Cytoskeletal vimentin regulates cell size and autophagy through mTORC1 signaling.

The nutrient-activated mTORC1 (mechanistic target of rapamycin kinase complex 1) signaling pathway determines cell size by controlling mRNA translation, ribosome biogenesis, protein synthesis, and autophagy. Here, we show that vimentin, a cytoskeletal intermediate filament protein that we have known to be important for wound healing and cancer progression, determines cell size through mTORC1 signaling, an effect that is also manifested at the organism level in mice. This vimentin-mediated regulation is manifested at all levels of mTOR downstream target activation and protein synthesis. We found that vimentin maintains normal cell size by supporting mTORC1 translocation and activation by regulating the activity of amino acid sensing Rag GTPase. We also show that vimentin inhibits the autophagic flux in the absence of growth factors and/or critical nutrients, demonstrating growth factor-independent inhibition of autophagy at the level of mTORC1. Our findings establish that vimentin couples cell size and autophagy through modulating Rag GTPase activity of the mTORC1 signaling pathway.

Passive coupling of membrane tension and cell volume during active response of cells to osmosis.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

A comparison of microfluidic methods for high-throughput cell deformability measurements.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.

Intelligent image-based deformation-assisted cell sorting with molecular specificity.

Although label-free cell sorting is desirable for providing pristine cells for further analysis or use, current approaches lack molecular specificity and speed. Here, we combine real-time fluorescence and deformability cytometry with sorting based on standing surface acoustic waves and transfer molecular specificity to image-based sorting using an efficient deep neural network. In addition to general performance, we demonstrate the utility of this method by sorting neutrophils from whole blood without labels.

Effects of vimentin on the migration, search efficiency, and mechanical resilience of dendritic cells.

Dendritic cells use amoeboid migration to pass through narrow passages in the extracellular matrix and confined tissue in search for pathogens and to reach the lymph nodes and alert the immune system. Amoeboid migration is a migration mode that, instead of relying on cell adhesion, is based on mechanical resilience and friction. To better understand the role of intermediate filaments in ameboid migration, we studied the effects of vimentin on the migration of dendritic cells. We show that the lymph node homing of vimentin-deficient cells is reduced in our in vivo experiments in mice. Lack of vimentin also reduces the cell stiffness, the number of migrating cells, and the migration speed in vitro in both 1D and 2D confined environments. Moreover, we find that lack of vimentin weakens the correlation between directional persistence and migration speed. Thus, vimentin-expressing dendritic cells move faster in straighter lines. Our numerical simulations of persistent random search in confined geometries verify that the reduced migration speed and the weaker correlation between the speed and direction of motion result in longer search times to find regularly located targets. Together, these observations show that vimentin enhances the ameboid migration of dendritic cells, which is relevant for the efficiency of their random search for pathogens.

Highly-Parallel Microfluidics-Based Force Spectroscopy on Single Cytoskeletal Motors.

Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, a highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods is introduced. Tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths, and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads are applied. Importantly, the 16 µm long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. The approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells.

Single-Cell Mechanics: Structural Determinants and Functional Relevance.

The mechanical phenotype of a cell determines its ability to deform under force and is therefore relevant to cellular functions that require changes in cell shape, such as migration or circulation through the microvasculature. On the practical level, the mechanical phenotype can be used as a global readout of the cell's functional state, a marker for disease diagnostics, or an input for tissue modeling. We focus our review on the current knowledge of structural components that contribute to the determination of the cellular mechanical properties and highlight the physiological processes in which the mechanical phenotype of the cells is of critical relevance. The ongoing efforts to understand how to efficiently measure and control the mechanical properties of cells will define the progress in the field and drive mechanical phenotyping toward clinical applications.

Metastatic malignancies in the parotid gland: A retrospective study.

The aim of this study was to compare treatment modalities, pathological and clinical characteristics, and outcomes in patients with metastasis in a parotid gland. The medical records of 34 patients who received treatment for metastasis in the parotid gland over a twenty-year period were evaluated. Patients with head and neck cutaneous squamous cell carcinoma (HNcSCC) metastasis were retrospectively reclassified using the P/N and N1S3 staging system. Patients with neck metastasis showed a significantly poorer prognosis (P = 0.025). Univariate analysis also revealed that extent of parotidectomy and type of neck dissection did not influence recurrence free survival (RFS) and overall survival (OS). When comparing the usefulness of the P/N and S1N3 staging systems, a positive correlation was observed between the P stage and the N1S3 stage in both RFS and OS. The extent of parotidectomy and concomitant neck dissection is still under discussion. Total parotidectomy and modified radical neck dissection did not improve RFS and OS. N1S3 is a less complex classification and possesses a higher predictive value when compared to the P/N staging system.

