55B is phosphorylated upon stimulation with various ligands and that Erk/MAPK mediates the TMEM55B phosphorylation. They have also revealed that a phosphorylation mimic mutant of TMEM55B enhances perinuclear lysosomal clustering compared to the wild-type TMEM55B. These findings suggest that TMEM55B phosphorylation by Erk/MAPK is responsible for regulating lysosomal positioning in response to external stimuli.

Dynein regulates Kv7.4 channel trafficking from the cell membrane.

The dynein motor protein transports proteins away from the cell membrane along the microtubule network. Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2-Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1-mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae.

The conserved autoimmune-disease risk gene TMEM39A regulates lysosome dynamics

TMEM39A encodes an evolutionarily conserved transmembrane protein and carries single-nucleotide polymorphisms associated with increased risk of major human autoimmune diseases, including multiple sclerosis. The exact cellular function of TMEM39A remains not well understood. Here, we report that TMEM-39, the sole Caenorhabditis elegans (C. elegans) ortholog of TMEM39A, regulates lysosome distribution and accumulation. Elimination of tmem-39 leads to lysosome tubularization and reduced lysosome mobility, as well as accumulation of the lysosome-associated membrane protein LMP-1. In mammalian cells, loss of TMEM39A leads to redistribution of lysosomes from the perinuclear region to cell periphery. Mechanistically, TMEM39A interacts with the dynein intermediate light chain DYNC1I2 to maintain proper lysosome distribution. Deficiency of tmem-39 or the DYNC1I2 homolog in C. elegans impairs mTOR signaling and activates the downstream TFEB-like transcription factor HLH-30. We propose evolutionarily conserved roles of TMEM39 family proteins in regulating lysosome distribution and lysosome-associated signaling, dysfunction of which in humans may underlie aspects of autoimmune diseases.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Coiled-Coil Registry Shifts in the F684I Mutant of Bicaudal D Result in Cargo-Independent Activation of Dynein Motility

Key questions in the dynein field are how cargo is recognized by dynein adaptors, such as Drosophila Bicaudal D (BicD) and how dynein is activated for processive motility by these adaptors. BicD is auto-inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. Here, we show that the full-length BicD/F684I mutant activates dynein processivity in a cargo-independent manner, in contrast to the wild type. To obtain mechanistic insights into activation, we determined the X-ray structure of the C-terminal domain of the BicD/F684I mutant, which reveals a coiled-coil registry shift; in the N-terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild-type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. Based on our results, we propose that a coiled-coil registry shift upon cargo binding activates BicD for dynein recruitment. Activation of dynein adapters such as BicD is a key regulatory step for dynein-dependent transport, since they are required to activate dynein for processive motility. Moreover, the human homolog of the BicD/F684I mutant (BicD2/F743I) exhibits diminished binding of cargo adaptor Nup358, implying that a coiled-coil registry shift may be a mechanism to modulate cargo selection for BicD2-dependent transport pathways. Our results provide new mechanistic insights into how the dynein adapter BicD2 recognizes its cargo Nup358. This Nup358/BicD2 pathway serves to position the nucleus with respect to the centrosome and is essential for a fundamental process in brain development that is required for radial glial progenitor cells to differentiate, which are precursors for the majority of neurons and glia cells in the vertebrate neocortex.

Opposing Motors Provide Mechanical and Functional Robustness in the Mammalian Spindle

The spindle is a macromolecular machine assembled from microtubules and motors to accurately segregate the genome at each cell division. How motors together give rise to the mammalian spindle's emergent architecture, mechanics, and function remains poorly understood. The motors dynein and Eg5 are each key to bipolar spindle formation: dynein mediates contractile microtubule minus-end clustering, and Eg5 drives extensile microtubule sliding. Yet when both are inhibited, the spindle can still establish its normal shape.

Adaptor Proteins for Opposing Motors Interact Simultaneously to Nuclear Pore Protein Nup358

Nup358 is a protein subunit of the nuclear pore complex that recruits the opposing microtubule motors kinesin-1 and dynein (via the dynein adaptor Bicaudal D2, BicD2) to the nuclear envelope. This pathway is important for positioning of the nucleus during early steps of mitotic spindle assembly and it is also essential for an important process in brain development. It is unknown whether dynein and kinesin-1 interact with Nup358 simultaneously or whether they compete. Here, we have reconstituted and characterized a minimal complex of kinesin-1 light chain 2 (KLC2) and Nup358.

The many modes of flagellar and ciliary beating: Insights from a physical analysis

The mechanism that allows the axoneme of eukaryotic cilia and flagella to produce both helical and planar beating is an enduring puzzle. The nine outer doublets of eukaryotic cilia and flagella are arranged in a circle. Therefore, each doublet pair with its associated dynein motors, should produce torque to bend the flagellum in a different direction. Sequential activation of each doublet pair should, therefore result in a helical bending wave. In reality, most cilia and flagella have a well‐defined bending plane and many exhibit an almost perfectly flat (planar) beating pattern. In this analysis we examine the physics that governs flagellar bending, and arrive at two distinct possibilities that could explain the mechanism of planar beating. Of these, the mechanism with the best observational support is that the flagellum behaves as two ribbons of doublets interacting with a central partition. We also examine the physics of torsion in flagella and conclude that torsion could play a role in transitioning from a planar to a helical beating modality in long flagella. Lastly, we suggest some tests that would provide theoretical and/or experimental evaluation of our proposals.

