Cytoskeletal Motor Research Articles

Nanoparticles engineered to bind cellular motors for efficient delivery

BackgroundDynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications.ResultsHere, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections.ConclusionsThe capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Sperm Dynein AAA1 and AAA2 Expression in Human Sperm : A Regulation in Sperm Preparation

Axoneme structures in sperm tail, is a supramolecular protein complex with motor protein and regulatory which playing a crucial role in determining sperm motility. Dynein, one of the three members of cytoskeletal motor protein, has a ring of six AAA+ which linked together into one large polypeptide that contribute to the formation of sperm flagella bending. The previously research reported that the first two AAA motor domains, AAA1 and AAA2, were a major site in ATP hydrolysis associated with motility in the flagellum. Intrauterine Insemination (IUI) as a management of infertility requires sperm preparation procedures, by Swim-up (SU) and Density Gradient Centrifugation (DGC), in order to enhance the quality regarding concentration and motility of the initial sperm. This study aimed to evaluate the efficiency of the DGC and SU methods in selecting sperm, based the expression of sperm dynein AAA1 and AAA2. Semen samples were obtained from men underwent sperm preparation for IUI and divided into two groups, normozoospermia and asthenozoospermia, according to World Health Organization 2010 guideline. Semen analysis was performed to measure the sperm motility and velocity, before and after sperm preparation. The axoneme was isolated from the obtained samples from SU and DGC methods, while the level of AAA1 and AAA2 was measured by ELISA. This study showed that the percentage of motile sperm and velocity of prepared sperm in both groups in prepared sperm (post-SU and post-DGC) was higher compared to whole semen. The expression of sperm dynein AAA1 of prepared sperm in normozoospermia group showed higher, while in asthenozoospermia group showed lower activities compared to whole semen. The expression of sperm dynein AAA2 of prepared sperm in both groups showed lower activities compared to whole semen. The sperm preparation enhanced the quality of sperm and may increase the expression of sperm dynein AAA1 compared to the whole semen, without the involvement of sperm dynein AAA2.

Energetic Costs, Precision, and Transport Efficiency of Molecular Motors.

An efficient molecular motor would deliver cargo to the target site at a high speed and in a punctual manner while consuming a minimal amount of energy. According to a recently formulated thermodynamic principle, referred to as the thermodynamic uncertainty relation, the travel distance of a motor and its variance are, however, constrained by the free energy being consumed. Here we use the principle underlying the uncertainty relation to quantify the transport efficiency of molecular motors for varying ATP concentration ([ATP]) and applied load (f). Our analyses of experimental data find that transport efficiencies of the motors studied here are semioptimized under the cellular condition. The efficiency is significantly deteriorated for a kinesin-1 mutant that has a longer neck-linker, which underscores the importance of molecular structure. It is remarkable to recognize that, among many possible directions for optimization, biological motors have evolved to optimize the transport efficiency in particular.

Overview of the mechanism of cytoskeletal motors based on structure.

In the last two decades, a wealth of structural and functional knowledge has been obtained for the three major cytoskeletal motor proteins, myosin, kinesin and dynein, which we review here. The cytoskeletal motor proteins myosin and kinesin are structurally similar in the core architecture of their motor domains and have similar force-producing mechanisms that are coupled with the chemical cycles of ATP binding, hydrolysis, Pi release and subsequent ADP release. The force is generated through conformational changes in the motor domain during Pi release and ATP binding in myosin and kinesin, respectively, and then converted into the rotation of the lever arm or neck linker (referred to as a power stroke) through the common structural pathways. On the other hand, the dynein cytoskeletal motor is an AAA+ protein and has a different structure and power stroke mechanism from those of myosins and kinesins. The linker protruding from the AAA+ ring of dynein swings according to the ATPase states, which, presumably, generates force to carry cargos within a cell. The communication mechanism between the track-binding and ATPase domains of dynein is unique because the two helices that presumably slide with respect to each other work as coordinators for these domains. Details of the mechanism underlying the power stroke and interdomain communication were revealed through recent progress in the structural studies of myosin, kinesin and dynein.

