Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells

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.