Evaluation of Microtubules Binary Collisions using Nano-Patterned Kinesins

Collective motion is a ubiquitous phenomenon manifested in a wide range of living systems ranging from bacterial swarming to animal flocking. The transition from a random distribution to an ordered state resulting in large scale patterns is a fascinating feature demonstrated in collective motion. The reconstituted biomolecular motor system of microtubules propelled by kinesin or dynein motor proteins has been envisioned as an ideal candidate to investigate underlies mechanism of collective motion in vitro. The earlier studies on microtubule/kinesin gliding assay suggest that the alignment of microtubules in binary collisions plays an indispensable factor in achieving collective motion. Therefore, in our ongoing research, we are studying the alignment interactions between microtubule filaments by controlling kinesin number and spacing using gold nanopillars fabricated on the SiO2. To assess the alignment interaction we studied the binary collision events of the microtubules and found colliding microtubules align with each other with high probability at low kinesin spacing. However, with the increase in kinesin spacing microtubules preferred to show overlapping in collisions. Additionally, we measured other parameters related to the binary collisions like the incoming collision angle (i.e., the angle between two approaching microtubules). We found that the distribution of microtubules incoming angle depends on kinesin spacing and arrangements. Our analysis show that at higher spacing microtubules prefer to collide orthogonally and resulted in overlapping events. We also investigated the effect of other parameters like microtubules flexural rigidity and found no significant effects on alignment interactions. Finally, we increased the concentration of microtubules and observed the emergence of bundles and stream patterns in collective motion depending on microtubule concentrations without any influence of depleting or binding agents. This study may provide new insights in understanding the collective motion in nature.

Dynein Adaptors Such as Drosophila Bicaudal (Dm BicD) Recognize Cargo and are Required to Activate Dynein for Processive Transport

In the absence of cargo, auto-inhibited BicD cannot recruit dynein, but the underlying mechanism of cargo-induced activation is elusive. Our single-molecule processivity assays show that auto-inhibition is abolished in the full-length Bicaudal mutant F684I, which activates dynein motility even in the absence of cargo. To investigate the structural basis for activation, we determined the X-ray structure of the C-terminal cargo-binding domain (CTD) of Dm BicD-CTD/F684I at 2.4 Å resolution. The structure revealed that the mutant has a homotypic coiled-coil registry, in which the two helices of the homodimer are aligned, whereas the wild-type structure has an asymmetric registry, in which the two chains are vertically shifted by ∼1 helical turn in a portion of the coiled-coil. In the mutant, N- terminal ∼20-residue region is disordered for one of the two chains. MD simulation and CD spectroscopy data suggested the structural flexibility of N-terminal region of Dm BicD-CTD/F684I and confirmed the intact protein is in the crystal and alpha-helical. We propose that a coiled-coil registry shift activates BicD for dynein recruitment. The registry shift could either be induced by the F684I mutation or by cargo-binding. Also, the human homolog of the BicD-CTD/F684I mutant (BicD2-CTD/F743I) showed diminished binding to its cargo Nup358. Thus, we propose that in mammalia, a coiled-coil registry shift modulates cargo selection for BicD2-dependent transport pathways, which are important for brain development and chromosome segregation. Furthermore, we investigated the mechanism for cargo recognition by BicD2. Our data from Circular Dicroism spectroscopy and small-angle X-ray scattering suggest that the BicD2 binding domain of Nup358, which is intrinsically disordered on its own, takes on a helical structure when binding to BicD2. This alpha-helix of Nup358 could be a structural feature for cargo recognition by BicD2.

Identification of Interface Residues of the Rab6/BicD2 Complex

Rab6 is a GTPase that is embedded in distinct secretory and Golgi-derived vesicles, where it recruits factors involved in vesicle transport and fusion. One of these proteins is the dynein adaptor BicD2, a homodimeric coiled-coil, which forms a complex with Rab6, thereby linking Rab6-decorated vesicles to the minus-end directed motor dynein for transport of the vesicles. How these two proteins interact remains unknown. In this work, a cluster of several Rab6 key interface residues was identified, which is important for the interaction with BicD2. We show that mutation of these residues abolishes binding to BicD2. These residues are also highly conserved and are located in a region that undergoes structural changes upon GTP-binding. Our preliminary data promises to reveal key mechanistic details about the poorly understood interaction between Rab6 and BicD2.

The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans

Cell scientist to watch – Anthony Roberts

ABSTRACT Anthony Roberts studied biochemistry at Imperial College London, UK. He then pursued a PhD in molecular and cellular biology with Peter Knight and Stan Burgess at the University of Leeds, UK, where he studied the mechanism of the dynein motor protein. After this, Anthony moved to Boston, USA, for his postdoctoral work with Samara Reck-Peterson, at the Harvard Medical School, focussing on cytoplasmic dynein regulation. In late 2014, he returned to London, UK to start his own lab at the Institute of Structural and Molecular Biology at Birkbeck, University of London, and University College London (UCL), where he is now a Wellcome Trust Senior Research Fellow and Proleptic Senior Lecturer. Anthony received the Biochemical Society Early Career Research Award in 2016, a Biotechnology and Biological Sciences Research Council New Investigator Award in 2017, and was elected to the EMBO Young Investigator Programme in 2018. His lab is focussing on the mechanisms of microtubule-based transport within cilia and flagella.