Modelling intracellular transport in crowded environments: effects of motor association to cargos.

In intracellular transports, motor proteins transport macromolecules as cargos to desired locations by moving on biopolymers such as microtubules. Recent experiments suggest that, while moving in crowded environments, cargos that can associate motor proteins during their translocation have larger run-length and association time compared to free motors. Here, we model the dynamics of a cargo that can associate at the most m free motors present on the microtubule track as obstacles to its motion. The proposed models display competing effects of association and crowding, leading to a peak in the run-length with the free-motor density. For m = 2 and 3, we show that this feature is governed by the largest eigenvalue of the transition matrix describing the cargo dynamics. In all the above cases, free motors are assumed to be present on the microtubule as stalled obstacles. We finally compare simulation results for the run-length for general scenarios where the free motors undergo processive motion in addition to binding and unbinding to or from the microtubule.

Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.

A Platform for Medium-Throughput Cell-Free Analyses of Microtubule-Interacting Proteins Using Mammalian Cell Lysates.

The microtubule (MT) cytoskeleton performs a variety of functions in cell division, cell architecture, neuronal differentiation, and ciliary beating. These functions are controlled by proteins that directly interact with MTs, commonly referred to as microtubule-associated proteins (MAPs). Out of the many proteins reported interact with MTs, only a some have been biochemically and functionally characterized so far. One of the limitations of classical in vitro assays and single-MT reconstitution approaches is that they are typically performed with purified proteins. As purification of proteins can be difficult and time-consuming, many previous studies have only focused on a few proteins, while systematic analyses of many different proteins by in vitro reconstitution assays were not possible. Here we present a detailed protocol using lysates of mammalian cells instead of purified proteins that overcomes this limitation. Those lysates contain all molecular components required for in vitro MT reconstitution including the endogenous tubulin and the recombinant MAPs, which form MT assemblies upon the injection of the lysates into a microscopy chamber. This allows to directly observe the dynamic behavior of growing MTs, as well as the fluorescently labeled associated proteins by total internal reflection fluorescence (TIRF) microscopy. Strikingly, all proteins tested so far were functional in our approach, thus providing the possibility to test virtually any protein of interest. This also opens the possibility to screen the impact of patient mutations on the MT binding behavior of MAPs in a medium-throughput manner. In addition, the lysate approach can easily be adapted to other applications that have predominantly been performed with purified proteins so far, such as investigating other cytoskeletal systems and cytoskeletal crosstalk, or to study structures of MAPs bound to MTs by cryo-electron microscopy. Our approach is thus a versatile, expandable, and easy-to-use method to characterize the impact of a broad spectrum of proteins on cytoskeletal behavior and function. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of lysates of human cells for TIRF reconstitution assays Basic Protocol 2: Quantification of GFP-tagged MAP concentration in cell lysates Support Protocol 1: Purification of KIF5B(N555/T92A) (dead kinesin) protein for TIRF reconstitution assays Support Protocol 2: Preparation of GMPCPP MT seeds for TIRF reconstitution assays Basic Protocol 3: TIRF-based MT-MAP reconstitution assays using cell lysates.

Distinct Sub- to Superdiffuse Insulin Granule Transport Behaviors in ß-Cells Are Strongly Affected by Granule Age.

Intracellular transport is a complex process that is difficult to describe by a single general model for motion. Here, we study the transport of insulin containing vesicles, termed granules, in live MIN6 cells. We characterize how the observed heterogeneity is affected by different intracellular factors by constructing a MIN6 cell line by CRISPR-CAS9 that constitutively expresses mCherry fused to insulin and is thus packaged in granules. Confocal microscopy imaging and single particle tracking of the granule transport provide long trajectories of thousands of single granule trajectories for statistical analysis. Mean squared displacement (MSD), angle correlation distribution, and step size distribution analysis allowed identifying five distinct granule transport subpopulations, from nearly immobile and subdiffusive to run-pause and superdiffusive. The subdiffusive subpopulation recapitulates the subordinated random walk we reported earlier (Tabei, 2013; ref 18). We show that the transport characteristics of the five subpopulations have a strong dependence on the age of insulin granules. The five subpopulations also reflect the effect of local microtubule and actin networks on transport in different cellular regions. Our results provide robust metrics to clarify the heterogeneity of granule transport and demonstrate the roles of microtubule versus actin networks with granule age since initial packaging in the Golgi.

Structure and dynamics of motor-driven microtubule bundles.

