Delineating the role of membrane blebs in a hybrid mode of cancer cell invasion in three-dimensional environments.

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional ß1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

Manifestations of static and dynamic heterogeneity in single molecule translational measurements in glassy systems.

Rotational-translational decoupling in systems near Tg, in which translational diffusion is apparently enhanced relative to rotation, has been observed in ensemble and single molecule experiments and has been linked to dynamic heterogeneity. Here, simulations of single molecules experiencing homogeneous diffusion and static and dynamic heterogeneous diffusion are performed to clarify the contributions of heterogeneity to such enhanced translational diffusion. Results show that time-limited trajectories broaden the distribution of diffusion coefficients in the presence of homogeneous diffusion but not when physically reasonable degrees of static heterogeneity are present. When dynamic heterogeneity is introduced, measured diffusion coefficients uniformly increase relative to input diffusion coefficients, and the widths of output distributions decrease, providing support for the idea that dynamic heterogeneity can drive apparent translational enhancement. Among simulations with dynamic heterogeneity, when the frequency of dynamic exchange is correlated with the initial diffusion coefficient, the measured diffusion coefficient behavior as a function of observation time matches that seen experimentally, the only set of simulations explored in which this occurs. Taken together with experimental results, this suggests that enhanced translational diffusion in glassy systems occurs through dynamic exchange consistent with wide underlying distributions of diffusion coefficients and exchange coupled to local spatiotemporal dynamics.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Correlating fragility and heterogeneous dynamics in polystyrene through single molecule studies.

Many macroscopic properties of polymers depend on their molecular weight, with one notable example being glass transition temperature: polymers with higher molecular weights typically have higher glass transition temperatures than their lower molecular weight polymeric and oligomeric counterparts. Polymeric systems close to their glass transition temperatures also exhibit interesting properties, showing both high (and molecular weight dependent) fragility and strong evidence of dynamic heterogeneity. While studies have detailed the correlations between molecular weight and fragility, studies clearly detailing correlations between molecular weight and degree of heterogeneous dynamics are lacking. In this study, we use single molecule rotational measurements to investigate the impact of molecular weight on polystyrene's degree of heterogeneity near its glass transition temperature. To this end, two types of fluorescent probes are embedded in films composed of polystyrene ranging from 0.6 to 1364.0 kg mol-1. We find correlation between polystyrene molecular weight, fragility, and degree of dynamic heterogeneity as reported by single molecule stretching exponents but do not find clear correlation between these quantities and time scales associated with dynamic exchange.

Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells.

The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of ∆Np63α, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the ∆Np63 promoter is sufficient for repression induced by H-RasV12. The suppression of ∆Np63α expression by these oncogenes concomitantly leads to an epithelial-to-mesenchymal transition (EMT). In addition, the depletion of ∆Np63α alone is sufficient to induce EMT. Both H-RasV12 expression and ∆Np63α depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ∆Np63α.

Which probes can report intrinsic dynamic heterogeneity of a glass forming liquid?

Using extrinsic probes to study a host system relies on the probes' ability to accurately report the host properties under study. Probes have long been used to characterize dynamic heterogeneity, the phenomenon in which a liquid near its glass transition exhibits distinct dynamics as a function of time and position, with molecules within nanometers of each other exhibiting dynamics that may vary by orders of magnitude. The spatial and temporal characteristics of dynamic heterogeneity demand the selection of probes using stringent criteria on their size and dynamics. In this report, we study the dynamic heterogeneity of the prototypical molecular glass former o-terphenyl by investigating single molecule rotation of two perylene dicarboximide probe molecules that differ in size and comparing this to results obtained previously with the probe BODIPY268. It is found that a probe's ability to accurately report dynamic heterogeneity in o-terphenyl depends on whether the reported distribution of dynamics overlaps with the intrinsic dynamics of the host, which is naturally related to the width of the intrinsic dynamics and the magnitude of dynamical shift in probe dynamics relative to the host. We show that a probe that rotates ≈15 times more slowly than the intrinsic dynamics of the host o-terphenyl senses the slowest ≈5% of the full dynamic heterogeneity whereas one that rotates ≈65 times more slowly than the host fails to report dynamic heterogeneity of the host.

Single molecule studies reveal temperature independence of lifetime of dynamic heterogeneity in polystyrene.

