Session N45: Focus Session: Cell Mechanics I

Cellular Tug-of-War: Forces at Work and DNA Stretching in Mitosis

In the microscopic world of the cell dominated by thermal noise, a cell must be able to successfully segregate its DNA with high fidelity in order to pass its genetic information on to its progeny. In this process of mitosis in eukaryotes, driving forces act on the cytoskeleton-based architecture called the mitotic spindle to promote this division. Our preliminary data demonstrates that the dynamics of this process in yeast cells is universal. Moreover, the dynamics suggest an increasing load as the chromosomes are pulled apart. To investigate this, we use three-dimensional imaging to track the dynamics of the poles of this architecture and the points of attachment to chromosomes simultaneously and with high spatial resolution. We analyze the relative motions of chromosomes as they are organized before segregation and as they are pulled apart, using this data to investigate the force-response behavior of this cytoskeleton-chromosome polymer system.   

\textit{in vivo} Measurements of Conformational Fluctuations of Chromosomal DNA in \textit{Escherichia Coli}

The cell is the site of active, motor-driven processes far from thermodynamic equilibrium. Therefore, the intracellular dynamics are complex and subject to a multitude of constraints and forces. We study the conformational fluctuations of chromosomal DNA \textit{in vivo} in live and dead \textit{E. coli} cells by Fluorescence Correlation Spectroscopy (FCS). The fluctuations move the DNA-bound fluorophores stochastically into the diffraction-limited excitation volume of a focused laser beam in a confocal microscope. From the time correlation functions of the fluorescence intensity, we obtain the mean square displacements of the DNA on a time scale from microseconds to seconds. We see a substantial decrease in the power spectral density (PSD) of the displacement fluctuations at frequencies below 10 Hz in the dead cells, compared to the live cells. The larger fluctuations in the living cells may indicate that the fluctuations on this time scale may be driven by active processes involving molecular motors that generate forces by ATP hydrolysis. A small difference in PSD between live and dead cells on shorter time scales suggests that the processes on corresponding short length scales rely primarily on thermally-driven diffusive mechanisms.   

Mediation of cell adhesion by the pericellular matrix

Cell adhesion requires a close proximity on the nanometer scale between the plasma membrane and the surrounding material (or neighboring cell). Yet, in many classic scenarios where cell adhesion is carefully regulated, including proliferation, migration, embryogenesis and cancer metastasis, the cell's surface is insulated by an invisible but microns thick polymer brush-like structure, called the pericellular matrix. Indeed the presence of the pericellular matrix has been correlated with increased migration and proliferation rates, where disruption of this bound polymer brush interferes with the efficacy of these processes. We present methods to characterize the pericellular matrix distribution, mechanics and mesh size and explore how cells orchestrate adhesion with the help of the pericellular matrix.   

Modifications of the structure of the pericellular matrix measured via optical force probe microscopy

The pericellular matrix is a large protein and polysaccharide rich polymer layer attached to the surface of many cells, and which often extends several microns out from the cell surface into the surrounding extracellular space. Here we study the intrinsic nature and modifications of the structure of the pericellular coat on rat chondrocytes with the use of optical force probe microscopy. Optical force probe studies allow us to make both dynamic force measurements as well as equilibrium force measurements throughout the coat. These force measurements are used to observe the structural change in the coat with the addition of exogenous aggrecan. Not only does addition of exogenous aggrecan dramatically swell our coat to well over twice in size, our analysis indicates that the addition of exogenous aggrecan decreases the mesh size throughout the coat. We speculate that the added aggrecan binds to available binding sites along the hyaluronan chain, both enlarging the coat's size as well as tightening up the opening within the coat. We further suggest that the available binding sites for the exogenous aggrecan are abundant in the outer edges of the coat, as both the dynamic and equilibrium forces in this region are changed. Here, both force measurements show that forces closest to the cell membrane remain relatively unchanged, while the forces in the outer region of the coat are increased. These results are consistent with those obtained with complementary measurements using quantitative particle exclusion assays.   

Quantitative particle exclusion assays of the pericellular coat reveal changing mesh size

We present a quantitative assay of the pericellular coat, a tethered polymer matrix that decorates the surface of numerous cell types. In these assays, we look at how passivated microspheres of varying diameter penetrate the cell coat. Distinct spatial distributions correspond to different particle sizes. These measurements confirm that the cell coat (on the chondrocyte RCJ-P cell line) has a spatially varying mesh size, in agreement with our independent assays performed with optical force probe microscopy. The data indicate that particles with diameters of 500 nm or greater do not penetrate the inner layer of the matrix, while particles smaller than 500 nm reach different regions, with the smallest reaching the cell surface. In an ongoing effort, we are developing a model for the observed statistical distribution of the beads. These experiments show that accessibility of the cell surface is strongly mediated by the presence of the cell coat, and they have important implications regarding the transport of molecules to the cell surface, protection from bacterial infection, drug delivery, as well as the way the cell interacts and adheres to the surrounding extracellular matrix.   

