Modularity of PRC1 composition and chromatin interaction define condensate properties.

Polycomb repressive complexes (PRCs) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells that are proposed to contribute to the maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using a reconstitution approach and single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. We find that the exact combination of PHC and CBX subunits determines condensate initiation, morphology, stability, and dynamics. Particularly, PHC2's polymerization activity influences condensate dynamics by promoting the formation of distinct domains that adhere to each other but do not coalesce. Live-cell imaging confirms CBX's role in condensate initiation and highlights PHC's importance for condensate stability. We propose that PRC1 composition can modulate condensate properties, providing crucial regulatory flexibility across developmental stages.

Architecture and flexibility of native kinetochores revealed by structural studies utilizing a thermophilic yeast.

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

Cytoskeleton crosstalk: Casting stable actin bundles with dynamic microtubule molds.

Actin-microtubule crosstalk diversifies cytoskeletal networks. A new study provides insight into how the microtubule polymerase CKAP5 mediates actin-microtubule crosstalk. CKAP5 directs the assembly of stable actin bundles on dynamic microtubules; in turn, the actin bundles align growing microtubules along their length.

Modularity of PRC1 Composition and Chromatin Interaction define Condensate Properties.

Polycomb repressive complexes (PRC) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells and the ability of PRC1 to form condensates has been proposed to contribute to maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. By reconstituting and imaging PRC1 with various subunit compositions, we find that the exact combination of PHC and CBX subunits determine the initiation, morphology, stability, and dynamics of condensates. In particular, the polymerization activity of PHC2 strongly influences condensate dynamics to promote formation of structures with distinct domains that adhere to each other but do not coalesce. Using live cell imaging, we confirmed that CBX properties are critical for condensate initiation and that PHC polymerization is important to maintain stable condensates. Together, we propose that PRC1 can fine-tune the degree and type of condensation by altering its composition which might offer important flexibility of regulatory function during different stages of development.

Interplay of self-organization of microtubule asters and crosslinking protein condensates.

The cytoskeleton is a major focus of physical studies to understand organization inside cells given its primary role in cell motility, cell division, and cell mechanics. Recently, protein condensation has been shown to be another major intracellular organizational strategy. Here, we report that the microtubule crosslinking proteins, MAP65-1 and PRC1, can form phase separated condensates at physiological salt and temperature without additional crowding agents in vitro. The size of the droplets depends on the concentration of protein. MAP65 condensates are liquid at first and can gelate over time. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. The droplet size directly controls the number of projections in the microtubule asters, demonstrating that the MAP65 concentration can control the organization of microtubules. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate, returning the MAP65 molecules to solution. This work implies that there is an interplay between condensate formation from microtubule-associated proteins, microtubule organization, and condensate dissolution that could be important for the dynamics of intracellular organization.

Real-Time Visualization of Microtubule and Protofilament-Scale Dynamics in Multi-Microtubule Arrays by Atomic Force Microscopy.

Microtubules, polymers of α, ß-tubulin heterodimers, are organized into multi-microtubule arrays for diverse cellular functions. The dynamic properties of microtubule arrays govern their structural and functional properties. While numerous insights into the biophysical mechanisms underlying microtubule organization have been gleaned from in vitro reconstitution studies, the assays are largely restricted to visualization of single or pairs of microtubules. Thus, the dynamic processes underlying the remodeling of multi-microtubule arrays remain poorly understood. Recent work shows that Atomic Force Microscopy (AFM) enables the visualization of nanoscale dynamics within multi-microtubule 2D arrays. In this assay, electrostatic interactions permit the non-specific adsorption of microtubule arrays to mica. AFM imaging in tapping mode, a gentle method of imaging, allows the visualization of microtubules and protofilaments without sample damage. The height information captured by AFM imaging enables the tracking of structural changes in microtubules and protofilaments within multi-microtubule arrays over time. The experimental data from the method described here reveal previously unseen modes of nanoscale dynamics in microtubule bundles formed by the microtubule-crosslinking protein PRC1 in the presence of the depolymerase MCAK. The observations demonstrate the potential of AFM imaging in transforming our understanding of the fundamental cellular process by which multi-microtubule arrays are dynamically assembled and disassembled. © 2023 Wiley Periodicals LLC. Basic Protocol: Sample preparation and real-time visualization of microtubule arrays by atomic force microscopy Alternate Protocol: Protocol for coating surface with poly-L-lysine and immobilizing microtubules.

GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Torques within and outside the human spindle balance twist at anaphase.

At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, using lattice light sheet microscopy, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are redundantly required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.

Motor guidance by long-range communication on the microtubule highway.

Coupling of motor proteins within arrays drives muscle contraction, flagellar beating, chromosome segregation, and other biological processes. Current models of motor coupling invoke either direct mechanical linkage or protein crowding, which rely on short-range motor-motor interactions. In contrast, coupling mechanisms that act at longer length scales remain largely unexplored. Here we report that microtubules can physically couple motor movement in the absence of detectable short-range interactions. The human kinesin-4 Kif4A changes the run length and velocity of other motors on the same microtubule in the dilute binding limit, when approximately 10-nm-sized motors are much farther apart than the motor size. This effect does not depend on specific motor-motor interactions because similar changes in Kif4A motility are induced by kinesin-1 motors. A micrometer-scale attractive interaction potential between motors is sufficient to recreate the experimental results in a biophysical model. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling affects not only binding kinetics but also motor mechanochemistry. Therefore, the model predicts that motors can sense and respond to motors bound several micrometers away on a microtubule. Our results are consistent with a paradigm in which long-range motor interactions along the microtubule enable additional forms of collective motor behavior, possibly due to changes in the microtubule lattice.

Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of KIF7, characterized by its striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in GLI, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. GLI increases KIF7 microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive GLI tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We retooled this coiled-coil-based GLI-KIF7 interaction to inhibit the nuclear and cilium localization of GLI. This strategy can potentially be exploited to downregulate erroneously activated GLI in human cancers.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy.

Microtubules are polymers of αß-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.

The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes implicated in both the complete demolition and fine size control of microtubule-based arrays are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here, we use atomic force microscopy (AFM) to image depolymerizing arrays at single-microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-scale cytoskeletal structures.

Engineering stability, longevity, and miscibility of microtubule-based active fluids.

Microtubule-based active matter provides insight into the self-organization of motile interacting constituents. We describe several formulations of microtubule-based 3D active isotropic fluids. Dynamics of these fluids is powered by three types of kinesin motors: a processive motor, a non-processive motor, and a motor which is permanently linked to a microtubule backbone. Another modification uses a specific microtubule crosslinker to induce bundle formation instead of a non-specific polymer depletant. In comparison to the already established system, each formulation exhibits distinct properties. These developments reveal the temporal stability of microtubule-based active fluids while extending their reach and the applicability.

Active Microphase Separation in Mixtures of Microtubules and Tip-Accumulating Molecular Motors.

Mixtures of filaments and molecular motors form active materials with diverse dynamical behaviors that vary based on their constituents' molecular properties. To develop a multiscale of these materials, we map the nonequilibrium phase diagram of microtubules and tip-accumulating kinesin-4 molecular motors. We find that kinesin-4 can drive either global contractions or turbulentlike extensile dynamics, depending on the concentrations of both microtubules and a bundling agent. We also observe a range of spatially heterogeneous nonequilibrium phases, including finite-sized radial asters, 1D wormlike chains, extended 2D bilayers, and system-spanning 3D active foams. Finally, we describe intricate kinetic pathways that yield microphase separated structures and arise from the inherent frustration between the orientational order of filamentous microtubules and the positional order of tip-accumulating molecular motors. Our work reveals a range of novel active states. It also shows that the form of active stresses is not solely dictated by the properties of individual motors and filaments, but is also contingent on the constituent concentrations and spatial arrangement of motors on the filaments.

The tail wags the tubulin.

The C-terminal tail of tubulin influences microtubule assembly and stability. In this issue of Developmental Cell, Chen et al. combine in vitro experiments using recombinant tubulin with molecular dynamics simulations to provide molecular-level insights into the importance of α-tubulin tail and its post-translational modifications in microtubule assembly and stability.

Differential regulation of single microtubules and bundles by a three-protein module.

A remarkable feature of the microtubule cytoskeleton is the coexistence of subpopulations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are the dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1 and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find that their collective activity promotes elongation of single microtubules while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (1) strong rescue activity of CLASP1, which overcomes the weaker effects of Kif4A on single microtubules, and (2) lower microtubule- and PRC1-binding affinity of CLASP1, which permits the dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule length, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.

Micron-scale geometrical features of microtubules as regulators of microtubule organization.

The organization of micron-sized, multi-microtubule arrays from individual microtubules is essential for diverse cellular functions. The microtubule polymer is largely viewed as a passive building block during the organization process. An exception is the 'tubulin code' where alterations to tubulin at the amino acid level can influence the activity of microtubule-associated proteins. Recent studies reveal that micron-scale geometrical features of individual microtubules and polymer networks, such as microtubule length, overlap length, contact angle, and lattice defects, can also regulate the activity of microtubule-associated proteins and modulate polymer dynamics. We discuss how the interplay between such geometrical properties of the microtubule lattice and the activity of associated proteins direct multiple aspects of array organization, from microtubule nucleation and coalignment to specification of array dimensions and remodeling of dynamic networks. The mechanisms reviewed here highlight micron-sized features of microtubules as critical parameters to be routinely investigated in the study of microtubule self-organization.

Meeting report - Mitotic spindle: from living and synthetic systems to theory.

Leading scientists from the field of mitotic spindle research gathered from 24-27 March 2019 to participate in the first 'Mitotic spindle: From living and synthetic systems to theory' conference. This meeting was held in Split, Croatia, organized by Nenad Pavin (Faculty of Science, University of Zagreb) and Iva Tolic (Ruder Boskovic Institute, Zagreb). Around 75 participants presented the latest advances in mitotic spindle research, ranging from live-cell imaging, in vitro reconstitution experiments and theoretical models of spindle assembly. The meeting successfully created an environment for interesting scientific discussions, initiation of new collaborations and development of fresh ideas. In this report, we will highlight and summarize new data challenging the established models of spindle architecture, advances in spindle reconstitution assays, discovery of new regulators of spindle size and shape as well as theoretical approaches for investigating motor protein function.

Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7.

The correct localization of Hedgehog effectors to the tip of primary cilia is critical for proper signal transduction. The conserved non-motile kinesin Kif7 defines a "cilium-tip compartment" by localizing to the distal ends of axonemal microtubules. How Kif7 recognizes microtubule ends remains unknown. We find that Kif7 preferentially binds GTP-tubulin at microtubule ends over GDP-tubulin in the mature microtubule lattice, and ATP hydrolysis by Kif7 enhances this discrimination. Cryo-electron microscopy (cryo-EM) structures suggest that a rotated microtubule footprint and conformational changes in the ATP-binding pocket underlie Kif7's atypical microtubule-binding properties. Finally, Kif7 not only recognizes but also stabilizes a GTP-form of tubulin to promote its own microtubule-end localization. Thus, unlike the characteristic microtubule-regulated ATPase activity of kinesins, Kif7 modulates the tubulin mechanochemical cycle. We propose that the ubiquitous kinesin fold has been repurposed in Kif7 to facilitate organization of a spatially restricted platform for localization of Hedgehog effectors at the cilium tip.