Polymeric systems close to their glass transition temperature are known to exhibit heterogeneous dynamics that evolve both over time and space, comparable to the dynamics of small molecule glass formers. It remains unclear how temperature influences the degree of heterogeneous dynamics in such systems. In the following report, a fluorescent perylene dicarboximide probe molecule that reflects the full breadth of heterogeneity of the host was used to examine the temperature dependence of the dynamic heterogeneity lifetime in polystyrene at several temperatures ranging from the glass transition to 10 K above this temperature via single molecule microscopy. Contrary to prior reports, no apparent temperature dependence of time scales associated with dynamic heterogeneity was detected; indeed, the probe molecules report characteristic dynamic heterogeneity lifetimes 100-300 times the average alpha-relaxation time (τα) of the polystyrene host at all temperatures studied.

Ideal probe single-molecule experiments reveal the intrinsic dynamic heterogeneity of a supercooled liquid.

The concept of dynamic heterogeneity and the picture of the supercooled liquid as a mosaic of environments with distinct dynamics that interchange in time have been invoked to explain the nonexponential relaxations measured in these systems. The spatial extent and temporal persistence of these regions of distinct dynamics have remained challenging to identify. Here, single-molecule fluorescence measurements using a probe similar in size and mobility to the host o-terphenyl unambiguously reveal exponential relaxations distributed in time and space and directly demonstrate ergodicity of the system down to the glass transition temperature. In the temperature range probed, at least 200 times the structural relaxation time of the host is required to recover ensemble-averaged relaxation at every spatial region in the system.

In†Situ Optical Imaging of the Growth of Conjugated Polymer Aggregates.

Understanding the mechanisms that contribute to conjugated polymer aggregate formation and growth may yield enhanced control of aggregate morphology and functional properties on the mesoscopic scale. In situ optical imaging of the growth of MEH-PPV aggregates in real time in controlled swollen films shows that growth occurs through multiple mechanisms and is more complex than previously described. Direct evidence is provided for both Ostwald ripening and aggregate coalescence as operative modes of aggregate growth in solvent swollen films. These growth mechanisms have a distinct and strong impact on the evolution of morphological order of growing aggregates: while Ostwald ripening allows preservation of highly ordered morphology, aggregate coalescence occurs with no preferential orientation, leading to attenuation in degree of ordering.

Confocal Rheology Probes the Structure and Mechanics of Collagen through the Sol-Gel Transition.

Fibrillar type I collagen-based hydrogels are commonly used in tissue engineering and as matrices for biophysical studies. Mechanical and structural properties of these gels are known to be governed by the conditions under which fibrillogenesis occurs, exhibiting variation as a function of protein concentration, temperature, pH, and ionic strength. Deeper understanding of how macroscopic structure affects viscoelastic properties of collagen gels over the course of fibrillogenesis provides fundamental insight into biopolymer gel properties and promises enhanced control over the properties of such gels. Here, we investigate type I collagen fibrillogenesis using confocal rheology-simultaneous confocal reflectance microscopy, confocal fluorescence microscopy, and rheology. The multimodal approach allows direct comparison of how viscoelastic properties track the structural evolution of the gel on fiber and network length scales. Quantitative assessment and comparison of each imaging modality and the simultaneously collected rheological measurements show that the presence of a system-spanning structure occurs at a time similar to rheological determinants of gelation. Although this and some rheological measures are consistent with critical gelation through percolation, additional rheological and structural properties of the gel are found to be inconsistent with this theory. This study clarifies how structure sets viscoelasticity during collagen fibrillogenesis and more broadly highlights the utility of multimodal measurements as critical test-beds for theoretical descriptions of complex systems.

Conformation-Dependent Photostability among and within Single Conjugated Polymers.

The relationship between photostability and conformation of 2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene (MEH-PPV) conjugated polymers was studied via excitation polarization modulation depth (M) measurements. Upon partial photobleaching, M distributions of collapsed, highly ordered MEH-PPV molecules shifted toward lower values. Conversely, M distributions of MEH-PPV molecules with random coil conformations moved toward higher values after partial photobleaching. Monte Carlo simulations of randomly distributed dipole moments along polymer chains subjected to partial photobleaching revealed that a statistical effect leads to an increase in peak M value. Decreases in M values seen experimentally in the population of MEH-PPV molecules with high M values, however, are due to conformation-dependent photostability within single MEH-PPV polymers. We show that, while folded MEH-PPV molecules are relatively more photostable than extended MEH-PPV molecules in an ensemble, extended portions of particular molecules are more photostable than folded domains within single MEH-PPV molecules.

