Publications by authors named "Karen E Kasza"

16 Publications

  • Page 1 of 1

Membrane curvature and connective fiber alignment in guinea pig round window membrane.

Acta Biomater 2021 Dec 24;136:343-362. Epub 2021 Sep 24.

Department of Mechanical Engineering, Columbia University, 220 Mudd Building 500 West 120th Street, New York, NY 10027, USA; Department of Otolaryngology - Head and Neck Surgery, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. Electronic address:

The round window membrane (RWM) covers an opening between the perilymph fluid-filled inner ear space and the air-filled middle ear space. As the only non-osseous barrier between these two spaces, the RWM is an ideal candidate for aspiration of perilymph for diagnostics purposes and delivery of medication for treatment of inner ear disorders. Routine access across the RWM requires the development of new surgical tools whose design can only be optimized with a thorough understanding of the RWM's structure and properties. The RWM possesses a layer of collagen and elastic fibers so characterization of the distribution and orientation of these fibers is essential. Confocal and two-photon microscopy were conducted on intact RWMs in a guinea pig model to characterize the distribution of collagen and elastic fibers. The fibers were imaged via second-harmonic-generation, autofluorescence, and Rhodamine B staining. Quantitative analyses of both fiber orientation and geometrical properties of the RWM uncovered a significant correlation between mean fiber orientations and directions of zero curvature in some portions of the RWM, with an even more significant correlation between the mean fiber orientations and linear distance along the RWM in a direction approximately parallel to the cochlear axis. The measured mean fiber directions and dispersions can be incorporated into a generalized structure tensor for use in the development of continuum anisotropic mechanical constitutive models that in turn will enable optimization of surgical tools to access the cochlea. STATEMENT OF SIGNIFICANCE: The Round Window Membrane (RWM) is the only non-osseous barrier separating the middle and inner ear spaces, and thus is an ideal portal for medical access to the cochlea. An understanding of RWM structure and mechanical response is necessary to optimize the design of surgical tools for this purpose. The RWM geometry and the connective fiber orientation and dispersion are measured via confocal and 2-photon microscopy. A region of the RWM geometry is characterized as a hyperbolic paraboloid and another region as a tapered parabolic cylinder. Predominant fiber directions correlate well with directions of zero curvature in the hyperbolic paraboloid region. Overall fiber directions correlate well with position along a line approximately parallel to the central axis of the cochlea's spiral.
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http://dx.doi.org/10.1016/j.actbio.2021.09.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8627469PMC
December 2021

Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension.

Biophys J 2021 10 20;120(19):4214-4229. Epub 2021 Jul 20.

Department of Mechanical Engineering, Columbia University, New York, New York. Electronic address:

Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar-polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin pattern influence the multiple cell behaviors, including cell intercalation, cell shape changes, and apical cell area fluctuations, that simultaneously occur during morphogenesis. Here, we developed two optogenetic tools, optoGEF and optoGAP, to activate or deactivate Rho1 signaling, respectively. We used these tools to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation and analyzed the effects on contractile cell behaviors. We show that uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar-polarized pattern of myosin at cell junctions on the timescale of 3-5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation but have distinct effects on cell area fluctuations and cell packings that are linked with changes in the medial and junctional myosin pools. These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar-polarized myosin pattern in the germband during axis elongation. Moreover, our results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles not only in organizing the cell rearrangements that are known to directly contribute to axis elongation but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.
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http://dx.doi.org/10.1016/j.bpj.2021.06.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8516680PMC
October 2021

Anisotropy links cell shapes to tissue flow during convergent extension.

Proc Natl Acad Sci U S A 2020 06 28;117(24):13541-13551. Epub 2020 May 28.

Department of Mechanical Engineering, Columbia University, New York, NY 10027;

Within developing embryos, tissues flow and reorganize dramatically on timescales as short as minutes. This includes epithelial tissues, which often narrow and elongate in convergent extension movements due to anisotropies in external forces or in internal cell-generated forces. However, the mechanisms that allow or prevent tissue reorganization, especially in the presence of strongly anisotropic forces, remain unclear. We study this question in the converging and extending germband epithelium, which displays planar-polarized myosin II and experiences anisotropic forces from neighboring tissues. We show that, in contrast to isotropic tissues, cell shape alone is not sufficient to predict the onset of rapid cell rearrangement. From theoretical considerations and vertex model simulations, we predict that in anisotropic tissues, two experimentally accessible metrics of cell patterns-the cell shape index and a cell alignment index-are required to determine whether an anisotropic tissue is in a solid-like or fluid-like state. We show that changes in cell shape and alignment over time in the germband predict the onset of rapid cell rearrangement in both wild-type and mutant embryos, where our theoretical prediction is further improved when we also account for cell packing disorder. These findings suggest that convergent extension is associated with a transition to more fluid-like tissue behavior, which may help accommodate tissue-shape changes during rapid developmental events.
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http://dx.doi.org/10.1073/pnas.1916418117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306759PMC
June 2020

Cellular defects resulting from disease-related myosin II mutations in .