Challenges in Estimating the Motility Parameters of Single Processive Motor Proteins

Cytoskeletal motor proteins are essential to the function of a wide range of intracellular mechano-systems. The biophysical characterization of their movement along their filamentous tracks is therefore of large importance. Toward this end, single-molecule, in vitro stepping-motility assays are commonly used to determine motor velocity and run length. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental conditions and the data analysis methods. Here, we investigate the movement of fluorescently labeled, processive, dimeric motor proteins and propose a unified algorithm to correct the measurements for finite filament length as well as photobleaching. Particular emphasis is put on estimating the statistical errors associated with the proposed evaluation method, as knowledge of these values is crucial when comparing measurements from different experiments. Testing our approach with simulated and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules, we show 1) that velocity distributions should be fitted by a t location-scale probability density function rather than by a normal distribution; 2) that the impossibility to measure events shorter than the image acquisition time needs to be taken into account; 3) that the interaction time and run length of the motors can be estimated independent of the filament length distribution; and 4) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. Moreover, our analysis reveals that controlling the temperature during the experiments with a precision below 1 K is of importance. We believe our method will not only improve the evaluation of experimental data, but also allow for better statistical comparisons between different populations of motor proteins (e.g., with distinct mutations or linked to different cargos) and filaments (e.g., in distinct nucleotide states or with different posttranslational modifications). Therefore, we include a detailed workflow for image processing and analysis (including MATLAB code), serving as a tutorial for the estimation of motility parameters in stepping-motility assays.

Role of Interactions and Correlations on Collective Dynamics of Molecular Motors Along Parallel Filaments

Cytoskeletal motors known as motor proteins are molecules that drive cellular transport along several parallel cytoskeletal filaments and support many biological processes. Experimental evidences suggest that they interact with the nearest molecules of their filament while performing mechanical work. To understand such mechanism theoretically, a new version of two-channel totally asymmetric simple exclusion process which incorporates interactions in a thermodynamically consistent way, is introduced. As the existing approaches for multi-channel systems deviate from analyzing the effect of inter and intra channel interactions, a new approach known as modified vertical cluster mean field is developed. The approach along with monte-carlo simulations successfully encounters some correlations and computes all complex dynamic properties of the system. Role of symmetry of interactions and inter-channel coupling is observed on triple points and particle maximal current. Surprisingly, for each coupling rate and most of the interaction splittings, there corresponds an optimal interaction strength for the maximal current which belongs to the case of weak repulsive interactions. Moreover, for a fixed interaction splitting, as coupling rate increases, the optimal strength decreases and tends towards the experimental predictions. Correlations are found to be short-range and weaker for repulsive and weak attractive interactions, while for stronger attractions they are long-range and stronger. All these findings are also discussed in the context of experimental observations.

Kinesin rotates unidirectionally and generates torque while walking on microtubules

Cytoskeletal motors drive many essential cellular processes. For example, kinesin-1 transports cargo in a step-wise manner along microtubules. To resolve rotations during stepping, we used optical tweezers combined with an optical microprotractor and torsion balance using highly birefringent microspheres to directly and simultaneously measure the translocation, rotation, force, and torque generated by individual kinesin-1 motors. While, at low adenosine 5'-triphosphate (ATP) concentrations, motors did not generate torque, we found that motors translocating along microtubules at saturating ATP concentrations rotated unidirectionally, producing significant torque on the probes. Accounting for the rotational work makes kinesin a highly efficient machine. These results imply that the motor's gait follows a rotary hand-over-hand mechanism. Our method is generally applicable to study rotational and linear motion of molecular machines, and our findings have implications for kinesin-driven cellular processes.

Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein.

Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein's molecular mechanism through the combination of structure-function and single-molecule studies. Single-molecule fluorescence assays have provided detailed insights into how dynein advances along its microtubule track in the absence of load, while optical tweezers have yielded insights into the force generation and stalling behavior of dynein. Here, using the S. cerevisiae expression system, we provide improved protocols for the generation of dynein mutants and for the expression and purification of the mutated and/or tagged proteins. To facilitate single-molecule fluorescence and optical trapping assays, we further describe updated, easy-to-use protocols for attaching microtubules to coverslip surfaces. The presented protocols together with the recently solved crystal structures of the dynein motor domain will further simplify and accelerate hypothesis-driven mutagenesis and structure-function studies on dynein.

