Rigid Microtubules Research Articles

The Microtubule End Binding Protein Mal3 Is Essential for the Dynamic Assembly of Microtubules during Magnaporthe oryzae Growth and Pathogenesis.

Cytoskeletal microtubules (MTs) play crucial roles in many aspects of life processes in eukaryotic organisms. They dynamically assemble physiologically important MT arrays under different cell conditions. Currently, aspects of MT assembly underlying the development and pathogenesis of the model plant pathogenic fungus Magnaporthe oryzae (M. oryzae) are unclear. In this study, we characterized the MT plus end binding protein MoMal3 in M. oryzae. We found that knockout of MoMal3 results in defects in hyphal polar growth, appressorium-mediated host penetration and nucleus division. Using high-resolution live-cell imaging, we further found that the MoMal3 mutant assembled a rigid MT in parallel with the MT during hyphal polar growth, the cage-like network in the appressorium and the stick-like spindle in nuclear division. These aberrant MT organization patterns in the MoMal3 mutant impaired actin-based cell growth and host infection. Taken together, these findings showed that M. oryzae relies on MoMal3 to assemble elaborate MT arrays for growth and infection. The results also revealed the assembly mode of MTs in M. oryzae, indicating that MTs are pivotal for M. oryzae growth and host infection and may be new targets for devastating fungus control.

Kinesin-driven active cytoskeletal composite restructuring.

The cytoskeleton is an intricate dynamic network that enables vital processes in the cell such as mitosis, movement, and growth. It is composed of protein filaments including semi-flexible actin filaments and rigid microtubules, as well as motor proteins, such as kinesin. The versatile restructurability of the cytoskeleton makes it highly intriguing for designing materials that combine tunability, flexibility, and resilience. Applications can span from soft tissue repair to biodegradable plastics and rubbers. Here, we create actin-microtubule composites, driven by kinesin motors, and adapted by different types of cross-linkers. We observe a range of restructuring of the composites, from formation of homogeneous clusters to heterogeneous threadlike networks. We quantify dynamics of our active composites by using particle image velocimetry (PIV) on time series videos of fluorescently labeled actin and microtubules, and by performing single particle tracking (SPT) on fluorescently labeled NeutrAvidin. Additionally, by imaging spherical hydrogels in the networks, then measuring the deformation of the spheres, we are able to measure forces generated by actin-microtubule composites being reconfigured by kinesin motors.

Polymer threadings and rigidity dictate the viscoelasticity of entangled ring-linear blends and their composites with rigid rod microtubules

Mixtures of polymers of varying topologies and stiffnesses display complex emergent rheological properties that often cannot be predicted from their single-component counterparts. For example, entangled blends of ring and linear polymers have been shown to exhibit enhanced shear thinning and viscosity, as well as prolonged relaxation timescales, compared to pure solutions of rings or linear chains. These emergent properties arise in part from the synergistic threading of rings by linear polymers. Topology has also been shown to play an important role in composites of flexible (e.g., DNA) and stiff (e.g., microtubules) polymers, whereby rings promote mixing while linear polymers induce demixing and flocculation of stiff polymers, with these topology-dependent interactions giving rise to highly distinct rheological signatures. To shed light on these intriguing phenomena, we use optical tweezers microrheology to measure the linear and nonlinear rheological properties of entangled ring-linear DNA blends and their composites with rigid microtubules. We show that linear viscoelasticity is primarily dictated by microtubules at lower frequencies, but their contributions become frozen out at frequencies above the DNA entanglement rate. In the nonlinear regime, we reveal that mechanical response features, such as shear thinning and stress softening, are mediated by entropic stretching, threading, and flow alignment of entangled DNA, as well as forced dethreading, disentanglement, and clustering. The contributions of each of these mechanisms depend on the strain rate as well as the entanglement density and stiffness of the polymers, leading to nonmonotonic rate dependences of mechanical properties that are most pronounced for highly concentrated ring-linear blends rather than DNA-microtubule composites.

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

Active dynamics and tunable mechanics of actin-microtubule composites

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II to drive key processes including migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with actomyosin activity is still nascent. I will discuss our recent studies investigating the dynamics, structure and microrheological properties of in vitro actin-microtubule composites driven by myosin II minifilaments.

Low Intensity Ultrasound as an Antidote to Taxane/Paclitaxel-induced Cytotoxicity.