Revealing and resolving degeneracies in stretching exponents in temporally heterogeneous environments.

Supercooled liquids are proposed to be dynamically heterogeneous, with regions exhibiting relaxation time scales that vary in space and time. Measurement of the distribution of such time scales could be an important test of various proposed theories of vitrification. Single molecule fluorescence experiments attempt to uncover this distribution, typically by embedding single molecule probes into these systems and monitoring their individual rotational relaxations from a computed autocorrelation function (ACF). These ACFs may exhibit stretched exponential decays, with the value of the stretching exponent assumed to report the set of dynamical environments explored by the probe. Here, we use simulated trajectories of rotation to investigate how the time scale of dynamic exchange relative to underlying relaxation time scales in the system affects probe ability to report the distribution relaxation of time scales present. We find that dynamically heterogeneous regions must persist for approximately 50 times the median relaxation time scale for a single molecule to accurately report the full distribution of time scales it has experienced. In systems with faster dynamic exchange, single molecule ACFs average over successive environments, limiting the reported heterogeneity of the system. This leads to degeneracies in stretching exponent for systems with different underlying relaxation time distributions. We show that monitoring single molecule median stretching exponent as a function of trajectory length or simultaneously measuring median stretching exponent and measured relaxation time distribution at a given trajectory length can resolve these degeneracies, revealing the underlying set of relaxation times as well as median exchange time.

Single molecule rotational probing of supercooled liquids.

Much of the interesting behavior that has been observed in supercooled liquids appears to be related to dynamic heterogeneity, the presence of distinct dynamic environments - with no apparent underlying structural basis - in these systems. To most directly interrogate these environments, proposed to span regions just a few nanometers across, molecular length scale probes are required. Single molecule fluorescent microscopy was introduced to the field a decade ago and has provided strong evidence of dynamic heterogeneity in supercooled systems. However, only more recently has the full set of challenges associated with interpreting results of these experiments been described. With a fuller understanding of these challenges in hand, single molecule measurements can be employed to provide a more precise picture of dynamic heterogeneity in supercooled liquids and other complex systems. In this tutorial review, experimental and data analysis details are presented for the most commonly employed single molecule approach to studying supercooled liquids, the measurement of rotational dynamics of single molecule probes. Guidance is provided in experimental set-up and probe selection, with a focus on choices that affect data interpretation and probe sensitivity to dynamic heterogeneity.

Collagen I self-assembly: revealing the developing structures that generate turbidity.

Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.

Extraction of rotational correlation times from noisy single molecule fluorescence trajectories.

Monitoring single molecule probe rotations is an increasingly common approach to studying dynamics of complex systems, including supercooled liquids. Even with advances in fluorophore design and detector sensitivity, such measurements typically exhibit low signal to noise and signal to background ratios. Here, we simulated and analyzed orthogonally decomposed fluorescence signals of single molecules undergoing rotational diffusion in a manner that mimics experimentally collected data of probes in small molecule supercooled liquids. The effects of noise, background, and trajectory length were explicitly considered, as were the effects of data processing approaches that may limit the impact of noise and background on assessment of environmental dynamics. In many cases, data treatment that attempts to remove noise and background were found to be deleterious. However, for short trajectories below a critical signal to background threshold, a thresholding approach that successfully removed data points associated with noise and spared those associated with signal allowed for assessment of environmental dynamics that was as accurate and precise as would be achieved in the absence of noise.

Heterogeneity in single-molecule observables in the study of supercooled liquids.

Bulk approaches to studying heterogeneous systems obscure important details, as they report average behavior rather than the distribution of behaviors in such environments. Small-molecule and polymeric supercooled liquids, which display heterogeneity in their dynamics without an underlying structural heterogeneity that sets those dynamics, are important constituents of this category of condensed matter systems. A variety of approaches have been devised to unravel ensemble averaging in supercooled liquids. This review focuses on the ultimate subensemble approach, single-molecule measurements, as they have been applied to the study of supercooled liquids. We detail how three key experimental observables (single-molecule probe rotation, translation, and fluorescence lifetime) have been employed to provide detail on dynamic heterogeneity in supercooled liquids. Special attention is given to the potential for, but also the challenges in, discriminating spatial and temporal heterogeneity and detailing the length scales and timescales of heterogeneity in these systems.

