Person:

Weitz, David

Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry.

Face-centered cubic single crystals of (σ=1.55 μm) diameter hard-sphere silica colloidal particles were prepared by sedimentation onto (100) and (110) oriented templates. The crystals had a wide interface with the overlaying liquid that was parallel to the template. The location of the interface was determined by confocal microscopic location of the particles, followed by identification of the crystalline and liquid phases by a bond-orientation order parameter. Fluctuations in the height of the interface about its average position were recorded for several hundred configurations. The interfacial stiffness (γ̃) was determined from the slope of the inverse squared Fourier components of the height profile vs the square of the wave number, according to the continuum capillary fluctuation method. The offset of the fit from the origin could quantitatively be accounted for by gravitational damping of the fluctuations. For the (100) interface, (γ̃ =(1.3±0.3)k_{B}T/σ^{2}); for the (110) interface, (γ̃ =(1.0±0.2)k_{B}T/σ^{2}). The interfacial stiffness of both interfaces was found to be isotropic in the plane. This is surprising for the (110), where crystallography predicts twofold symmetry. Sedimentation onto a (111) template yielded a randomly stacked hexagonal crystal with isotropic (γ̃ =0.66k_{B}T/σ^{2}). This value, however, is less reliable than the two others due to imperfections in the crystal.

Colloidal capsules can sustain an external osmotic pressure; however, for a sufficiently large pressure, they will ultimately buckle. This process can be strongly influenced by structural inhomogeneities in the capsule shells. We explore how the time delay before the onset of buckling decreases as the shells are made more inhomogeneous; this behavior can be quantitatively understood by coupling shell theory with Darcy’s law. In addition, we show that the shell inhomogeneity can dramatically change the folding pathway taken by a capsule after it buckles.

We performed a series of deformation experiments on a monodisperse, hard-sphere colloidal glass while simultaneously following the 3D trajectories of roughly 50,000 individual particles with a confocal microscope. In each experiment, we deformed the glass in pure shear at a constant strain rate (1 − 5 × 10−5s−1) to maximum macroscopic strains (5 − 10%), then reversed the deformation at the same rate to return to zero macroscopic strain. We also measured 3D particle trajectories in an identically-prepared quiescent glass in which the macroscopic strain was always zero. We find that shear transformation zones exist and are active in both sheared and quiescent colloidal glasses, revealed by a distinctive four-fold signature in spatial autocorrelations of the local shear strain. With increasing shear, the population of local shear transformations develops more quickly than in a quiescent glass, and many of these transformations are irreversible. When the macroscopic strain is reversed, we observe partial elastic recovery, followed by plastic deformation of the opposite sign, required to compensate for the irreversibly transformed regions. The average diameter of the shear transformation zones at maximum strain is 2.3 particle diameters.

Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity.

Collagen is the most abundant extracellular-network-forming protein in animal biology and is important in both natural and artificial tissues, where it serves as a material of great mechanical versatility. This versatility arises from its almost unique ability to remodel under applied loads into anisotropic and inhomogeneous structures. To explore the origins of this property, we develop a set of analysis tools and a novel experimental setup that probes the mechanical response of fibrous networks in a geometry that mimics a typical deformation profile imposed by cells in vivo. We observe strong fiber alignment and densification as a function of applied strain for both uncrosslinked and crosslinked collagenous networks. This alignment is found to be irreversibly imprinted in uncrosslinked collagen networks, suggesting a simple mechanism for tissue organization at the microscale. However, crosslinked networks display similar fiber alignment and the same geometrical properties as uncrosslinked gels, but with full reversibility. Plasticity is therefore not required to align fibers. On the contrary, our data show that this effect is part of the fundamental non-linear properties of fibrous biological networks.

Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. Methodology/principal findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D (\beta-strands) of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress. Conclusions/significance: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

Background: Highly malignant gliomas are characterized by rapid growth, extensive local tissue infiltration and the resulting overall dismal clinical outcome. Gaining any additional insights into the complex interaction between this aggressive brain tumor and its microenvironment is therefore critical. Currently, the standard imaging modalities to investigate the crucial interface between tumor growth and invasion in vitro are light and confocal laser scanning microscopy. While immensely useful in cell culture, integrating these modalities with this cancer's clinical imaging method of choice, i.e. MRI, is a non-trivial endeavour. However, this integration is necessary, should advanced computational modeling be able to utilize these in vitro data to eventually predict growth behaviour in vivo. We therefore argue that employing the same imaging modality for both the experimental setting and the clinical situation it represents should have significant value from a data integration perspective. In this case study, we have investigated the feasibility of using a specific form of MRI, i.e. magnetic resonance microscopy or MRM, to study the expansion dynamics of a multicellular tumor spheroid in a collagen type I gel. Methods: An U87mEGFR human giloblastoma multicellular spheroid (MTS) containing approximately 4·103 cells was generated and pipetted into a collagen I gel. The sample was then imaged using a T2-weighted 3D spoiled gradient echo pulse sequence on a 14T MRI scanner over a period of 12 hours with a temporal resolution of 3 hours at room temperature. Standard histopathology was performed on the MRM sample, as well as on control samples. Results: We were able to acquire three-dimensional MR images with a spatial resolution of 24 × 24 × 24 μm3. Our MRM data successfully documented the volumetric growth dynamics of an MTS in a collagen I gel over the 12-hour period. The histopathology results confirmed cell viability in the MRM sample, yet displayed distinct patterns of cell proliferation and invasion as compared to control. Conclusion: In this study, we demonstrate that a specific form of MRI, i.e. magnetic resonance microscopy or MRM, can be used to study the dynamic growth of a multicellular tumor spheroid (MTS) with a single cell scale spatial resolution that approaches the level of light microscopy. We argue that MRM can be employed as a complementary non-invasive tool to characterize microscopic MTS expansion, and thus, together with integrative computational modeling, may allow bridging of the experimental and clinical scales more readily.

Random motion within the cytoplasm gives rise to molecular diffusion; this motion is essential to many biological processes. However, in addition to thermal Brownian motion, the cytoplasm also undergoes constant agitation caused by the activity of molecular motors and other nonequilibrium cellular processes. Here, we discuss recent work that suggests this activity can give rise to cytoplasmic motion that has the appearance of diffusion but is significantly enhanced in its magnitude and which can play an important biological role, particularly in cytoskeletal assembly.

We present a confocal microscopy study of 1.55micrometers monodisperse silica hard spheres as they sediment and crystallize at the bottom wall of a container. If the particles sediment onto a featureless flat wall, the two bottom layers crystallize simultaneously and layerwise growth follows. If the wall is replaced by a hexagonal template, only layerwise growth occurs. Our results complement earlier numerical simulations and experiments on other colloidal systems.