Collaboration between Distinct Rab Small GTPase Trafficking Circuits Mediates Bacterial Clearance from the Bladder Epithelium

Rab small GTPases control membrane trafficking through effectors that recruit downstream mediators such as motor proteins. Subcellular trafficking typically involves multiple Rabs, with each specific step mediated by a distinct Rab protein. We describe a collaboration between two distinct Rab-protein-orchestrated trafficking circuits in bladder epithelial cells (BECs) that expels intracellular uropathogenic Escherichia coli (UPEC) from their intracellular niche. RAB11a and RAB27b and their trafficking circuitry are simultaneously involved in UPEC expulsion. While RAB11a recruits its effector RAB11FIP3 and cytoskeletal motor Dynein, RAB27b mobilizes the effector MyRIP and motor Myosin VIIa to mediate bacterial expulsion. This collaboration is coordinated by deposition of the exocyst complex on bacteria-containing vesicles, an event triggered by the innate receptor Toll-like receptor 4. Both RAB11a and RAB27b are recruited and activated by the exocyst complex components SEC6/SEC15. Thus, the cell autonomous defense system can mobilize and coalesce multiple subcellular trafficking circuitries to combat infections.

Alignment of actin filament streams driven by myosin motors in crowded environments

BackgroundCellular dynamics depend on cytoskeletal filaments and motor proteins. Collective movements of filaments driven by motor proteins are observed in the presence of dense filaments in in vitro systems. As multiple macromolecules exist within cells and the physiological ionic conditions affect their interactions, crowding might contribute to ordered cytoskeletal architecture because of collective behavior. MethodsUsing an in vitro reconstituted system, we observed the emergence of stripe patterns resulting from collective actin filament streaming driven by myosin motors in the presence of the crowding agent, methylcellulose (MC). ResultsAlthough at high KCl concentrations (150mM), actin filaments tended to dissociate from a myosin-coated surface, 1% MC prevented this dissociation and enabled filament movement on myosin molecules. At concentrations of actin filaments above 0.2mg/mL, the moving filaments accumulated and progressively formed long, dense bands. The bands were spaced at about 10-μm intervals. Increasing the KCl concentration up to 300mM resulted in narrowing of the spacing between the aligned bands. On the other hand, low KCl concentrations (≤25mM) induced broad streams, where actin filaments exhibited bidirectional movement. ConclusionsThese results suggest that crowded environments can promote spatial patterning of the actin cytoskeleton, depending on the intensity of the myosin driving force and filament velocity, both modulated by the ionic strength. General significanceThe mutual contribution of packing and driving forces provides insight into cytoskeleton organization in living cells, in which various macromolecules mingle.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

The force-generating mechanism of dynein differs from the force-generating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.

Microtubule Motors in Establishment of Epithelial Cell Polarity

Epithelial cells play a key role in insuring physiological homeostasis by acting as a barrier between the outside environment and internal organs. They are also responsible for the vectorial transport of ions and fluid essential to the function of many organs. To accomplish these tasks, epithelial cells must generate an asymmetrically organized plasma membrane comprised of structurally and functionally distinct apical and basolateral membranes. Adherent and occluding junctions, respectively, anchor cells within a layer and prevent lateral diffusion of proteins in the outer leaflet of the plasma membrane and restrict passage of proteins and solutes through intercellular spaces. At a fundamental level, the establishment and maintenance of epithelial polarity requires that signals initiated at cell-substratum and cell-cell adhesions are transmitted appropriately and dynamically to the cytoskeleton, to the membrane-trafficking machinery, and to the regulation of occluding and adherent junctions. Rigorous descriptive and mechanistic studies published over the last 50 years have provided great detail to our understanding of epithelial polarization. Yet still, critical early steps in morphogenesis are not yet fully appreciated. In this review, we discuss how cytoskeletal motor proteins, primarily kinesins, contribute to coordinated modification of microtubule and actin arrays, formation and remodeling of cell adhesions to targeted membrane trafficking, and to initiating the formation and expansion of an apical lumen.