Proc Natl Acad Sci U S A 2019 10 15;116(44):22205-22211. Epub 2019 Oct 15.

Howard Hughes Medical Institute, Sloan Kettering Institute, New York, NY 10065;

The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. Here we use the embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, we show that engineering human -related disease mutations into myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.
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http://dx.doi.org/10.1073/pnas.1909227116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825282PMC
October 2019

Manipulating the Patterns of Mechanical Forces That Shape Multicellular Tissues.

Physiology (Bethesda) 2019 11;34(6):381-391

Department of Mechanical Engineering, Columbia University, New York, New York.

During embryonic development, spatial and temporal patterns of mechanical forces help to transform unstructured groups of cells into complex, functional tissue architectures. Here, we review emerging approaches to manipulate these patterns of forces to investigate the mechanical mechanisms that shape multicellular tissues, with a focus on recent experimental studies of epithelial tissue sheets in the embryo of the model organism .
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http://dx.doi.org/10.1152/physiol.00018.2019DOI Listing
November 2019

Biophysical control of the cell rearrangements and cell shape changes that build epithelial tissues.

Curr Opin Genet Dev 2018 08 10;51:88-95. Epub 2018 Aug 10.

Department of Mechanical Engineering, Columbia University, 500 W. 120th Street, New York, NY 10027, USA. Electronic address:

Epithelial cell rearrangements and cell shape changes are fundamental mechanisms by which cells build and shape elaborate and diverse tissue architectures from simple tissue sheets. These cell behaviors are regulated by a complex interplay between physical and biochemical mechanisms, many of which have been uncovered in recent studies in Drosophila. While the regulation of these cell behaviors is still under investigation, emerging technologies are being used to gain experimental control over these behaviors, opening new possibilities for designing and engineering tissue structures. Analysis of the biophysical mechanisms governing cell shape and movement will be crucial for understanding morphogenesis and for harnessing this knowledge to build tissues of precise shapes and structures for basic science and engineering applications.
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http://dx.doi.org/10.1016/j.gde.2018.07.005DOI Listing
August 2018

In-vitro perforation of the round window membrane via direct 3-D printed microneedles.

Biomed Microdevices 2018 06 8;20(2):47. Epub 2018 Jun 8.

Department of Mechanical Engineering, Columbia University, 220 Mudd Building 500 West 120th Street, New York, NY, 10027, USA.

The cochlea, or inner ear, is a space fully enclosed within the temporal bone of the skull, except for two membrane-covered portals connecting it to the middle ear space. One of these portals is the round window, which is covered by the Round Window Membrane (RWM). A longstanding clinical goal is to reliably and precisely deliver therapeutics into the cochlea to treat a plethora of auditory and vestibular disorders. Standard of care for several difficult-to-treat diseases calls for injection of a therapeutic substance through the tympanic membrane into the middle ear space, after which a portion of the substance diffuses across the RWM into the cochlea. The efficacy of this technique is limited by an inconsistent rate of molecular transport across the RWM. A solution to this problem involves the introduction of one or more microscopic perforations through the RWM to enhance the rate and reliability of diffusive transport. This paper reports the use of direct 3D printing via Two-Photon Polymerization (2PP) lithography to fabricate ultra-sharp polymer microneedles specifically designed to perforate the RWM. The microneedle has tip radius of 500 nm and shank radius of 50 μ m, and perforates the guinea pig RWM with a mean force of 1.19 mN. The resulting perforations performed in vitro are lens-shaped with major axis equal to the microneedle shank diameter and minor axis about 25% of the major axis, with mean area 1670 μ m. The major axis is aligned with the direction of the connective fibers within the RWM. The fibers were separated along their axes without ripping or tearing of the RWM suggesting the main failure mechanism to be fiber-to-fiber decohesion. The small perforation area along with fiber-to-fiber decohesion are promising indicators that the perforations would heal readily following in vivo experiments. These results establish a foundation for the use of Two-Photon Polymerization lithography as a means to fabricate microneedles to perforate the RWM and other similar membranes.
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http://dx.doi.org/10.1007/s10544-018-0287-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091873PMC
June 2018

Cell volume change through water efflux impacts cell stiffness and stem cell fate.

Proc Natl Acad Sci U S A 2017 10 25;114(41):E8618-E8627. Epub 2017 Sep 25.

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138;

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.
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http://dx.doi.org/10.1073/pnas.1705179114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642688PMC
October 2017

Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation.

Proc Natl Acad Sci U S A 2014 Aug 28;111(32):11732-7. Epub 2014 Jul 28.

