Intracellular mechanical fingerprint reveals cell type specific mechanical tuning

Reviewer #1 (Public Review):

Summary:

In this MS, Muenker and colleagues, explore the intracellular mechanics of a range of animal adherent cells. The study is based on the use of an optical tweezer set up, which allows to apply oscillatory forces on endocytosed/phagocytosed glass beads with a large frequency range (from ~1 to 1000 Hz) , allowing to probe cytoplasm material properties at multiple time scales. By switching off the laser trap, the authors also record the positional fluctuations of beads, to extract passive rheological signatures. The combination of both methods allow to fit 6 parameters (from power law fits) that allow to characterize the viscous and elastic nature of the cytoplasm material as well as an effective active energy driven by cellular metabolism. Using these methodologies, the authors first establish/confirm, using HeLa cells, that the cytoplasm is more solid like at short frequencies, and more fluid like at higher frequencies, and that these material states depend on both microtubules and actin cytoskeleton. The manuscript then go on to explore how these parameters evolve in other 6 cell types including muscles, highly migratory and epithelial cells. These results show for instance that muscle cells are much stiffer, while migratory cells are more fluid like with an increased active energy. Finally using statistical methods and principal component analysis, the authors establish some mechanical fingerprints (activity, fluidity and resistance) that allow to distinguish cell's mechanical state and relate it to their particular functions.

Strengths:

Overall this is a very well-executed work, which provides a large body of rigorous numbers and data to understand the regulation of cytoplasm mechanics and its relation to cell state/function.

Weaknesses:

A limit of the paper is that the biological mechanisms by which intracellular mechanics is modulated (e.g. among cell types) remains unexplored and only briefly discussed. Yet this limit is greatly offset by the rigor of the approach.

Reviewer #2 (Public Review):

Summary:

By analyzing cells' frequency-dependent viscoelastic properties and intracellular activity through microrheology, Münker et al simplify the complex active mechanical state into six key parameters that constitute the mechanical fingerprint. They apply this concept to cells treated with cytoskeleton-inhibiting drugs. Additionally, a comprehensive statistical analysis across various cell types shows how cells coordinate their mechanical properties within a defined phase-space marked by activity, mechanical resistance, and fluidity.

Strengths:

(1) The distribution of the six parameters: they have been well characterized based on established theories, and they can be used to understand cell-type-specific biomechanical differences. The examples of muscle cells and immune cells were profound and informative.
(2) Efforts to perform dimension reduction of parameter space into activity (E), fluidity (C1) and resistance (A) are insightful and will be helpful for future characterization of cell mechanics.

Weaknesses:

(1) The most difficult part of the method is the part with actin polymerization inhibition with cytochalasin B. The data shows that viscoelastic parameters as well as active energy parameters are unaffected by cytochalasin B. It is reasonable to expect that elasticity will reduce and fluidity will increase upon application of such a drug. The stiffness-reducing effect was observed only when CB was used with nocodazole most likely because of phagocytosis of the bead, which is governed by microtubule. The use of other actin-depolymerizing drugs such as latrunculin A would be needed to test actin's role in mechanical fingerprints. If actin's role is only explained by accompanying microtubule inhibition, it is not a convenient system to directly test the mechano-adaptation process.
(2) Depolymerization of MT with nocodazole did not reduce the solid-like property A. Adding discussion and comparison with other papers in the literature using nocodazole will be helpful in understanding why.
(3) Overall, the usefulness of the concept of mechanical fingerprints and comparisons with other cell mechanics studies (from other groups) will make this manuscript stronger.