Connecting the large-scale emergent behaviors of active cytoskeletal materials to the microscopic properties of their constituents is a challenge due to a lack of data on the multiscale dynamics and structure of such systems. We approach this problem by studying the impact of depletion attraction on bundles of microtubules and kinesin-14 molecular motors. For all depletant concentrations, kinesin-14 bundles generate comparable extensile dynamics. However, this invariable mesoscopic behavior masks the transition in the microscopic motion of microtubules. Specifically, with increasing attraction, we observe a transition from bi-directional sliding with extension to pure extension with no sliding. Small-angle X-ray scattering shows that the transition in microtubule dynamics is concurrent with a structural rearrangement of microtubules from an open hexagonal to a compressed rectangular lattice. These results demonstrate that bundles of microtubules and molecular motors can display the same mesoscopic extensile behaviors despite having different internal structures and microscopic dynamics. They provide essential information for developing multiscale models of active matter.

Competition between physical search and a weak-to-strong transition rate-limits kinesin binding times.

The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions. In this work, we leverage theory and experiments to identify and quantify the rate-limiting mechanism of the initial association between a cargo-bound kinesin motor and a microtubule track. Recent advances in optical tweezers provide binding times for several lengths of kinesin motors trapped at varying distances from a microtubule, empowering the investigation of competing models. We first explore a diffusion-limited model of binding. Through Brownian dynamics simulations and simulation-based inference, we find this simple diffusion model fails to explain the experimental binding times, but an extended model that accounts for the ADP state of the molecular motor agrees closely with the data, even under the scrutiny of penalizing for additional model complexity. We provide quantification of both kinetic rates and biophysical parameters underlying the proposed binding process. Our model suggests that a typical binding event is limited by ADP state rather than physical search. Lastly, we predict how these association rates can be modulated in distinct ways through variation of environmental concentrations and physical properties.

Thermally Drawn-Based Microtubule Soft Continuum Robot for Cardiovascular Intervention.

Cardiovascular disease is becoming the leading cause of human mortality. In order to address this, flexible continuum robots have emerged as a promising solution for miniaturizing and automating vascular interventional equipment for diagnosing and treating cardiovascular diseases. However, existing continuum robots used for vascular intervention face challenges such as large cross-sectional sizes, inadequate driving force, and lack of navigation control, preventing them from accessing cerebral blood vessels or capillaries for medical procedures. Additionally, the complex manufacturing process and high cost of soft continuum robots hinder their widespread clinical application. In this study, we propose a thermally drawn-based microtubule soft continuum robot that overcomes these limitations. The proposed robot has cross-sectional dimensions several orders of magnitude smaller than the smallest commercially available conduits, and it can be manufactured without any length restrictions. By utilizing a driving strategy based on liquid kinetic energy advancement and external magnetic field for steering, the robot can easily navigate within blood vessels and accurately reach the site of the lesion. This innovation holds the potential to achieve controlled navigation of the robot throughout the entire blood vessel, enabling in situ diagnosis and treatment of cardiovascular diseases.

Helical motion and torque generation by microtubule motors.

Microtubule motors play key roles in cellular functions, such as transport, mitosis and cell motility. Fueled by ATP hydrolysis, they convert chemical energy into mechanical work, which enables their movement on microtubules. While their motion along the long axis of microtubules has been studied extensively, some motors display an off-axis component, which results in helical motion around microtubules and the generation of torque in addition to linear forces. Understanding these nuanced movements expands our comprehension of motor protein dynamics and their impact on cellular processes.

Tubulin posttranslational modifications through the lens of new technologies.

The Tubulin Code revolutionizes our understanding of microtubule dynamics and functions, proposing a nuanced system governed by tubulin isotypes, posttranslational modifications (PTMs) and microtubule-associated proteins (MAPs). Tubulin isotypes, diverse across species, contribute structural complexity, and are thought to influence microtubule functions. PTMs encode dynamic information on microtubules, which are read by several microtubule interacting proteins and impact on cellular processes. Here we discuss recent technological and methodological advances, such as in genome engineering, live cell imaging, expansion microscopy, and cryo-electron microscopy that reveal new elements and levels of complexity of the tubulin code, including new modifying enzymes and nanopatterns of PTMs on individual microtubules. The Tubulin Code's exploration holds transformative potential, guiding therapeutic strategies and illuminating connections to diseases like cancer and neurodegenerative disorders, underscoring its relevance in decoding fundamental cellular language.

Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Quantum Dot-Fab' Conjugates as Compact Immunolabels for Microtubule Imaging and Cell Classification.

Antibodies and their conjugates of fluorescent labels are widely applied in life sciences research and clinical pathology. Among diverse label types, compact quantum dots (QDs) provide advantages of multispectral multiplexing, bright signals in the deep red and infrared, and low steric hindrance. However, QD-antibody conjugates have random orientation of the antigen-binding domain which may interfere with labeling and are large (20-30 nm) and heterogeneous, which limits penetration into biospecimens. Here, we develop conjugates of compact QDs and Fab' antibody fragments as primary immunolabels. Fab' fragments are conjugated site-specifically through sulfhydryl groups distal to antigen-binding domains, and the multivalent conjugates have small and homogeneous sizes (∼12 nm) near those of full-sized antibodies. Their performance as immunolabels for intracellular antigens is evaluated quantitatively by metrics of microtubule labeling density and connectivity in fixed cells and for cytological identification in fixed brain specimens, comparing results with probes based on spectrally-matched dyes. QD-Fab' conjugates outperformed QD conjugates of full-sized antibodies and could be imaged with bright signals with 1-photon and 2-photon excitation. The results demonstrate a requirement for smaller bioaffinity agents and site-specific orientation for the success of nanomaterial-based labels to enhance penetration in biospecimens and minimize nonspecific staining.

Structure-based discovery and rational design of microtubule-targeting agents.

Microtubule-targeting agents (MTAs) have demonstrated remarkable efficacy as antitumor, antifungal, antiparasitic, and herbicidal agents, finding applications in the clinical, veterinary, and agrochemical industry. Recent advances in tubulin and microtubule structural biology have provided powerful tools that pave the way for the rational design of innovative small-molecule MTAs for future basic and applied life science applications. In this mini-review, we present the current status of the tubulin and microtubule structural biology field, the recent impact it had on the discovery and rational design of MTAs, and exciting avenues for future MTA research.

Autonomous assembly and disassembly of gliding molecular robots regulated by a DNA-based molecular controller.

In recent years, there has been a growing interest in engineering dynamic and autonomous systems with robotic functionalities using biomolecules. Specifically, the ability of molecular motors to convert chemical energy to mechanical forces and the programmability of DNA are regarded as promising components for these systems. However, current systems rely on the manual addition of external stimuli, limiting the potential for autonomous molecular systems. Here, we show that DNA-based cascade reactions can act as a molecular controller that drives the autonomous assembly and disassembly of DNA-functionalized microtubules propelled by kinesins. The DNA controller is designed to produce two different DNA strands that program the interaction between the microtubules. The gliding microtubules integrated with the controller autonomously assemble to bundle-like structures and disassemble into discrete filaments without external stimuli, which is observable by fluorescence microscopy. We believe this approach to be a starting point toward more autonomous behavior of motor protein-based multicomponent systems with robotic functionalities.

Ultraviolet Superradiance from Mega-Networks of Tryptophan in Biological Architectures.

Networks of tryptophan (Trp)─an aromatic amino acid with strong fluorescence response─are ubiquitous in biological systems, forming diverse architectures in transmembrane proteins, cytoskeletal filaments, subneuronal elements, photoreceptor complexes, virion capsids, and other cellular structures. We analyze the cooperative effects induced by ultraviolet (UV) excitation of several biologically relevant Trp mega-networks, thus giving insights into novel mechanisms for cellular signaling and control. Our theoretical analysis in the single-excitation manifold predicts the formation of strongly superradiant states due to collective interactions among organized arrangements of up to >105 Trp UV-excited transition dipoles in microtubule architectures, which leads to an enhancement of the fluorescence quantum yield (QY) that is confirmed by our experiments. We demonstrate the observed consequences of this superradiant behavior in the fluorescence QY for hierarchically organized tubulin structures, which increases in different geometric regimes at thermal equilibrium before saturation, highlighting the effect's persistence in the presence of disorder. Our work thus showcases the many orders of magnitude across which the brightest (hundreds of femtoseconds) and darkest (tens of seconds) states can coexist in these Trp lattices.

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

The emerging tools for precisely manipulating microtubules.

Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions.

Structure of the native γ-tubulin ring complex capping spindle microtubules.

Microtubule (MT) filaments, composed of α/ß-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/ß-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/ß-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.

Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α-tubulin and ß-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/ß-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.