Molecular alterations in metaphase chromosomes induced by bleomycin.

Chromosomes are intranuclear structures, their main function is to store and transmit genetic information during cell division. They are composed of tightly packed DNA in the form of chromatin, which is constantly exposed to various damaging factors. The resulting changes in DNA can have serious consequences (e.g. mutations) if they are not repaired or repaired incorrectly. In this article, we studied chromosomes isolated from human cervical cancer cells (HeLa) exposed to a genotoxic drug causing both single- and double-strand breaks. Specifically, we used bleomycin to induce DNA damage. We followed morphological and chemical changes in chromosomes upon damage induction. Atomic force microscopy was used to visualize the morphology of chromosomes, while Raman microspectroscopy enabled the detection of changes in the chemical structure of chromatin with the resolution close to the diffraction limit. Additionally, we extracted spectra corresponding to chromosome I or chromatin from hyperspectral Raman maps with convolutional neural networks (CNN), which were further analysed with the principal component analysis (PCA) algorithm to reveal molecular markers of DNA damage in chromosomes. The applied multimodal approach revealed simultaneous morphological and molecular changes, including chromosomal aberrations, alterations in DNA conformation, methylation pattern, and increased protein expression upon the bleomycin treatment at the level of the single chromosome.

Dynamics of cell rounding during detachment.

Animal cells undergo repeated shape changes, for example by rounding up and respreading as they divide. Cell rounding can be also observed in interphase cells, for example when cancer cells switch from a mesenchymal to an ameboid mode of cell migration. Nevertheless, it remains unclear how interphase cells round up. In this article, we demonstrate that a partial loss of substrate adhesion triggers actomyosin-dependent cortical remodeling and ERM activation, which facilitates further adhesion loss causing cells to round. Although the path of rounding in this case superficially resembles mitotic rounding in involving ERM phosphorylation, retraction fiber formation, and cortical remodeling downstream of ROCK, it does not require Ect2. This work provides insights into the way partial loss of adhesion actives cortical remodeling to drive cell detachment from the substrate. This is important to consider when studying the mechanics of cells in suspension, for example using methods like real-time deformability cytometry (RT-DC).

Analysis of Biomechanical Properties of Hematopoietic Stem and Progenitor Cells Using Real-Time Fluorescence and Deformability Cytometry.

Stem cell mechanics, determined predominantly by the cell's cytoskeleton, plays an important role in different biological processes such as stem cell differentiation or migration. Several methods to measure mechanical properties of cells are currently available, but most of them are limited in the ability to screen large heterogeneous populations in a robust and efficient manner-a feature required for successful translational applications. With real-time fluorescence and deformability cytometry (RT-FDC), mechanical properties of cells in suspension can be screened continuously at rates of up to 1,000 cells/s-similar to conventional flow cytometers-which makes it a suitable method not only for basic research but also for a clinical setting. In parallel to mechanical characterization, RT-FDC allows to measure specific molecular markers using standard fluorescence labeling. In this chapter, we provide a detailed protocol for the characterization of hematopoietic stem and progenitor cells (HSPCs) in heterogeneous mobilized peripheral blood using RT-FDC and present a specific morpho-rheological fingerprint of HSPCs that allows to distinguish them from all other blood cell types.

High-throughput single-cell mechanical phenotyping with real-time deformability cytometry.

Mechanical properties of cells can serve as a label-free marker of cell state and function and their alterations have been implicated in processes such as cancer metastasis, leukocyte activation, or stem cell differentiation. Over recent years, new techniques for single-cell mechanical characterization at high throughput have been developed to accelerate discovery in the field of mechanical phenotyping. One such technique is real-time deformability cytometry (RT-DC), a robust technology based on microfluidics that performs continuous mechanical characterization of cells in a contactless manner at rates of up to 1000 cells per second. This tremendous throughput allows for comparison of large sample numbers and precise characterization of heterogeneous cell populations. Additionally, parameters acquired in RT-DC measurements can be used to determine the apparent Young's modulus of individual cells. In this chapter, we present practical aspects important for the implementation of the RT-DC methodology, including a description of the setup, operation principles, and experimental protocols. In the latter, we describe a variety of preparation procedures for samples originating from different sources including 2D and 3D cell cultures as well as blood and tissue-derived primary cells, and discuss obstacles that may arise during their measurements. Finally, we provide insights into standard data analysis procedures and discuss the method's performance in light of other available techniques.