Reversible and irreversible deformations of bacterial cell walls

Bacterial cell walls determine their shape and hold their large internal pressure. In spite of their biological importance, a full understanding of their structure and mechanics is lacking. Here, we shed new light on the nature of the deformations of bacterial cell walls by showing, theoretically and experimentally, that these can be either elastically (reversibly) or plastically (irreversibly) deformed, depending on the timescales involved. Our data suggests that irreversible bending of the cell wall arises due to an asymmetric insertion of new material, responding to the mechanical stresses.   

Mechanical Properties of Primary Cilia

Recent studies have shown that the primary cilium, long thought to be a vestigial cellular appendage with no function, is involved in a multitude of sensory functions. One example, interesting from both a biophysical and medical standpoint, is the primary cilium of kidney epithelial cells, which acts as a mechanosensitive flow sensor. Genetic defects in ciliary function can cause, e.g., polycystic kidney disease (PKD). The material properties of these non-motile, microtubule-based 9$+$0 cilia, and the way they are anchored to the cell cytoskeleton, are important to know if one wants to understand the mechano-electrochemical response of these cells, which is mediated by their cilia. We have probed the mechanical properties, boundary conditions, and dynamics of the cilia of MDCK cells using optical traps and DIC/fluorescence microscopy. We found evidence for both elastic relaxation of the cilia themselves after bending and for compliance in the intracellular anchoring structures. Angular and positional fluctuations of the cilia reflect both thermal excitations and cellular driving forces.   

The influence of myosin-generated force to the intracellular microrheology in living cells

The mechanics of cells are governed by cytoskeletal filaments and molecular motors forming a dynamic mechanical entity. A recent experimental study by Mizuno et al. showed local shear modulus of a synthesized cytoskeletal network could increase as a result of myosin-generated internal stresses. To examine whether similar behaviors could take place in living cells we combined active and passive microrheology to measure the myosin-generated fluctuating force and intracellular shear modulus in HeLa cells. While our experiment showed an increase in the fluctuations of the shear modulus with increasing motor forces, the experiment did not find a direct correlation between the mean intracellular shear modulus and the motor-generated fluctuating force. Based on Mizuno et al's assumption shear modulus is increasing as local tensions, the difference between the results obtained by the intracellular behavior and the synthesized cytoskeletal network could be due to the existence of a steady-state intracellular tension that is stronger than the motor-generated fluctuating force.   

Cellular pressure and volume regulation and implications for cell mechanics

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death and migration. Volume and shape regulation also directly impacts the mechanics of the cell and multi-cellular tissues. Recent experiments found that during mitosis, eukaryotic cells establish a preferred steady volume and pressure, and the steady volume and pressure can robustly adapt to large osmotic shocks. Here we develop a mathematical model of cellular pressure and volume regulation, incorporating essential elements such as water permeation, mechano-sensitive channels, active ion pumps and active stresses in the actomyosin cortex. The model can fully explain the available experimental data, and predicts the cellular volume and pressure for several models of cell cortical mechanics. Furthermore, we show that when cells are subjected to an externally applied load, such as in an AFM indentation experiment, active regulation of volume and pressure leads to complex cellular response. We found the cell stiffness highly depends on the loading rate, which indicates the transport of water and ions might contribute to the observed viscoelasticity of cells.   

Direct mechanical measurements of cytoskeleton-mediated intercellular fluid flow

Cell behavior in tissues is intimately tied to forces generated by cytoskeletal contractions. Contraction generated tensions are balanced by deformations in the cell's microenvironment, by internal cytoskeletal structures, and by the incompressible cytosolic fluid contained by the cell membrane. However, contraction generated pressures cannot be supported by the cytosol if the cell membrane is adequately permeable. Small, non-selective pores called gap junctions connect cells in a layer, allowing small molecules to pass between cells. The ability of contraction driven fluid movement to transmit forces across gap junctions and the ability of cells to respond to this movement is unexplored. To study the mechanics of intercellular fluid flow, we apply biologically relevant pressures to large regions of cells in a monolayer with a micro-indentation system. We directly measure indentation force and volume as a function of time to determine fluid flow rates and associated stresses between cells. Preliminary results will be presented.   

Cell stretching in extensional flows for assaying cell mechanics

There is growing evidence that cell deformability is a useful indicator of cell state and may be a label-free biomarker of metastatic potential, degree of differentiation, and leukocyte activation. In order for deformability measurements to be clinically valuable given the heterogeneity of biological samples, there exists a need for a high-throughput assay of this biophysical property. We developed a robust method for obtaining high-throughput ($>$1,000 cells/sec) single-cell mechanical measurements which employs coupled hydrodynamic lift forces and curvature-induced secondary flows to uniformly position cells in flow, extensional flow stretching, high-speed imaging, and automated image analysis to extract diameter and deformability parameters. Using this method we have assayed numerous in vitro models of cellular transformations and clinical fluids where malignant cells manifest. We found transformations associated with increased motility or invasiveness increased deformability and the presence of large and deformable cells within clinical pleural fluids correlated well with cytological diagnoses of malignancy. This agrees with the hypothesis that cancerous cells are deformable by necessity--to be able to transverse tight endothelial gaps and invade tissues.