Daphne Weihs

Particle tracking in living cells: a review of the mean square displacement method and beyond

The focus of many particle tracking experiments in the last decade has been active systems, such as living cells. In active systems, the particles undergo simultaneous active and thermally driven transport. In contrast to thermally driven transport, particle motion driven by active processes cannot directly be correlated to the rheology of the probed region. The rheology in particle tracking experiments is typically obtained through the mean square displacements (MSD) of the trajectories. Hence, the MSD and its functional form remain the only basic tools to evaluate and compare living cells or other active systems. However, the mechano-structural characteristics of the intracellular environment and the mechanisms driving particle transport cannot be revealed by the MSD alone. Hence, approaches for advanced analysis of particle trajectories have been introduced recently. Here, we present a broad review of the extensive intracellular particle tracking experiments that have been carried out on a wide variety of cell types. Those works utilize the MSD, revealing similarities and differences relating to cell type and experimental setup. We also highlight several advanced trajectory-and displacement-based analysis methods and illustrate their capabilities using particle tracking data obtained from two cancer cell lines. We show that combining these analysis methods with the MSD can reveal additional information on intracellular structure and the existence and nature of active processes driving particle motion in cells.

Intracellular Mechanics and Activity of Breast Cancer Cells Correlate with Metastatic Potential

Mechanics of cancer cells are directly linked to their metastatic potential, or ability to produce a secondary tumor at a distant site. Metastatic cells survive in the circulatory system in a non-adherent state, and can squeeze through barriers in the body. Such considerable structural changes in cells rely on rapid remodeling of internal structure and mechanics. While external mechanical measurements have demonstrated enhanced pliability of cancer cells with increased metastatic potential, little is known about dynamics of their interior and we expect that to change significantly in metastatic cells. We perform a comparative study, using particle-tracking to evaluate the intracellular mechanics of living epithelial breast cells with varying invasiveness. Particles in all examined cell lines exhibit super-diffusion with a scaling exponent of 1.4 at short lag times, likely related to active transport by fluctuating microtubules and their associated molecular motors. Specifics of probe-particle transport differ between the cell types, depending on the cytoskeleton network-structure and interactions with it. Our study shows that the internal microenvironment of the highly metastatic cells evaluated here is more pliable and their cytoskeleton is less dense than the poorly metastatic and benign cells. We thus reveal intracellular structure and mechanics that can support the unique function and invasive capabilities of highly metastatic cells.

Microstructures in the aqueous solutions of a hybrid anionic fluorocarbon/hydrocarbon surfactant

The aqueous solutions of the anionic hybrid fluorocarbon/hydrocarbon surfactant sodium 1-oxo-1[4-(tridecafluorohexyl)phenyl]-2-hexanesulfate (FC6HC4) shows peculiar rheological behavior. At 25 degrees C the viscosity vs concentration curve goes successively through a maximum and a minimum, while the viscosity vs temperature curve of the 10 wt% aqueous FC6HC4 solution goes through a marked maximum at 36 degrees C [Tobita et al., Langmuir 13 (1997) 5054]. In an attempt to explain these properties the microstructure of aqueous solutions of FC6HC4 has been investigated by means of digital light microscopy, transmission electron microscopy at cryogenic temperature (cryo-TEM), rheology, and self-diffusion NMR. At 20 degrees C, the increase of the FC6HC4 concentration was found to result in a progressive change of structure of the surfactant assemblies from mainly spherical micelles at 0.5 wt% to mainly cylindrical micelles at 10 wt%. At intermediate concentrations small disk-like micelles and small complete and incomplete vesicles coexisting with cylindrical micelles were visualized. The occurrence of stretched cylindrical micelles is responsible for the effect of the surfactant concentration on the solution viscosity. Cryo-TEM, rheology, and self-diffusion NMR all suggest that an increase of the temperature brings about a growth of the assemblies present in the 10 wt% solution of FC6HC4. The structure of the assemblies present at the temperature where the viscosity is a maximum could not be elucidated by cryo-TEM because of the probable occurrence of an on-the-grid phase transformation, the result of blotting during specimen preparation. Nevertheless, the results show that the observed large assemblies break up at higher temperature to give rise to a more labile bicontinuous structure that consists of multi-connected disordered lamellae, with many folds and creases, and that may well be the L3 phase.