Howard Hughes Medical Institute and Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10065

Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. We show that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis.
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http://dx.doi.org/10.1073/pnas.1400520111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136583PMC
August 2014

Imaging techniques for measuring the materials properties of cells.

Cold Spring Harb Protoc 2011 Apr 1;2011(4):pdb.top107. Epub 2011 Apr 1.

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http://dx.doi.org/10.1101/pdb.top107DOI Listing
April 2011

Magnetic twisting cytometry.

Cold Spring Harb Protoc 2011 Apr 1;2011(4):pdb.prot5599. Epub 2011 Apr 1.

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http://dx.doi.org/10.1101/pdb.prot5599DOI Listing
April 2011

Dynamics and regulation of contractile actin-myosin networks in morphogenesis.

Curr Opin Cell Biol 2011 Feb 3;23(1):30-8. Epub 2010 Dec 3.

Howard Hughes Medical Institute, Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10065, USA.

Contractile actin-myosin networks generate forces that drive cell shape changes and tissue remodeling during development. These forces can also actively regulate cell signaling and behavior. Novel features of actin-myosin network dynamics, such as pulsed contractile behaviors and the regulation of myosin localization by tension, have been uncovered in recent studies of Drosophila. In vitro studies of single molecules and reconstituted protein networks reveal intrinsic properties of motor proteins and actin-myosin networks, while in vivo studies have provided insight into the regulation of their dynamics and organization. Analysis of the complex behaviors of actin-myosin networks will be crucial for understanding force generation in actively remodeling cells and the coordination of cell shape and movement at the tissue level.
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http://dx.doi.org/10.1016/j.ceb.2010.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320050PMC
February 2011

Elasticity in ionically cross-linked neurofilament networks.

Biophys J 2010 May;98(10):2147-53

Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

Neurofilaments are found in abundance in the cytoskeleton of neurons, where they act as an intracellular framework protecting the neuron from external stresses. To elucidate the nature of the mechanical properties that provide this protection, we measure the linear and nonlinear viscoelastic properties of networks of neurofilaments. These networks are soft solids that exhibit dramatic strain stiffening above critical strains of 30-70%. Surprisingly, divalent ions such as Mg(2+), Ca(2+), and Zn(2+) act as effective cross-linkers for neurofilament networks, controlling their solidlike elastic response. This behavior is comparable to that of actin-binding proteins in reconstituted filamentous actin. We show that the elasticity of neurofilament networks is entropic in origin and is consistent with a model for cross-linked semiflexible networks, which we use to quantify the cross-linking by divalent ions.
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http://dx.doi.org/10.1016/j.bpj.2010.01.062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872258PMC
May 2010

Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.

PLoS One 2009 18;4(3):e4928. Epub 2009 Mar 18.

Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004928PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654154PMC
July 2009

Chapter 19: Mechanical response of cytoskeletal networks.

Methods Cell Biol 2008 ;89:487-519

Department of Physics and Institute for Biophysical Dynamics, University of Chicago, Illinois 60637, USA.

The cellular cytoskeleton is a dynamic network of filamentous proteins, consisting of filamentous actin (F-actin), microtubules, and intermediate filaments. However, these networks are not simple linear, elastic solids; they can exhibit highly nonlinear elasticity and a thermal dynamics driven by ATP-dependent processes. To build quantitative mechanical models describing complex cellular behaviors, it is necessary to understand the underlying physical principles that regulate force transmission and dynamics within these networks. In this chapter, we review our current understanding of the physics of networks of cytoskeletal proteins formed in vitro. We introduce rheology, the technique used to measure mechanical response. We discuss our current understanding of the mechanical response of F-actin networks, and how the biophysical properties of F-actin and actin cross-linking proteins can dramatically impact the network mechanical response. We discuss how incorporating dynamic and rigid microtubules into F-actin networks can affect the contours of growing microtubules and composite network rigidity. Finally, we discuss the mechanical behaviors of intermediate filaments.
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http://dx.doi.org/10.1016/S0091-679X(08)00619-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456006PMC
February 2009

The cell as a material.

Curr Opin Cell Biol 2007 Feb 15;19(1):101-7. Epub 2006 Dec 15.

Department of Physics & DEAS, Harvard University, Cambridge, MA 02138, USA.

To elucidate the dynamic and functional role of a cell within the tissue it belongs to, it is essential to understand its material properties. The cell is a viscoelastic material with highly unusual properties. Measurements of the mechanical behavior of cells are beginning to probe the contribution of constituent components to cell mechanics. Reconstituted cytoskeletal protein networks have been shown to mimic many aspects of the mechanical properties of cells, providing new insight into the origin of cellular behavior. These networks are highly nonlinear, with an elastic modulus that depends sensitively on applied stress. Theories can account for some of the measured properties, but a complete model remains elusive.
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http://dx.doi.org/10.1016/j.ceb.2006.12.002DOI Listing
February 2007
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