Reviewer #3 (Public Review):

Summary:

Cells and tissues are viscoelastic materials. However, metabolic processes that underly survival, growth and migration render the cell as an active matter at non-equilibrium. These two facts contribute to the difficulty of probing mechanical properties especially with sub-cellular resolution. However, the concept that the mechanical phenotype can be indicative of normal physiology necessitates approaches of defining the cellular phenotype. Here, Muenker et al evokes a powerful argument for mapping intracellular mechanics using optical tweezer- active microrheology. They present a suite of parameters towards a definition of a mechanical fingerprint. This is a compelling idea. There are some concerns as detailed below

Strengths:

These are technically challenging experiments and the authors provide systematic approaches to probe a system at non-equilibrium.

Weaknesses:

The importance of the mechanical fingerprint is diluted due to some missing controls needed for biological relevance.

Author response:

Reviewer 1:

A limit of the paper is that the biological mechanisms by which intracellular mechanics is modulated (e.g. among cell types) remains unexplored and only briefly discussed. Yet this limit is greatly offset by the rigor of the approach.

We thank the reviewer for the valuable feedback. The question regarding the biological mechanisms responsible for the different mechanical properties is, indeed, a highly important and interesting issue. In line with the reviewer, we consider this so important that it requires an extra, dedicated research focus, which is far beyond the scope of this article. By introducing the concept of the mechanical fingerprint, we provide in this work the framework to systematically investigate biological mechanisms but also the functional relevance of the intracellular mechanical properties in future studies. In the revised manuscript, we’ll elaborate on the discussion.

Reviewer 2:

The most difficult part of the method is the part with actin polymerization inhibition with cytochalasin B. The data shows that viscoelastic parameters as well as active energy parameters are unaffected by cytochalasin B. It is reasonable to expect that elasticity will reduce and fluidity will increase upon application of such a drug. The stiffness-reducing effect was observed only when CB was used with nocodazole most likely because of phagocytosis of the bead, which is governed by microtubule. The use of other actin-depolymerizing drugs such as latrunculin A would be needed to test actin’s role in mechanical fingerprints. If actin’s role is only explained by accompanying microtubule inhibition, it is not a convenient system to directly test the mechano-adaptation process.

We thank the reviewer for the time and the instructive feedback. Our finding that actin depolymerization has no effect on the intracellular mechanics may appear unfamiliar, as many rheological studies performed on the cell’s cortex highlight the importance of actin on the mechanical properties of the whole cell. However, as the actin network is reported to be very sparse away from the cortex it is not impossible that the mechanical properties may be dominated by other structures in the cytoplasm. Indeed, our findings are consisted with other studies that see no strong effect of actin depolymerization on the interphase intracellular mechanics (e.g. https://doi.org/10.1016/j.bpj.2023.04.011 or https://doi.org/10.1038/s41567-021-01368-z). Still, we fully agree with the reviewers that this is an important point. In a revised version we aim to investigate the effect of other actin-depolymerizing drugs and will try to perform immunostaining to visualize and further illuminate the potential compensation mechanism between actin and MT.

Depolymerization of MT with nocodazole did not reduce the solid-like property A. Adding discussion and comparison with other papers in the literature using nocodazole will be helpful in understanding why.

Again, we agree with the reviewer and propose to further study this point by performing additional immunostainings and by elaborating on the discussion, also including the results of other studies.

Reviewer 3:

The importance of the mechanical fingerprint is diluted due to some missing controls needed for biological relevance.

We thank the reviewer for his valuable time and feedback. This comment is in line with the point already raised by reviewer 1 and highlights the important question of how the intracellular mechanical properties are related to the actual cell function. We fully agree with the reviewers that at this point we can only report on differences, but cannot claim a biological function that is depending on the fingerprint. Although we think the alignment between function and the mechanical fingerprints allows the hypothesis that the biological system is tuning its mechanical properties for a specific function, we do not want to make any claim in this direction at the current state of our research. Hence, to answer these intriguing questions, carefully designed control experiments are required, as pointed out by the reviewer. However, this direction is not the scope of this manuscript. Here, we establish the tools we’ll use in future studies to address these highly relevant questions. Therefore, we propose to discuss these important future directions in a revised manuscript.