The taxane family of compounds, including Taxol/paclitaxel and Taxotere/docetaxel, are surprisingly successful drugs used in combination or alone for the treatment of most major solid tumors, especially metastatic cancer. The drugs are commonly used in regimen with other agents (often platinum drugs) as frontline treatment, or used as a single agent in a dose dense regimen for recurrent cancer. The major side effects of taxanes are peripheral neuropathy, alopecia, and neutropenia, which are grave burden for patients and limit the full potential of the taxane drugs. Especially in the current treatment protocol for peripheral neuropathy, taxane dosage is reduced once the symptoms present, resulting in the loss of full or optimal cancer killing activity.Substantial efforts have been made to address the problem of cytotoxic side effects of taxanes, though strategies remain very limited. Following administration of the taxane compound by infusion, taxane binds to cellular microtubules and is sequestered within the cells for several days. Taxane stabilizes and interferes with microtubule function, leading to ultimate death of cancer cells, but also damages hair follicles, peripheral neurons, and hemopoietic stem cells. Currently, cryo-treatment is practiced to limit exposure and side effects of the drug during infusion, though the effectiveness is uncertain or limited.A recent laboratory finding may provide a new strategy to counter taxane cytotoxicity, that a brief exposure to low density ultrasound waves was sufficient to eliminate paclitaxel cytotoxicity cells in culture by transiently breaking microtubule filaments, which were then relocated to lysosomes for disposal. Thus, ultrasonic force to break rigid microtubules is an effective solution to counter taxane cytotoxicity. The discovery and concept of low intensity ultrasound as an antidote may have the potential to provide a practical strategy to counter paclitaxel-induced peripheral neuropathy and alopecia that resulted from chemotherapy.Taxanes are a class of important drugs used in chemotherapy to treat several major cancers. This article reviews a new laboratory discovery that ultrasound can be used as an antidote for the peripheral cytotoxicity of taxane drugs and discusses the potential development and application of low intensity ultrasound to prevent side effects in chemotherapeutic treatment of cancer patients.

Motor-Driven Restructuring of Cytoskeleton Composites Leads to Tunable Time-Varying Elasticity.

The composite cytoskeleton, comprising interacting networks of semiflexible actin and rigid microtubules, generates forces and restructures by using motor proteins such as myosins to enable key processes including cell motility and mitosis. Yet, how motor-driven activity alters the mechanics of cytoskeleton composites remains an open challenge. Here, we perform optical tweezers microrheology and confocal imaging of composites with varying actin-tubulin molar percentages (25-75, 50-50, and 75-25), driven by light-activated myosin II motors, to show that motor activity increases the elastic plateau modulus by over 2 orders of magnitude by active restructuring of both actin and microtubules that persists for hours after motor activation has ceased. Nonlinear microrheology measurements show that motor-driven restructuring increases the force response and stiffness and suppresses actin bending. The 50-50 composite exhibits the most dramatic mechanical response to motor activity due to the synergistic effects of added stiffness from the microtubules and sufficient motor substrate for pronounced activity.

Breaking malignant nuclei as a non-mitotic mechanism of taxol/paclitaxel.

Discovered in a large-scale screening of natural plant chemicals, Taxol/paclitaxel and the taxane family of compounds are surprisingly successful anti-cancer drugs, used in treatment of the majority of solid tumors, and especially suitable for metastatic and recurrent cancer. Paclitaxel is often used in combination with platinum agents and is administrated in a dose dense regimen to treat recurrent cancer.The enthusiasm and clinical development were prompted by the discovery that Taxol binds beta-tubulins specifically found within microtubules and stabilizes the filaments, and consequently inhibits mitosis. However, questions on how paclitaxel suppresses cancer persist, as other specific mitotic inhibitors are impressive in pre-clinical studies but fail to achieve significant clinical activity. Thus, additional mechanisms, such as promoting mitotic catastrophe and impacting non-mitotic targets, have been proposed and studied. A good understanding of how paclitaxel, and additional new microtubule stabilizing agents, kill cancer cells will advance the clinical application of these common chemotherapeutic agents.A recent study provides a potential non-mitotic mechanism of paclitaxel action, that paclitaxel-induced rigid microtubules act to break malleable cancer nuclei into multiple micronuclei. Previous studies have established that cancer cells have a less sturdy, more pliable nuclear envelope due to the loss or reduction of lamin A/C proteins. Such changes in nuclear structure provide a selectivity for paclitaxel to break the nuclear membrane and kill cancer cells over non-neoplastic cells that have a sturdier nuclear envelope.The formation of multiple micronuclei appears to be an important aspect of paclitaxel in the killing of cancer cells, either by a mitotic or non-mitotic mechanism. Additionally, by binding to microtubule, paclitaxel is readily sequestered and concentrated within cells.This unique pharmacokinetic property allows the impact of paclitaxel on cells to persist for several days, even though the circulating drug level is much reduced following drug administration/infusion. The retention of paclitaxel within cells likely is another factor contributing to the efficacy of the drugs.Overall, the new understanding of Taxol/paclitaxel killing mechanism—rigid microtubule-induced multiple micronucleation—will likely provide new strategies to overcome drug resistance and for rational drug combination.