SnapShot: Subcellular mRNA Localization

Many cells localize mRNAs to discrete locations in the cytoplasm. Coupled to local translation, this process affords precise spatial and temporal control of protein function. This SnapShot provides an overview of the key events in subcellular mRNA localization and highlights recent progress in understanding how cytoskeletal motors orchestrate mRNA trafficking.

Microtubule motors involved in nuclear movement during skeletal muscle differentiation.

Nuclear positioning is a determining event in several cellular processes, such as fertilization, cell migration, and cell differentiation. The structure and function of muscle cells, which contain hundreds of nuclei, have been shown to rely in part on proper nuclear positioning. Remarkably, in the course of muscle differentiation, nuclear movements along the myotube axis might represent the event required for the even positioning of nuclei in the mature myofiber. Here we analyze nuclear behavior, time in motion, speed, and alignment during myotube differentiation and temporal interference of cytoskeletal microtubule-related motors. Using specific inhibitors, we find that nuclear movement and alignment are microtubule dependent, with 19 microtubule motor proteins implicated in at least one nuclear behavior. We further focus on Kif1c, Kif5b, kif9, kif21b, and Kif1a, which affect nuclear alignment. These results emphasize the different roles of molecular motors in particular mechanisms.

Molecular Characterization of a Membrane-Associated Cytoskeletal Motor Family

Many large morphological changes in the plasma and intracellular membranes are powered by the actions of cytoskeletal motors. These motors include myosin-Is, which are the widely expressed members of the myosin superfamily that bind directly to membranes, linking them to the actin cytoskeleton. To better understand the molecular roles of myosin-I isoforms, and the mechanisms by which they modulate membrane dynamics, we performed a range of single-molecule biophysical, structural, biochemical, and cell biological experiments to determine force-generating, membrane-binding, and motile properties of these motors. We discovered that there is remarkable diversity among myosin-I isoforms in their ability to alter their ATPase activities in response to mechanical forces, and we are learning how structural features within the motor domain lead to chemomechanical tuning. We have also learned much about the biophysics of the attachment of myosin-I to membrane bilayers in the presence and absence of mechanical load. Our understanding of the biophysical properties of myosin-I isoforms has allowed us to further explore their roles in membrane dynamics via multi-motor in vitro assays that reconstitute membrane motility and tubulation. This work is supported by grants from NIH/NIGMS (GM057247 and GM087253).

Myo1C and Endophilin Promote Kinesin-Driven Tubulation at Engineered Cytoskeletal Intersections

In cells, kinesin-driven membrane tubulation along microtubules (MT) typically initiates in regions with a locally high actin filament (AF) density, suggesting that membrane deformations may result from the cooperative activities of kinesin and myosin motors at cytoskeletal intersections. While kinesin-1 motors are likely the primary drivers of tubule extension, membrane-binding myosin-I motors have also been proposed to participate in tubulation and membrane deformation in vivo. We hypothesize that Myo1c anchors membrane vesicles at AF/MT intersections, providing a resistive force that enables effective kinesin-1-driven tubulation. We tested this proposal by attaching kinesin-1 and Myo1c motors to giant unilamellar vesicles (GUVs); a truncated, His-tagged kinesin-1 construct (K560) was bound to NTA(Ni)-functionalized lipids and full length Myo1c was bound to PtdIns(4,5)P2 incorporated into the membrane. GUVs were introduced into chambers with engineered cytoskeletal arrays of sparse MTs bridging stripes densely-coated with short AFs. In the absence of Myo1c, kinesin-driven vesicles paused periodically at AF/MT intersections, but vesicle deformation was not observed. In contrast, GUVs with bound kinesin-1 and Myo1c motors paused at AF/MT intersections and deformed readily along MTs. However, extended tubules of diffraction-limited diameter were rarely observed. Next, we tested whether the addition of a BAR domain protein would facilitate vesicle tubulation by adding the membrane-binding domain of endophilin-A1, which is necessary for many endocytic trafficking tubulation events in vivo. We noted a significant, concentration-dependent, increase in the frequency and length of GUV tubulation events in the presence of endophilin. Importantly, this increased tubulation frequency required both kinesin-1 and Myo1c. These data provide insights into the interplay between cytoskeletal motors and BAR domain proteins in facilitating membrane tubulation. This work was supported by NIH grants GM087253 to E.M.O. and E.L.F.H. and T32-AR053461 to B.B.M.