Plasticity Variable Collagen-PEG Interpenetrating Networks Modulate Cell Spreading.

The extracellular matrix protein collagen I has been used extensively in the field of biomaterials due to its inherent biocompatibility and unique viscoelastic and mechanical properties. Collagen I self-assembly into fibers and networks is environmentally sensitive to gelation conditions such as temperature, resulting in gels with distinct network architectures and mechanical properties. Despite this, collagen gels are not suitable for many applications given their relatively low storage modulus. We have prepared collagen-poly(ethylene glycol) [PEG] interpenetrating network (IPN) hydrogels to reinforce the collagen network, which also induces changes to network plasticity, a recent focus of study in cell-matrix interactions. Here, we prepare collagen/PEG IPNs varying collagen concentration and collagen gelation temperature to assess changes in microarchitecture and mechanical properties of these networks. By tuning these parameters, IPNs with a range of stiffness, plasticity and pore size are obtained. Cell studies suggest that matrix plasticity is a key determinant of cell behavior, including cell elongation, on these gels. This work presents a natural/synthetic biocompatible matrix that retains the unique structural properties of collagen networks with increased storage modulus and tunable plasticity. The described IPN materials will be of use for applications in which control of cell spreading is desirable, as only minimal changes in sample preparation lead to changes in cell spreading and circularity. Additionally, this study contributes to our understanding of the connection between collagen self-assembly conditions and matrix structural and mechanical properties and presents them as useful tools for the design of other collagen based biomaterials. STATEMENT OF SIGNIFICANCE: We developed a collagen-poly(ethylene glycol) interpenetrating network (IPN) platform that retains native collagen architecture and biocompatibility but provides higher stiffness and tunable plasticity. With minor changes in collagen gelation temperature or concentration, IPN gels with a range of plasticity, storage modulus, and pore size can be obtained. The tunable plasticity of the gels is shown to modulate cell spreading, with a greater proportion of elongated cells on the most plastic of IPNs, supporting the assertion that matrix plasticity is a key determinant of cell spreading. The material can be of use for situations where control of cell spreading is desired with minimal intervention, and the findings herein may be used to develop similar collagen based IPN platforms.

Strain stiffening in collagen I networks.

Biopolymer gels exhibit strain stiffening that is generally not seen in synthetic gels. Here, we investigate the strain-stiffening behavior in collagen I gels that demonstrate elasticity derived from a variety of sources including crosslinking through telopeptides, bundling through low-temperature gelation, and exogenous crosslinking with genipin. In all cases, it is found that these gels exhibit strain stiffening; in general, onset of strain stiffening occurs earlier, yield strain is lower, and degree of strain stiffening is smaller in higher concentration gels and in those displaying thick fibril bundles. Recovery after exposure to high strains is substantial and similar in all gels, suggesting that much of the stiffening comes from reversible network deformations. A key finding of this study is that collagen I gels of identical storage and loss moduli may display different nonlinear responses and different capacities to recover from high strain.

Single molecule probe reports of dynamic heterogeneity in supercooled ortho-terphenyl.

The rotational dynamics of three perylene diimide dyes are studied on the single molecule (SM) level in ortho-terphenyl (OTP) near the glass transition temperature (T(g)). At all temperatures probed, spanning 1.03-1.06 T(g), each of the three probes exhibits rotational correlation times, τ(c), that span more than a decade, consistent with the presence of spatially heterogeneous dynamics in OTP. No trend is found as a function of temperature, but a trend as a function of probe is observed: Average probe rotational correlation time scales inversely with breadth of SM τ(c) distribution, with faster probes exhibiting broader τ(c) distributions. This implies that dynamic exchange occurs on and below time scales associated with probe rotation. Extrapolating FWHM of rotational relaxation times to the structural relaxation time of the host shows that the τ(c) distribution would span nearly two decades in the limit of no probe temporal averaging. Comparison with SM measurements in glycerol suggests that OTP demonstrates a greater degree of spatially heterogeneous dynamics in this temperature range than does glycerol.

DISC-3D: dual-hydrogel system enhances optical imaging and enables correlative mass spectrometry imaging of invading multicellular tumor spheroids.

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging.