Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells

Abstract

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer–polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions (“sticking”) following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE’s opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer–polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

Single‐Molecule FRET of Membrane Transport Proteins

“…For the last two decades, [1] smFRET techniques have been extensively used to study the properties of molecular machines, [2] intrinsically disordered proteins (IDPs),[ 3 , 4 , 5 , 6 , 7 , 8 ] protein folding processes,[ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ] protein‐ligand[ 17 , 18 , 19 ] and protein‐nucleic acid interactions,[ 20 , 21 , 22 , 23 ] as well as other structure‐function relationships and dynamic processes. [ 24 , 25 , 26 ] SmFRET is a particularly powerful and versatile tool to gain molecular and mechanistic insights because of its high spatial resolution (2–10 nm) combined with a wide range of accessible timescales (ns‐minutes).…”

Implications of the Actin Cytoskeleton on the Multi-Step Process of [PSI+] Prion Formation

“…We found that actin depolymerization drugs, such as latrunculin A, made localization less biased to the periphery ( Figure 5 B), suggesting that actin may play a role in how these aggregates are localized. Yet, it has been shown that actin depolymerization drugs also reduce molecular crowding [ 41 ], suggesting that the observed change in localization in the presence of latrunculin A may correspond with changes in molecular crowding rather than a direct effect of the actin cytoskeleton.…”

Glass Dynamics Deep in the Energy Landscape

“…The nature of chemical bonding, chemical reactivity, photochemistry, and quantum phenomena in materials trapped deep in the energy landscape provide still more challenges despite having been exploited technologically for decades. Likewise the fact that analogous theoretical issues arise in the study of biomolecules and in the active matter that makes up cells , motivates still more work.…”