Optical control of fast and processive engineered myosins in vitro and in living cells.

Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality.

NuSeT: A deep learning tool for reliably separating and analyzing crowded cells.

Segmenting cell nuclei within microscopy images is a ubiquitous task in biological research and clinical applications. Unfortunately, segmenting low-contrast overlapping objects that may be tightly packed is a major bottleneck in standard deep learning-based models. We report a Nuclear Segmentation Tool (NuSeT) based on deep learning that accurately segments nuclei across multiple types of fluorescence imaging data.

Concerted localization-resets precede YAP-dependent transcription.

Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis.

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density.

A Mutation in Histone H2B Represents a New Class of Oncogenic Driver.

By examination of the cancer genomics database, we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K), found particularly in bladder and head and neck cancers, that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease.

Satb1 integrates DNA binding site geometry and torsional stress to differentially target nucleosome-dense regions.

The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity.

Physical confinement induces malignant transformation in mammary epithelial cells.

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules.

Strong triaxial coupling and anomalous Poisson effect in collagen networks.

While cells within tissues generate and sense 3D states of strain, the current understanding of the mechanics of fibrous extracellular matrices (ECMs) stems mainly from uniaxial, biaxial, and shear tests. Here, we demonstrate that the multiaxial deformations of fiber networks in 3D cannot be inferred solely based on these tests. The interdependence of the three principal strains gives rise to anomalous ratios of biaxial to uniaxial stiffness between 8 and 9 and apparent Poisson's ratios larger than 1.

A fluorogenic array for temporally unlimited single-molecule tracking.

We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules.

Mechanisms of Plastic Deformation in Collagen Networks Induced by Cellular Forces.

Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts.

Origins of chemoreceptor curvature sorting in Escherichia coli.

Bacterial chemoreceptors organize into large clusters at the cell poles. Despite a wealth of structural and biochemical information on the system's components, it is not clear how chemoreceptor clusters are reliably targeted to the cell pole. Here, we quantify the curvature-dependent localization of chemoreceptors in live cells by artificially deforming growing cells of Escherichia coli in curved agar microchambers, and find that chemoreceptor cluster localization is highly sensitive to membrane curvature.

ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements.

Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner.

Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC.

Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation.

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated.

Rapid disorganization of mechanically interacting systems of mammary acini.

Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype.

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.

The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them.

Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors.

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold.

A physical sciences network characterization of non-tumorigenic and metastatic cells.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols.

A single-molecule analysis reveals morphological targets for cellulase synergy.

The mechanisms of enzyme activity on solid substrates are not well understood. Unlike enzyme catalysis in aqueous solutions, enzyme activity on surfaces is complicated by adsorption steps and structural heterogeneities that make enzyme-substrate interactions difficult to characterize. Cellulase enzymes, which catalyze the depolymerization of cellulose, show binding specificities for different cellulose surface morphologies, but the influence of these specificities on the activity of multienzyme mixtures has remained unclear.

mMaple: a photoconvertible fluorescent protein for use in multiple imaging modalities.

Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells.

Molecular architecture and assembly principles of Vibrio cholerae biofilms.

In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters.

Scanning angle interference microscopy reveals cell dynamics at the nanoscale.

Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.

What does physics have to do with cancer?

Large-scale cancer genomics, proteomics and RNA-sequencing efforts are currently mapping in fine detail the genetic and biochemical alterations that occur in cancer. However, it is becoming clear that it is difficult to integrate and interpret these data and to translate them into treatments. This difficulty is compounded by the recognition that cancer cells evolve, and that initiation, progression and metastasis are influenced by a wide variety of factors.