Low intensity ultrasound perturbs cytoskeleton dynamics

Therapeutic ultrasound is widely employed in clinical applications but its mechanism of action remains unclear. Here we report prompt fluidization of a cell and dramatic acceleration of its remodeling dynamics when exposed to low intensity ultrasound. These physical changes are caused by very small strains (10-5) at ultrasonic frequencies (106 Hz), but are closely analogous to those caused by relatively large strains (10-1) at physiological frequencies (100 Hz). Moreover, these changes are reminiscent of rejuvenation and aging phenomena that are well-established in certain soft inert materials. As such, we suggest cytoskeletal fluidization together with resulting acceleration of cytoskeletal remodeling events as a mechanism contributing to the salutary effects of low intensity therapeutic ultrasound.

Metastatic cancer cells tenaciously indent impenetrable, soft substrates

We present here the first evidence of mechanical penetration by a metastatic cancer cell. During metastasis, the invasive cancer-cell penetrates tissue and extracellular matrix, changes shape and applies force. These applied forces, in turn, depend on substrate stiffness and degradability. The initial stage of metastatic penetration comprises substrate indentation, which, however, has not yet been studied. Hence, we evaluate the evolution of indentation, focusing on differences relating to the metastatic potential (MP) of the cells and substrate stiffness. We found that metastatic cells attain a mushroom-like morphology and then, over several hours, repeatedly indent the substrate in a manner suggestive of a special role for the nucleus. Cells with higher MP have previously been shown to be softer internally and externally than those with lower MP yet, paradoxically, applied stronger forces. Cells of higher MP develop stronger forces on gels stiff enough to provide grip handles yet soft enough to indent, whereas benign cells did not indent substrates at all. These findings provide insight into the central role of physical forces in the initial stages of metastatic penetration and reveal new targets for treatment.

Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells

Metastasis formation is a major cause of mortality in cancer patients and includes tumor cell relocation to distant organs. A metastatic cell invades through other cells and extracellular matrix by biochemical attachment and mechanical force application. Force is used to move on or through a 2- or 3-dimensional (3D) environment, respectively, or to penetrate a 2D substrate. We have previously shown that even when a gel substrate is impenetrable, metastatic breast cancer cells can still indent it by applying force. Cells typically apply force through the acto-myosin network, which is mechanically connected to the nucleus. We develop a 3D image-analysis to reveal relative locations of the cell elements, and show that as cells apply force to the gel, a coordinated process occurs that involves cytoskeletal remodeling and repositioning of the nucleus. Our approach shows that the actin and microtubules reorganize in the cell, bringing the actin to the leading edge of the cell. In parallel, the nucleus is transported behind the actin, likely by the cytoskeleton, into the indentation dimple formed in the gel. The nucleus volume below the gel surface correlates with indentation depth, when metastatic breast cancer cells indent gels deeply. However, the nucleus always remains above the gel in benign cells, even when small indentations are observed. Determining mechanical processes during metastatic cell invasion can reveal how cells disseminate in the body and can uncover targets for diagnosis and treatment.

Effects of cytoskeletal disruption on transport, structure, and rheology within mammalian cells

Quantification of cellular responses to stimuli is challenging. Cells respond to changing external conditions through internal structural and compositional and functional modifications, thereby altering their transport and mechanical properties. By properly interpreting particle-tracking microrheology, we evaluate the response of live cells to cytoskeletal disruption mediated by the drug nocodazole. Prior to administering the drug, the particles exhibit an apparently diffusive behavior that is actually a combination of temporally heterogeneous ballistic and caged motion. Selectively depolymerizing microtubules with the drug causes actively crawling cells to halt, providing a means for assessing drug efficacy, and making the caged motion of the probes readily apparent.

