@article {48858, title = {Cell volume change through water efflux impacts cell stiffness and stem cell fate.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Sep 25}, pages = {E8618-27}, abstract = {

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.

}, issn = {1091-6490}, doi = {10.1073/pnas.1705179114}, author = {Guo, Ming and Pegoraro, Adrian F and Mao, Angelo and Zhou, Enhua H and Arany, Praveen R and Han, Yulong and Burnette, Dylan T and Jensen, Mikkel H and Kasza, Karen E and Moore, Jeffrey R and Mackintosh, Frederick C and Fredberg, Jeffrey J and Mooney, David J and Lippincott-Schwartz, Jennifer and Weitz, David A} } @article {47619, title = {Intracellular and extracellular forces drive primary cilia movement.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, year = {2015}, month = {2015 Feb 3}, pages = {1410-5}, abstract = {

Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.

}, keywords = {Animals, Centrosome, Cilia, Dogs, Madin Darby Canine Kidney Cells, Microscopy, Electron, Movement}, issn = {1091-6490}, doi = {10.1073/pnas.1421845112}, author = {Battle, Christopher and Ott, Carolyn M and Burnette, Dylan T and Lippincott-Schwartz, Jennifer and Schmidt, Christoph F} } @article {47629, title = {A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.}, journal = {The Journal of cell biology}, volume = {205}, year = {2014}, month = {2014 Apr 14}, pages = {83-96}, abstract = {

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers{\textquoteright} attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

}, keywords = {Actin Cytoskeleton, Actinin, Actins, Animals, Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Shape, Cercopithecus aethiops, COS Cells, Focal Adhesions, Humans, Mechanotransduction, Cellular, Models, Biological, Nonmuscle Myosin Type IIA, Pressure, Time Factors, Transfection}, issn = {1540-8140}, doi = {10.1083/jcb.201311104}, author = {Burnette, Dylan T and Shao, Lin and Ott, Carolyn and Pasapera, Ana M and Fischer, Robert S and Baird, Michelle A and Der Loughian, Christelle and Delanoe-Ayari, Helene and Paszek, Matthew J and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {45153, title = {A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.}, journal = {Journal of Cell Biology}, volume = {205}, year = {2014}, month = {2014 Apr 14}, pages = {83-96}, abstract = {

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers{\textquoteright} attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

}, keywords = {Actin Cytoskeleton, Actinin, Actins, Animals, Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Shape, Cercopithecus aethiops, COS Cells, Focal Adhesions, Humans, Mechanotransduction, Cellular, Models, Biological, Nonmuscle Myosin Type IIA, Pressure, Time Factors, Transfection}, issn = {1540-8140}, doi = {10.1083/jcb.201311104}, author = {Burnette, Dylan T and Shao, Lin and Ott, Carolyn and Pasapera, Ana M and Fischer, Robert S and Baird, Michelle A and Der Loughian, Christelle and Delanoe-Ayari, Helene and Paszek, Matthew J and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {47633, title = {Superresolution imaging with standard fluorescent probes.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {60}, year = {2013}, month = {2013}, pages = {Unit 21.8.}, abstract = {

For more than 100 years, the ultimate resolution of a light microscope (\~{} 200 nm) has been constrained by the fundamental physical phenomenon of diffraction, as described by Ernst Abbe in 1873. While this limitation is just as applicable to today{\textquoteright}s light microscopes, it is the combination of high-end optics, clever methods of sample illumination, and computational techniques that has enabled researchers to access information at an order of magnitude greater resolution than once thought possible. This combination, broadly termed superresolution microscopy, has been increasingly practical for many labs to implement from both a hardware and software standpoint, but, as with many cutting-edge techniques, it also comes with limitations. One of the current drawbacks to superresolution microscopy is the limited number of probes and conditions that have been suitable for imaging. Here, a technique termed bleaching/blinking-assisted localization microscopy (BaLM) makes use of the inherent blinking and bleaching properties of almost all fluorophores as a means to generate superresolution images.

}, keywords = {Animals, Cercopithecus aethiops, COS Cells, Microscopy, Fluorescence, Signal-To-Noise Ratio}, issn = {1934-2616}, doi = {10.1002/0471143030.cb2108s60}, author = {Millis, Bryan A and Burnette, Dylan T and Lippincott-Schwartz, Jennifer and Kachar, Bechara} } @article {47661, title = {Bayesian localization microscopy reveals nanoscale podosome dynamics.}, journal = {Nature methods}, volume = {9}, year = {2012}, month = {2012 Feb}, pages = {195-200}, abstract = {

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

}, keywords = {Bayes Theorem, Cell Line, Tumor, Humans, Nanotechnology}, issn = {1548-7105}, doi = {10.1038/nmeth.1812}, author = {Cox, Susan and Rosten, Edward and Monypenny, James and Jovanovic-Talisman, Tijana and Burnette, Dylan T and Lippincott-Schwartz, Jennifer and Jones, Gareth E and Heintzmann, Rainer} } @article {47660, title = {Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Dec 27}, pages = {21081-6}, abstract = {

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

}, keywords = {Animals, Cercopithecus aethiops, COS Cells, Fluorescent Dyes, Green Fluorescent Proteins, Image Interpretation, Computer-Assisted, Microscopy, Fluorescence, Photobleaching, Software}, issn = {1091-6490}, doi = {10.1073/pnas.1117430109}, author = {Burnette, Dylan T and Sengupta, Prabuddha and Dai, Yuhai and Lippincott-Schwartz, Jennifer and Kachar, Bechara} } @article {47671, title = {A role for actin arcs in the leading-edge advance of migrating cells.}, journal = {Nature cell biology}, volume = {13}, year = {2011}, month = {2011 Apr}, pages = {371-81}, abstract = {

Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion.

}, keywords = {Actins, Cell Movement, Cytoskeleton, Epithelial Cells, Focal Adhesions, Myosin Type II, Pseudopodia, Recombinant Fusion Proteins}, issn = {1476-4679}, doi = {10.1038/ncb2205}, author = {Burnette, Dylan T and Manley, Suliana and Sengupta, Prabuddha and Sougrat, Rachid and Davidson, Michael W and Kachar, Bechara and Lippincott-Schwartz, Jennifer} }