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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. [Display omitted] • Nucleosomal arrays and PRC1 act synergistically to form condensates • PRC1 pre-wetting of chromatin initiates switch-like transition to form condensates • Modularity of PRC1 complex composition fine-tunes condensate properties • PHC polymerization alters condensate properties forming distinct long-lived domains Through reconstitution and imaging, Niekamp et al. reveal that the Polycomb repressive complex 1 (PRC1) and chromatin act synergistically to form condensates. They show that specific combinations of PHC and CBX subunits determine condensate initiation, morphology, stability, and dynamics, which could provide important flexibility in regulatory functions across developmental stages. [ABSTRACT FROM AUTHOR]

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility. [ABSTRACT FROM AUTHOR]

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ~1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ~4-nm nucleotide-dependent conformational change in the coiledcoil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales. [ABSTRACT FROM AUTHOR]

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer‐binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule‐binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled‐coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule‐independent hyperactive ATPase activity, and nucleotide‐independent low affinity for microtubules. Cryo‐electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide‐dependent conformational changes occur normally in one‐half of the AAA ring, but are disrupted in the other half. The large‐scale linker conformational change observed in the wild‐type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring. Synopsis: The movement of dynein on microtubules requires communication between enzymatic, polymer‐binding, and mechanical elements. Our results reveal how dynein's coiled‐coil stalk plays a critical role in coordinating such domain movements. Stalk mutants disrupt unidirectional motion along microtubules, show nucleotide‐independent low affinity for microtubules, and lack microtubule‐regulation of ATPase activity.Cryo‐electron microscopy structures of one mutant show that nucleotide‐dependent conformational changes are disrupted in one half of the AAA ring while the other half is minimally affected; the partial ring conformational change results in unregulated ATP hydrolysis and blocks the linker conformational change that drives motility.Our structural and functional results suggest a model for how the stalk domain modulates conformational changes around the AAA ring, which are initiated by nucleotide binding at dynein's main ATP site (AAA1). [ABSTRACT FROM AUTHOR]