Mammalian Cytoplasmic Dynein Research Articles

Nano-particles carried by multiple dynein motors self-regulate their number of actively participating motors.

The application of nanotechnology in medicine is expected to spread rapidly. Many substances are now being researched for drug delivery and, more especially, cancer therapy and dealing with various viruses such as HIV, Herpes simplex virus, and adenoviruses. Since it is the center of gene maintenance and expression regulation, the cell nucleus is a valuable target for viruses of the type outlined above as well as drug delivery systems. The microtubule-associated motor protein dynein is primarily responsible for spontaneously occurring intracellular transport to the nucleus. In this case, we are inspired by viruses (for example, HIV) that recruit dyneins to actively move towards the nucleus. This type of transport is known as “active transport,” and it is distinguished by its directionality and persistence motion toward the nucleus, making it more effective... In this project we aim to mimic the highly efficient transportation properties of native cargoes by rationally designing universally decorated nano-particles (NPs) that could be used for drug delivery applications. We investigate the motion of artificial NPs which grafted with flexible polymers, each ending with a nuclear localization signal (NLS) peptide, thereby allowing recruitment of mammalian cytoplasmic dynein. This unique NP can self-regulate the number of participating dynein motor proteins to optimize their motility properties - engendering persistent motion towards the cell nucleus while being able to go around obstacles that are abundant in the cytoplasm or on the microtubules. Based on these benefits, the use of active transport rather than passive transport has a promising future for therapeutic reasons, both in terms of treatment efficiency and reduced side effects, as the amounts of drug/DNA necessary for therapy are reduced.

A model for the chemomechanical coupling of the mammalian cytoplasmic dynein molecular motor.

Available single-molecule data have shown that some mammalian cytoplasmic dynein dimers move on microtubules with a constant step size of about 8.2nm. Here, a model is presented for the chemomechanical coupling of these mammalian cytoplasmic dynein dimers. In contrast to the previous models, a peculiar feature of the current model is that the rate constants of ATPase activity are independent of the external force. Based on this model, analytical studies of the motor dynamics are presented. With only four adjustable parameters, the theoretical results reproduce quantitatively diverse available single-molecule data on the force dependence of stepping ratio, velocity, mean dwell time, and dwell-time distribution between two mechanical steps. Predicted results are also provided for the force dependence of the number of ATP molecules consumed per mechanical step, indicating that under no or low force the motors exhibit a tight chemomechanical coupling, and as the force increases the number of ATPs consumed per step increases greatly.

Activation of mammalian cytoplasmic dynein in multimotor motility assays

Long-range intracellular transport is facilitated by motor proteins, such as kinesin-1 and cytoplasmic dynein, moving along microtubules (MTs). These motors often work in teams for the transport of various intracellular cargos. Although transport by multiple kinesin-1 motors has been studied extensively in the past, collective effects of cytoplasmic dynein are less well understood. On the level of single molecules, mammalian cytoplasmic dynein is not active in the absence of dynactin and adaptor proteins. However, when assembled into a team bound to the same cargo, processive motility has been observed. The underlying mechanism of this activation is not known. Here, we found that in MT gliding motility assays the gliding velocity increased with dynein surface density and MT length. Developing a mathematical model based on single-molecule parameters, we were able to simulate the observed behavior. Integral to our model is the usage of an activation term, which describes a mechanical activation of individual dynein motors when being stretched by other motors. We hypothesize that this activation is similar to the activation of single dynein motors by dynactin and adaptor proteins.This article has an associated First Person interview with the first author of the paper.

First person – Gina Monzon

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Gina Monzon is co-first author on ‘Activation of mammalian cytoplasmic dynein in multimotor motility assays’, published in Journal of Cell Science. Gina is a PhD student in the lab of Ludger Santen at Saarland University, Saarbrücken, Germany, studying theoretical modelling of intracellular microtubule-associated transport by kinesin and dynein motor proteins.