Revised Model, with Experimental Verification, for Motor Densities in Gliding Assays

Gliding assays are used to investigate cytoskeletal polymers and motor proteins in vitro, including measurements of cytoskeletal polymer stiffness and the cooperative effects of motors. A theoretical model (Duke et al., 1995) developed to extract the biophysical quantities of interest from gliding assays has been used extensively, yet the model remains incompletely validated. In gliding assay experiments using kinesin and microtubules, we found inconsistencies in model-dependent measurements of average kinesin spacing along microtubules, a key parameter for motor cooperativity and bending stiffness interpretations. We traced these inconsistencies to an implicit approximation, an infinite processivity for kinesin. We report an updated theoretical model for gliding assays incorporating the finite processivity of kinesin, and report experimental verification of the active motor density. Deviations between densities inferred from the original model and updated model range from under 25% for short microtubules (0.5 micrometers long) to greater than four-fold for long microtubules (more than 2 micrometers long). We use these revised motor densities to measure the length-dependence of microtubule bending stiffness (persistence length).

Dynein's Direction-Dependent Microtubule-Binding Strength is Controlled via a Tension-Induced Sliding of Dynein's Stalk Helices Mediated by the Coiled-Coil Strut

Cytoskeletal motor protein motility requires coordination of ATPase and filament-binding cycles. Mechanical tension strongly influences these processes, and likely regulates motor stepping as external forces resist motor movement and intramolecular tension develops between motor domains. In cytoplasmic dynein, an AAA+ ATPase, applied tension affects microtubule (MT)-binding strength anisotropically -backward tension induces stronger binding- while in the absence of tension, reconfiguration of the coiled-coil ‘stalk’ (which connects the AAA+ and MT-binding domains) is known to alter MT affinity. Using optical tweezers, mutagenesis, and chemical cross-linking, we show that preventing relative motion of the stalk helices or deleting the ‘strut’ (which emerges from the AAA+ domain and contacts the stalk) both eliminate tension-based regulation of MT-binding strength. Thus, tension alters dynein's MT-binding strength by inducing sliding of the stalk helices, and the strut is a key mediator of this process.

Defective Axonal Transport and Alzheimer's Disease Correlations: A Molecular Motor Point of View

Alzheimer's disease (AD) is the principal factor for dementia during old age; it is characterized by a progressive impairment of cognitive skills. Several genetic and environmental factors contribute for the AD onset. Recently, several evidences were unraveled that correlated defective axonal transport (AT) caused by abnormal axonal swellings to the pathogenesis of AD. AT is an active process that utilizes the cellular cytoskeleton as a road network for the cytoskeletal motor proteins (dyneins and kinesins superfamilies). There are two types of AT: the anterograde and the retrograde axonal transport. Dynein molecular motors support the retrograde transport to cell bodies by walking to the minus end of microtubules while the kinesin motors drive the anterograde axonal transport towards the synapses by walking the microtubules to the plus end. The three major components of the neuronal cytoskeleton are microtubules, actin and intermediate filaments. One major feature about AT that has been challenging scientists is its bidirectional transport character and how it is efficiently organized. It is critical for axonal function the vesicular transport for long distances and this transport occurs mainly through the microtubule (MT) tracks. In this work, we model this bidirectional transport using a variant of TASEP (Totally Asymmetric Simple Exclusion Process) which consists of two parallel one dimensional lattices with periodic boundary conditions (two lanes model). One lane represents the transport through the filament and the other lane mimics a diffusive environment. The filament lane also takes into account the filament dynamics. The results are discussed in terms of the efficiency of the bidirectional transport.