Macromolecular crowding: Sensing without a sensor

Holt, Liam J.; Delarue, Morgan

doi:10.1016/j.ceb.2023.102269

Cited by 7 publications

(2 citation statements)

References 67 publications

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“…In living cells, factors such as macromolecular crowding 29 and allosteric modulators 30 , in addition to the KD, would become relevant. Nevertheless, the concentration dependence on the dimer pool is an order of magnitude higher in 14-mer relative to the 12-mer.…”

Section: Discussionmentioning

confidence: 99%

Hub stability in the calcium calmodulin-dependent protein kinase II

Chien,

Puhl,

Vogel

et al. 2024

Commun Biol

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The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the β hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the β holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αβ heterooligomers for their dynamic localization within synapses in neurons.

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Section: Discussionmentioning

confidence: 99%

Hub stability in the calcium calmodulin-dependent protein kinase II

Chien,

Puhl,

Vogel

et al. 2024

Commun Biol

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“…In E. coli, for example, they occupy up to 44% of the cell interior. − Such macromolecular crowding − has diverse effects on physicochemical processes: It shifts equilibria ,− and cooperativity of chemical reactions, can inhibit or enhance enzyme-catalyzed reactions, − and affects metabolic channeling, − gene regulation, , and cell growth . Yet, the most apparent and extensively studied but not fully understood effect is the slowdown of intracellular diffusion.…”

Section: Diffusion Of Metabolites From Nmr Experimentsmentioning

confidence: 99%

Size Sensitivity of Metabolite Diffusion in Macromolecular Crowds

Raczyłło,

Gołowicz,

Skóra

et al. 2024

Nano Lett.

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Metabolites play crucial roles in cellular processes, yet their diffusion in the densely packed interiors of cells remains poorly understood, compounded by conflicting reports in existing studies. Here, we employ pulsed-gradient stimulated-echo NMR and Brownian/Stokesian dynamics simulations to elucidate the behavior of nano-and subnanometer-sized tracers in crowded environments. Using Ficoll as a crowder, we observe a linear decrease in tracer diffusivity with increasing occupied volume fraction, persisting�somewhat surprisingly�up to volume fractions of 30−40%. While simulations suggest a linear correlation between diffusivity slowdown and particle size, experimental findings hint at a more intricate relationship, possibly influenced by Ficoll's porosity. Simulations and numerical calculations of tracer diffusivity in the E. coli cytoplasm show a nonlinear yet monotonic diffusion slowdown with particle size. We discuss our results in the context of nanoviscosity and discrepancies with existing studies.

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Small organic osmolytes accelerate actin filament assembly and stiffen filaments

Demosthene,

Kravchuk,

Harmon

et al. 2024

Cytoskeleton

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Actin filament assembly and mechanics are crucial for maintenance of cell structure, motility, and division. Actin filament assembly occurs in a crowded intracellular environment consisting of various types of molecules, including small organic molecules known as osmolytes. Ample evidence highlights the protective functions of osmolytes such as trimethylamine‐N‐oxide (TMAO), including their effects on protein stability and their ability to counteract cellular osmotic stress. Yet, how TMAO affects individual actin filament assembly dynamics and mechanics is not well understood. We hypothesize that, owing to its protective nature, TMAO will enhance filament dynamics and stiffen actin filaments due to increased stability. In this study, we investigate osmolyte‐dependent actin filament assembly and bending mechanics by measuring filament elongation rates, steady‐state filament lengths, and bending persistence lengths in the presence of TMAO using total internal reflection fluorescence microscopy and pyrene assays. Our results demonstrate that TMAO increases filament elongation rates as well as steady‐state average filament lengths, and enhances filament bending stiffness. Together, these results will help us understand how small organic osmolytes modulate cytoskeletal protein assembly and mechanics in living cells.

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Macromolecular crowding: Sensing without a sensor

Cited by 7 publications

References 67 publications

Hub stability in the calcium calmodulin-dependent protein kinase II

Hub stability in the calcium calmodulin-dependent protein kinase II

Size Sensitivity of Metabolite Diffusion in Macromolecular Crowds

Small organic osmolytes accelerate actin filament assembly and stiffen filaments

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