The Effect of Temperature on Microtubule-Based Transport by Cytoplasmic Dynein and Kinesin-1 Motors

Cytoplasmic dynein and kinesin are both microtubule-based molecular motors but are structurally and evolutionarily unrelated. Under standard conditions, both move with comparable unloaded velocities toward either the microtubule minus (dynein) or plus (most kinesins) end. This similarity is important because it is often implicitly incorporated into models that examine the balance of cargo fluxes in cells and into models of the bidirectional motility of individual cargos. We examined whether this similarity is a robust feature, and specifically whether it persists across the biologically relevant temperature range. The velocity of mammalian cytoplasmic dynein, but not of mammalian kinesin-1, exhibited a break from simple Arrhenius behavior below 15°C—just above the restrictive temperature of mammalian fast axonal transport. In contrast, the velocity of yeast cytoplasmic dynein showed a break from Arrhenius behavior at a lower temperature (∼8°C). Our studies implicate cytoplasmic dynein as a more thermally tunable motor and therefore a potential thermal regulator of microtubule-based transport. Our theoretical analysis further suggests that motor velocity changes can lead to qualitative changes in individual cargo motion and hence net intracellular cargo fluxes. We propose that temperature can potentially be used as a noninvasive probe of intracellular transport.

Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces

Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by kinesin-1 and kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.

Identification of mutations in DYNC2LI1, a member of the mammalian cytoplasmic dynein 2 complex, expands the clinical spectrum of Jeune/ATD ciliopathies

Ciliopathies are caused by defects in formation, maintenance and function of the primary cilium and underlying genes affect the dynein motor, intraflagellar transport complexes, or the basal body. In a patient of non-consanguineous parents presenting an intermediate phenotype between asphyxiating thoracic dystrophy and Ellis-van Crefeld syndrome we performed exome sequencing. Variants were selected based on potential ciliary function as identified in a yeast two-hybrid screen with NEK1, a basal body protein involved in short rib-polydactyly type Majeweski (SRPSII). We identified compound heterozygous nonsense (p.R208X) and missense (p.T221I) mutations in DYNC2LI1 segregating in the family. DYNC2LI1 is ubiquitously expressed and interacts with DYNC2H1 to form the dynein 2 complex important for retrograde intraflagellar transport. The hypothetical protein caused by the nonsense mutation lacks the coiled-coil domain involved in protein interaction and dimerization. The mutation p.T221I affects a highly conserved nucleoside triphosphate hydrolase domain responsible for GTPase driven dynein protein localization. Mutations in both DYNC2LI1 interacting partners DYNC2H1 and NEK1 are associated with ATD and SRPSs. We screened further patients of our short stature cohort and identified in two siblings heterozygous mutations in DYNC2LI1 (p.M1T) and its interaction partner DYNC2H1 (p.K495T). The DYNC2H1 mutation was previously reported by El Hokayem et al. compound heterozygous with a splice site mutation in a patient with SRPSII. Our results might indicate a possible digenic diallelic inheritance in our patients. This is the first report of mutations in DYNC2LI1 as part of the dynein 2 complex further expanding the clinical spectrum of ciliopathies.

Self-Regulation of Cytoplasmic Dynein through its Unconventional Force Response

Cytoplasmic dynein is essential for a wide range of cellular activities, including intracellular transport, cell division, and cell migration in most of eukaryotic organisms. To achieve these various functions, dynein activity must be tightly controlled. However, the motility of mammalian cytoplasmic dynein has been controversial in previous studies, which makes it unclear how dynein activity is regulated and tuned for a specific function. Here, using electron microscopy and DNA nanostructure-based motility assays, we investigated how the control of dynein activity is achieved. We showed that single dynein molecules diffused along microtubules in an autoinhibited state, in which two motor heads were stacked together. This state was released when multiple dynein molecules worked together on a single cargo or when dynein was pulled by an optical tweezer, suggesting that individual dynein molecules in the team were activated through destabilization of the stacked conformation by mechanical strain generated between dynein molecules. We confirmed this force-dependent activation mechanism by observing the movement of a chimeric dynein fused with a inactive kinesin, which we assume acts as a load. This mechanism would function at a fundamental level of dynein regulation that does not require an external regulator, thereby serving as a stable basis for the higher-level regulation of dynein-driven transport.

