Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion

Reviewer #1 (Public review):

Summary:

The authors investigate the mechanism behind the widely observed but poorly understood phenomenon of reversible vimentin disassembly upon hypotonic challenge. Using permeabilized COS-7 cells expressing vimentin-mEos3.2, the authors demonstrate that vimentin disassembly is not due to lower osmotic pressure but rather due to decreased intracellular ionic strength. They propose a model in which vimentin filament stability is predicted by the protein's net charge and support this idea through approaches that involve (i) manipulating buffer ionic strength, (ii) manipulating buffer pH, or (iii) introducing charged amino acids into the linker of the exogenously expressed vimentin-mEos3.2.

Strengths & Weaknesses:

While the discovery is intriguing and presents an interesting concept, significant shortcomings in experimental design and numerous inconsistencies prevent it from reaching the high standards expected. The lack of reproducibility, inadequate controls, and insufficient quantification make the findings feel very preliminary. Additionally, the authors need to address the apparent discrepancies between their current results and their previous work implicating calpains and altered calcium levels in vimentin disassembly upon hypotonic challenge (which has led to much confusion in the field). This discrepancy should be thoroughly addressed in the discussion with the authors citing their prior work and explaining why it was incorrect.

An additional concern is the relevance of the findings to vimentin biology inside cells. The most important insight in this work is the observation that an isotonic buffer balanced with non-electrolytes (glucose or sorbitol) is sufficient to drive vimentin disassembly. The authors show that vimentin disassembly is not due to changes in osmotic pressure but rather due to a change in the concentration of critical dissolved ions, specifically the number of charged states on vimentin. What is missing is when and how this is controlled within cells under physiological conditions - not just when cells are permeabilized with detergents (conditions that cells rarely survive). Without this deeper dive into vimentin states within cells and how it is controlled, the paper seems very narrow in its focus.

Reviewer #2 (Public review):

The reviewed manuscript "Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion" presents exciting results on the mechanisms governing the assembly and disassembly of the vimentin cytoskeleton. They show, using live-cell imaging, that changes in the intracellular ionic strength induce rapid and dramatic changes to the integrity of the vimentin cytoskeleton. Interestingly, mutants of vimentin with net positive or negative charges display notably different responses to hypotonic stress (and thus changes to the intracellular ionic strength). Even more interesting, the ionic strength-driven mechanism seems to generalize to the several other intermediate filaments explored here. These results are of high interest to the broader cytoskeleton field. A major caveat is that essentially every experiment in the paper is n=1, showing example images of a single cell. The experiments were not repeated, and the results were not quantified. Purported differences between experimental variables/conditions lack statistical significance. Generalization of the ionic strength-based mechanism is hindered by the fact that only one cell type was tested for each cytoskeletal protein. Another caveat is that the fluorescently tagged vimentin used thoroughly in this work is exogenous and overexpressed; it is unclear if the observed effects would also occur at endogenous concentrations of vimentin. As it is currently presented, it is my opinion that all four main figures in this work - although interesting and quite likely correct - should be interpreted as preliminary data by readers.

Reviewer #3 (Public review):

Summary:

This report analyzes the structure of vimentin, GFAP, and keratin intermediate filament networks in cells that have been subjected to hypotonic stress and other treatments that either alter the ionic strength of the cytoplasm or change the charge density of the intermediate filament.

Strengths:

These experiments expand on the work of references 8 and 9, which showed that the vimentin network rapidly dissociates after hypotonic shock. The cellular imaging uses sophisticated super-resolution techniques and produces some striking images.

Weaknesses:

A fundamental weakness of this study lies in the interpretation, and lack of biochemical evidence for the provocative hypothesis raised that the assembly state of intermediate filaments in a cell is governed by coulomb interactions between charged filaments. Several essential experiments need to be done before this striking hypothesis can be plausibly supported.

(1) First the assembly and disassembly of vimentin filaments needs to be done in vitro systems. If the hypothesis is correct then these same effects will happen with purified vimentin intermediate filaments. These proteins are readily purified from bacterial expression systems and there's a wealth of biophysical data on them, so verifying the predictions of this cell-based model can be realistically tested with purified proteins.

(2) Interpretation of these results explicitly avoids a role for post-translational modifications of vimentin, which are well described and related to filament assembly state. For example, reference 9, which is one of the first discoveries that the vimentin network dissociates under osmotic stress explicitly shows that the vimentin network remains intact if either calcium influx or calpain proteolytic activity is prevented. Hypoosmotic stress will still lead to the same dilution, but the filaments remain intact, apparently contradicting the major interpretation of this paper.

(3) The third issue is that the role of polyelectrolyte effects and especially the importance of divalent cations is scarcely mentioned in the interpretation. There are many studies of how strongly vimentin intermediate filaments interact with divalent cations, in a manner that is relatively insensitive to ionic strength, and these effects need to be taken into account to interpret the cellular data or the hypothesis that coulomb repulsion is the major driver for vimentin disassembly.