Triggering Cation-Induced Contraction of Cytoskeleton Networks via Microfluidics.

The dynamic morphology and mechanics of the cytoskeleton is determined by interacting networks of semiflexible actin filaments and rigid microtubules. Active rearrangement of networks of actin and microtubules can not only be driven by motor proteins but by changes to ionic conditions. For example, high concentrations of multivalent ions can induce bundling and crosslinking of both filaments. Yet, how cytoskeleton networks respond in real-time to changing ion concentrations, and how actin-microtubule interactions impact network response to these changing conditions remains unknown. Here, we use microfluidic perfusion chambers and two-color confocal fluorescence microscopy to show that increasing magnesium ions trigger contraction of both actin and actin-microtubule networks. Specifically, we use microfluidics to vary the Mg2+ concentration between 2 and 20 mM while simultaneously visualizing the triggered changes to the overall network size. We find that as Mg2+ concentration increases both actin and actin-microtubule networks undergo bulk contraction, which we measure as the shrinking width of each network. However, surprisingly, lowering the Mg2+concentration back to 2 mM does not stop or reverse the contraction but rather causes both networks to contract further. Further, actin networks begin to contract at lower Mg2+ concentrations and shorter times than actin-microtubule networks. In fact, actin-microtubule networks only undergo substantial contraction once the Mg2+ concentration begins to lower from 20 mM back to 2 mM. Our intriguing findings shed new light on how varying environmental conditions can dynamically tune the morphology of cytoskeleton networks and trigger active contraction without the use of motor proteins.

Filament Rigidity Vies with Mesh Size in Determining Anomalous Diffusion in Cytoskeleton.

The diffusion of microscopic particles through the cell, important to processes such as viral infection, gene delivery, and vesicle transport, is largely controlled by the complex cytoskeletal network, comprised of semiflexible actin filaments and rigid microtubules, that pervades the cytoplasm. By varying the relative concentrations of actin and microtubules, the cytoskeleton can display a host of different structural and dynamic properties that, in turn, impact the diffusion of particles through the composite network. Here, we couple single-particle tracking with differential dynamic microscopy to characterize the transport of microsphere tracers diffusing through composite in vitro networks with varying ratios of actin and microtubules. We analyze multiple complementary metrics for anomalous transport to show that particles exhibit anomalous subdiffusion in all networks, which our data suggest arises from caging by networks. Further, subdiffusive characteristics are markedly more pronounced in actin-rich networks, which exhibit similarly more prominent viscoelastic properties compared to microtubule-rich composites. While the smaller mesh size of actin-rich composites compared to microtubule-rich composites plays an important role in these results, the rigidity of the filaments comprising the network also influences the anomalous characteristics that we observe. Our results suggest that as microtubules in our composites are replaced with actin filaments, the decreasing filament rigidity competes with increasing network connectivity to drive anomalous transport.

Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule composites

The cytoskeleton precisely tunes its mechanics by altering interactions between semiflexible actin filaments, rigid microtubules, and crosslinking proteins. We use optical tweezers microrheology and confocal microscopy to characterize how varying crosslinking motifs impact the mesoscale mechanics and mobility of actin-microtubule composites. We show that, upon subtle changes in crosslinking patterns, composites can exhibit two distinct classes of force response – primarily elastic versus more viscous. For example, a composite in which actin and microtubules are crosslinked to each other but not to themselves is markedly more elastic than one in which both filaments are independently crosslinked. Notably, this distinction only emerges at mesoscopic scales in response to nonlinear forcing, whereas varying crosslinking motifs have little impact on the microscale mechanics and mobility. Our unexpected scale-dependent results not only inform the physics underlying key cytoskeleton processes and structures, but, more generally, provide valuable perspective to materials engineering endeavors focused on polymer composites.

Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites.