The Mechanism of the Transition from Diffusive to Directed Movement in Mammalian Cytoplasmic Dynein

Cytoplasmic dynein is a minus-end-directed molecular motor involving in various cellular functions. Mammalian cytoplasmic dynein has been reported to exhibit unidirectional movements for several micrometers in vivo (Kobayashi, 2009). However, some in vitro studies have reported that single molecules of cytoplasmic dynein/dynactin complex showed biased diffusive movements along microtubules (Ross, 2006; Miura, 2010), and a recent study has demonstrated that an mRNP complex bound by a few dyneins displayed bidirectional diffusive movements (Amrute-Nayak, 2012). To reveal the mechanism of the transition from diffusive to directed movement in cytoplasmic dynein, we designed recombinant dyneins using HEK293 cell expressing system. An artificially dimerized, tail-truncated human cytoplasmic dynein 1 (DHC380) showed unidirectional movement, whereas full-length dyneins purified from the same system displayed biased diffusion towards the microtubule minus end. Furthermore, using several other recombinant dyneins that have different head-to-head distances between two motor heads, we discovered that the dynein with longer head-to-head distance contained larger diffusive component. We also observed that multiple full-length dyneins bound to Qdot moved unidirectionally along microtubules, while single dyneins carrying Qdot exhibited diffusive movements. These observations imply that the directed movement of dynein is influenced strongly by the head-to-head distances and the motor number, possibly leading to the regulation of dynein by these factors in the cell.

Reconstitution of the human cytoplasmic dynein complex

Cytoplasmic dynein is the major motor protein responsible for microtubule minus-end-directed movements in most eukaryotic cells. It transports a variety of cargoes and has numerous functions during spindle assembly and chromosome segregation. It is a large complex of about 1.4 MDa composed of six different subunits, interacting with a multitude of different partners. Most biochemical studies have been performed either with the native mammalian cytoplasmic dynein complex purified from tissue or, more recently, with recombinant dynein fragments from budding yeast and Dictyostelium. Hardly any information exists about the properties of human dynein. Moreover, experiments with an entire human dynein complex prepared from recombinant subunits with a well-defined composition are lacking. Here, we reconstitute a complete cytoplasmic dynein complex using recombinant human subunits and characterize its biochemical and motile properties. Using analytical gel filtration, sedimentation-velocity ultracentrifugation, and negative-stain electron microscopy, we demonstrate that the smaller subunits of the complex have an important structural function for complex integrity. Fluorescence microscopy experiments reveal that while engaged in collective microtubule transport, the recombinant human cytoplasmic dynein complex is an active, microtubule minus-end-directed motor, as expected. However, in contrast to recombinant dynein of nonmetazoans, individual reconstituted human dynein complexes did not show robust processive motility, suggesting a more intricate mechanism of processivity regulation for the human dynein complex. In the future, the comparison of reconstituted dynein complexes from different species promises to provide molecular insight into the mechanisms regulating the various functions of these large molecular machines.

2A1546 哺乳類細胞質ダイニンが一方向性を獲得する機構について(分子モーターII:ダイニン,口頭発表,日本生物物理学会第50回年会(2012年度))

2A1546 哺乳類細胞質ダイニンが一方向性を獲得する機構について(分子モーターII:ダイニン,口頭発表,日本生物物理学会第50回年会(2012年度))

2M1424 哺乳類細胞質ダイニンが示す拡散と一方向性の運動の転換について(分子モーター3,第49回日本生物物理学会年会)

2M1424 哺乳類細胞質ダイニンが示す拡散と一方向性の運動の転換について(分子モーター3,第49回日本生物物理学会年会)

Nucleotide-dependent behavior of single molecules of cytoplasmic dynein on microtubules in vitro