Origin of active transport in breast-cancer cells

Living cells are active systems, continuously adapting to respond to their environment. The dynamic cytoskeleton and especially the molecular motors acting on it provide the cell with its remodeling capabilities and allow active transport within the cell. While active transport in living cells has been well-documented, the underlying mechanisms have not been determined. Hence, we systematically target the cytoskeleton, molecular motors, and ATP energy related processes to determine their roles in particle transport. We perform intracellular particle tracking in low and high metastatic potential (MP) breast-cancer cells and analyze particle motion through the mean square displacement and other powers of the displacement. From those experiments, we show that the motion in both cell types is actively driven by fluctuating microtubules and their associated molecular motors. In the two cells, however, the relative importance of the mechanisms varies. In the low MP cells, particles are mostly transported by motors and likely remain on a single microtubule. In those cells, microtubule fluctuations cause intermittent jumps and actin typically hinders motion in the dense intracellular microenvironment. In contrast, particle motion in the high MP cells is driven by indirect motor-transport or by jumping between microtubules and is highly impacted by fluctuations of the microtubules. Thus, we are able to provide insight into mechanisms driving active transport in living cells, using intracellular particle tracking.

Time-dependent micromechanical responses of breast cancer cells and adjacent fibroblasts to electric treatment.

Direct-current, low-intensity, electric fields were suggested as a minimally invasive treatment for various cancers. The tumor microenvironment may affect treatment efficacy, albeit it has not generally been considered when evaluating novel anti-cancer treatments. We evaluate the effects of electric treatment on epithelial, breast-cancer cells, co-cultured with non-cancerous fibroblasts, a simplified model for the tumor-microenvironment. We evaluate changes in morphology, cytoskeleton, and focus on dynamic intracellular structure and mechanics. Multiple-particle tracking was used within living cells to quantify time-dependent structural and mechanical changes. Cancer cells suffer severe cell death and exhibit transient rounding and changes in internal structural and mechanics. Interestingly, treating cancer cells in co-culture with fibroblasts delays and reduces their responses to treatment. Our particle-tracking data indicates a mechanism relating the observed changes in intracellular transport to transient changes in the microtubule network and its motors. In contrast, fibroblasts are only minimally affected by treatment, separately or in co-culture. To conclude, intracellular mechanics reveal time-dependent responses after treatment, unavailable by bulk measurements. This time-dependence could provide a window of opportunity for continued treatment. We demonstrate the importance of evaluating anti-cancer treatments within their microenvironment, which can affect response magnitude and time-course.

Cytoskeleton and plasma-membrane damage resulting from exposure to sustained deformations: A review of the mechanobiology of chronic wounds

The purpose of this review paper is to summarize the current knowledge on cell-scale mechanically-inflicted deformation-damage, which is at the frontier of cell mechanobiology and biomechanics science, specifically in the context of chronic wounds. The dynamics of the mechanostructure of cells and particularly, the damage occurring to the cytoskeleton and plasma-membrane when cells are chronically deformed (as in a weight-bearing static posture) is correlated to formation of the most common chronic wounds and injuries, such as pressure ulcers (injuries). The first occurrence is microscopic injury which onsets as damage in individual cells and then progresses macroscopically to the tissue-scale. Here, we specifically focus on sub-catastrophic and catastrophic damage to cells that can result from mechanical loads that are delivered statically or at physiological rates; this results in apoptosis at prolonged times or necrosis, rapidly. We start by providing a basic background of cell mechanics and dynamics, focusing on the plasma-membrane and the cytoskeleton, and discuss approaches to apply and estimate deformations in cells. We then consider the effects of different levels of mechanical loads, i.e. low, high and intermediate, and describe the expected damage in terms of time-scales of application and in terms of cell response, providing experimental examples where available. Finally, we review different theoretical and computational modeling approaches that have been used to describe cell responses to sustained deformation. We highlight the insights that those models provide to explain, for example, experimentally observed variabilities in cell damage and death under loading.