The cytoskeleton is able to precisely tune its structure and mechanics through interactions between semiflexible actin filaments, rigid microtubules and a suite of crosslinker proteins. However, the role that each of these components, as well as the interactions between them, plays in the dynamics of the composite cytoskeleton remains an open question. Here, we use optical tweezers microrheology and fluorescence confocal microscopy to reveal the surprising ways in which actin crosslinking tunes the viscoelasticity and mobility of actin-microtubule composites from steady-state to the highly nonlinear regime. While previous studies have shown that increasing crosslinking in actin networks increases elasticity and stiffness, we instead find that composite stiffness displays a striking non-monotonic dependence on actin crosslinking - first increasing then decreasing to a response similar to or even lower than un-linked composites. We further show that actin crosslinking has an unexpectedly strong impact on the mobility of microtubules; and it is in fact the microtubule mobility - dictated by crosslinker-driven rearrangements of actin filaments - that controls composite stiffness. This result is at odds with conventional thought that actin mobility drives cytoskeleton mechanics. More generally, our results demonstrate that - when crosslinking composite materials to confer strength and resilience - more is not always better.

Diffusion and Conformational Dynamics of Single DNA Molecules Crowded by Cytoskeletal Proteins

The high concentrations of proteins crowding cells greatly influence intracellular DNA dynamics. These crowders, ranging from small mobile proteins to large cytoskeletal filaments such as semiflexible actin and rigid microtubules, can hinder diffusion and induce conformational changes in DNA. While previous studies have mainly focused on the effect of small mobile crowders on DNA transport, we examine the impact of crowding by actin filaments and microtubules. Further, because actin filaments and microtubules are formed by polymerization of actin monomers and tubulin dimers, respectively, we also investigate the role that the polymerization state of each protein plays in DNA transport and in the time-varying conformational changes of single DNA molecules diffusing in in vitro networks of polymerized and monomeric actin and tubulin. We find that crowding by actin monomers slows DNA diffusion while tubulin crowding actually increases diffusion coefficients. Monomeric actin crowding DNA diffusion, more than when actin is polymerized, while crowding by tubulin dimers increases DNA diffusion more than when tubulin is polymerized (microtubules). Further, we find unexpected relationships between DNA coil size and diffusion when crowded. All crowding conditions lead to some degree of DNA compaction, but less compaction enables faster dynamics.

Enhanced Dynamics of Confined Cytoskeletal Filaments Driven by Asymmetric Motors

Cytoskeletal filaments and molecular motors facilitate the micron-scale force generation necessary for the distribution of organelles and the restructuring of the cytoskeleton within eukaryotic cells. Although the mesoscopic structure and the dynamics of such filaments have been studied in vitro and in vivo, their connection with filament polarity-dependent motor-mediated force generation is not well understood. Using 2D Brownian dynamics simulations, we study a dense, confined mixture of rigid microtubules (MTs) and active springs that have arms that cross-link neighboring MT pairs and move unidirectionally on the attached MT. We simulate depletion interactions between MTs using an attractive potential. We show that dimeric motors, with a motile arm on only one of the two MTs, produce large polarity-sorted MT clusters, whereas tetrameric motors, with motile arms on both microtubules, produce bundles. Furthermore, dimeric motors induce, on average, higher velocities between antialigned MTs than tetrameric motors. Our results, where MTs move faster near the confining wall, are consistent with experimental observations in Drosophila oocytes where enhanced microtubule activity is found close to the confining plasma membrane.

Covalent immobilization of microtubules on glass surfaces for molecular motor force measurements and other single-molecule assays.

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins.

Selective Formation of a Linear-Shaped Bundle of Microtubules

By using rigid microtubules (MTs) prepared by polymerization with guanylyl-(alpha, beta)-methylene-diphosphonate GMPCPP, giant straight-shaped MT bundles were selectively obtained through a dynamic self-assembly process. We demonstrate the effect of the rigidity on the shape and motility of MT bundle composed of GMPCPP-polymerized MTs (GMPCPP-MTs) compared with control MTs that were polymerized with GTP and stabilized with paclitaxel.

Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.

Microtubule retraction into the uropod and its role in T cell polarization and motility.

Spherical circulating T cells must polarize to extravasate. We have found that the polarization process includes a drastic reconfiguration of the tubulin cytoskeleton. In spherical T cells, the nucleus is surrounded by microtubules radiating from the microtubule organizing center (MTOC). During polarization the uropod (a slender posterior appendage) forms at the site of the MTOC. As the uropod buds out, the MTOC is carried in its distal tip. The attached microtubules retract into the uropod lumen, collapsing like the spokes of an umbrella into a compact sheaf. Experiments with microtubule inhibitors show that the retracted microtubules do not support the uropod or produce motive force. Instead, the data suggest that retraction of the relatively rigid microtubules into the streamlined uropod increases T cell deformability, thereby facilitating migration through constricted spaces. Microtubule retraction, therefore, may prove to be a strategy for accelerating extravasation without disassembly of the microtubule-based transport system.