We visualized the nucleotide-dependent behavior of single molecules of mammalian native cytoplasmic dynein using fragments of dynactin p150 with or without its N-terminal microtubule binding domain. The results indicate that the binding affinity of dynein for microtubules is high in AMP-PNP, middle in ADP or no nucleotide, and low in ADP·Pi conditions. It is also demonstrated that the microtubule binding domain of dynactin p150 maintains the association of dynein with microtubules without altering the motile property of dynein in the weak binding state. In addition, we observed bidirectional movement of dynein in the presence of ATP as well as in ADP/Vi condition, suggesting that the bidirectional movement is driven by diffusion rather than active transport.

Cytoplasmic dynein nomenclature.

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.

The Roadblock Light Chain Binds a Novel Region of the Cytoplasmic Dynein Intermediate Chain

Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.

Light chains of mammalian cytoplasmic dynein: Identification and characterization of a family of LC8 light chains. Cell Motil. Cytoskeleton 49:229‐240 (2001)

AbstractDue to an oversight, the dynein light chain, roadblock‐1, was labeled as roadblock‐2 in our paper.

Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2.

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo.

Light chains of mammalian cytoplasmic dynein: identification and characterization of a family of LC8 light chains

Cytoplasmic dynein is a large multisubunit motor protein that moves various cargoes toward the minus ends of microtubules. In addition to the previously identified heavy, intermediate, and light intermediate chains, it has recently been recognized that cytoplasmic dynein also has several light chain subunits with apparent molecular weights between 8-20 kDa. To systematically identify the light chains of purified rat brain cytoplasmic dynein, peptide sequences were obtained from each light chain band resolved by gel electrophoresis. Both members of the tctex1 light chain family, tctex1 and rp3, were identified in a single band. Only one member of the roadblock family, roadblock-2, was found. Two members of the LC8 family were resolved as separate bands, the previously identified LC8 subunit, and a second novel cytoplasmic dynein family member, LC8b. The tissue distribution of these two dynein LC8 subunits differed, although LC8b was the major family member in brain. Database searches found that both LC8a and LC8b were also present in several mammalian species, and a third mammalian LC8 sequence, LC8c was found in the human database. The amino acid sequences of both LC8a and LC8b were completely conserved in mammals. LC8a and LC8b differ in only six of the 89 amino acids. The amino acid differences between LC8a and LC8b were located near the N-terminus of the molecules, and most were in the outward facing alpha-helices of the LC8 dimer. When the mammalian LC8a sequence was compared to the LC8 sequences found in six other animal species including Xenopus and Drosophila, there was, on average, 94% sequence identity. More variation was found in LC8 sequences obtained from plants, fungi, and parasites. LC8c differed from the other two human LC8 sequences in that it has amino acid substitutions in the intermediate chain binding domain at the C-terminal of the molecule. The position of amino acid substitutions of the three mammalian LC8 family members is consistent with the hypothesis that they bind to different proteins.

Biochemical and Molecular Analysis of the Mammalian Cytoplasmic Dynein Intermediate Chain

Cytoplasmic dynein is a multisubunit protein complex responsible for the intracellular movement of membranous organelles and other cargo along microtubules. The heavy chains contain the motor domains, while the intermediate chain and other subunits are important for binding to cargo. There are at least five different intermediate chain polypeptides, the products of alternative splicing of two genes. The cytoplasmic dynein intermediate chains are also phosphorylated. The expression of the different intermediate chain mRNAs is characterized by reverse transcription-polymerase chain reactions using oligonucleotide primers appropriate for the alternative splicing sites. The presence of the different intermediate chain polypeptide isoforms is determined by two-dimensional gel analysis of cytoplasmic dynein samples. The phosphorylation state of the polypeptides is determined by treatment of immunoprecipitated cytoplasmic dynein with protein phosphatase and analysis of changes in polypeptide spot distribution after two-dimensional gel electrophoresis.

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K m, ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K m, ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.