Person: Krishnan, Ramaswamy
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Krishnan
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Ramaswamy
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Krishnan, Ramaswamy
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Publication Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors(MDPI, 2017) Bartolák-Suki, Erzsébet; Suki, Béla; Imsirovic, Jasmin; Nishibori, Yuichiro; Krishnan, RamaswamyMitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging.Publication Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines(Frontiers Media S.A., 2017) Rajakylä, Kaisa; Krishnan, Ramaswamy; Tojkander, SariCancer cells are surrounded by a mechanically and biochemically distinct microenvironment that undergoes dynamic changes throughout the neoplastic progression. During this progression, some cancer cells acquire abnormal characteristics that potentiate their escape from the primary tumor site, to establish secondary tumors in distant organs. Recent studies with several human cancer cell lines have shown that the altered physical properties of tumor cells, such as their ability to apply high traction forces to the surroundings, are directly linked with their potential to invade and metastasize. To test the hypothetical interconnection between actomyosin-mediated traction forces and invasion potential within 3D-microenvironment, we utilized two canine mammary tumor cell lines with different contractile properties. These cell lines, canine mammary tumor (CMT)-U27 and CMT-U309, were found to have distinct expression patterns of lineage-specific markers and organization of actin-based structures. In particular, CMT-U309 carcinoma cells were typified by thick contractile actomyosin bundles that exerted high forces to their environment, as measured by traction force microscopy. These high contractile forces also correlated with the prominent invasiveness of the CMT-U309 cell line. Furthermore, we found high contractility and 3D-invasion potential to be dependent on the activity of 5′AMP-activated protein kinase (AMPK), as blocking AMPK signaling was found to reverse both of these features. Taken together, our findings implicate that actomyosin forces correlate with the invasion potential of the studied cell lines.Publication Regulation of microtubule dynamics by DIAPH3 influences amoeboid tumor cell mechanics and sensitivity to taxanes(Nature Publishing Group, 2015) Morley, Samantha; You, Sungyong; Pollan, Sara; Choi, Jiyoung; Zhou, Bo; Hager, Martin H.; Steadman, Kenneth; Spinelli, Cristiana; Rajendran, Kavitha; Gertych, Arkadiusz; Kim, Jayoung; Adam, Rosalyn; Yang, Wei; Krishnan, Ramaswamy; Knudsen, Beatrice S.; Di Vizio, Dolores; Freeman, Michael R.Taxanes are widely employed chemotherapies for patients with metastatic prostate and breast cancer. Here, we show that loss of Diaphanous-related formin-3 (DIAPH3), frequently associated with metastatic breast and prostate cancers, correlates with increased sensitivity to taxanes. DIAPH3 interacted with microtubules (MT), and its loss altered several parameters of MT dynamics as well as decreased polarized force generation, contractility, and response to substrate stiffness. Silencing of DIAPH3 increased the cytotoxic response to taxanes in prostate and breast cancer cell lines. Analysis of drug activity for tubulin-targeted agents in the NCI-60 cell line panel revealed a uniform positive correlation between reduced DIAPH3 expression and drug sensitivity. Low DIAPH3 expression correlated with improved relapse-free survival in breast cancer patients treated with chemotherapeutic regimens containing taxanes. Our results suggest that inhibition of MT stability arising from DIAPH3 downregulation enhances susceptibility to MT poisons, and that the DIAPH3 network potentially reports taxane sensitivity in human tumors.Publication The actin regulator zyxin reinforces airway smooth muscle and accumulates in airways of fatal asthmatics(Public Library of Science, 2017) Rosner, Sonia R.; Pascoe, Christopher D.; Blankman, Elizabeth; Jensen, Christopher C.; Krishnan, Ramaswamy; James, Alan L.; Elliot, John G.; Green, Francis H.; Liu, Jeffrey C.; Seow, Chun Y.; Park, Jin-Ah; Beckerle, Mary C.; Paré, Peter D.; Fredberg, Jeffrey; Smith, Mark A.Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms–and their failure in asthma–remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.Publication Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly(eLife Sciences Publications, Ltd, 2015) Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, PekkaAdhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001Publication Airway and Parenchymal Strains during Bronchoconstriction in the Precision Cut Lung Slice(Frontiers Media S.A., 2016) Hiorns, Jonathan E.; Bidan, Cécile M.; Jensen, Oliver E.; Gosens, Reinoud; Kistemaker, Loes E. M.; Fredberg, Jeffrey; Butler, Jim P.; Krishnan, Ramaswamy; Brook, Bindi S.The precision-cut lung slice (PCLS) is a powerful tool for studying airway reactivity, but biomechanical measurements to date have largely focused on changes in airway caliber. Here we describe an image processing tool that reveals the associated spatio-temporal changes in airway and parenchymal strains. Displacements of sub-regions within the PCLS are tracked in phase-contrast movies acquired after addition of contractile and relaxing drugs. From displacement maps, strains are determined across the entire PCLS or along user-specified directions. In a representative mouse PCLS challenged with 10−4M methacholine, as lumen area decreased, compressive circumferential strains were highest in the 50 μm closest to the airway lumen while expansive radial strains were highest in the region 50–100 μm from the lumen. However, at any given distance from the airway the strain distribution varied substantially in the vicinity of neighboring small airways and blood vessels. Upon challenge with the relaxant agonist chloroquine, although most strains disappeared, residual positive strains remained a long time after addition of chloroquine, predominantly in the radial direction. Taken together, these findings establish strain mapping as a new tool to elucidate local dynamic mechanical events within the constricting airway and its supporting parenchyma.Publication Functional Validation of an Alpha-Actinin-4 Mutation as a Potential Cause of an Aggressive Presentation of Adolescent Focal Segmental Glomerulosclerosis: Implications for Genetic Testing(Public Library of Science, 2016) Feng, Di; Steinke, Julia M.; Krishnan, Ramaswamy; Birrane, Gabriel; Pollak, MartinGenetic testing in the clinic and research lab is becoming more routinely used to identify rare genetic variants. However, attributing these rare variants as the cause of disease in an individual patient remains challenging. Here, we report a patient who presented with nephrotic syndrome and focal segmental glomerulosclerosis (FSGS) with collapsing features at age 14. Despite treatment, her kidney disease progressed to end-stage within a year of diagnosis. Through genetic testing, an Y265H variant with unknown clinical significance in alpha-actinin-4 gene (ACTN4) was identified. This variant has not been seen previously in FSGS patients nor is it present in genetic databases. Her clinical presentation is different from previous descriptions of ACTN4 mediated FSGS, which is characterized by sub-nephrotic proteinuria and slow progression to end stage kidney disease. We performed in vitro and cellular assays to characterize this novel ACTN4 variant before attributing causation. We found that ACTN4 with either Y265H or K255E (a known disease-causing mutation) increased the actin bundling activity of ACTN4 in vitro, was associated with the formation of intracellular aggregates, and increased podocyte contractile force. Despite the absence of a familial pattern of inheritance, these similar biological changes caused by the Y265H and K255E amino acid substitutions suggest that this new variant is potentially the cause of FSGS in this patient. Our studies highlight that functional validation in complement with genetic testing may be required to confirm the etiology of rare disease, especially in the setting of unusual clinical presentations.Publication A microfluidic chamber-based approach to map the shear moduli of vascular cells and other soft materials(Nature Publishing Group UK, 2017) Suki, Béla; Hu, Yingying; Murata, Naohiko; Imsirovic, Jasmin; Mondoñedo, Jarred R.; de Oliveira, Claudio L. N.; Schaible, Niccole; Allen, Philip G.; Krishnan, Ramaswamy; Bartolák-Suki, ErzsébetThere is growing interest in quantifying vascular cell and tissue stiffness. Most measurement approaches, however, are incapable of assessing stiffness in the presence of physiological flows. We developed a microfluidic approach which allows measurement of shear modulus (G) during flow. The design included a chamber with glass windows allowing imaging with upright or inverted microscopes. Flow was controlled gravitationally to push culture media through the chamber. Fluorescent beads were conjugated to the sample surface and imaged before and during flow. Bead displacements were calculated from images and G was computed as the ratio of imposed shear stress to measured shear strain. Fluid-structure simulations showed that shear stress on the surface did not depend on sample stiffness. Our approach was verified by measuring the moduli of polyacrylamide gels of known stiffness. In human pulmonary microvascular endothelial cells, G was 20.4 ± 12 Pa and decreased by 20% and 22% with increasing shear stress and inhibition of non-muscle myosin II motors, respectively. The G showed a larger intra- than inter-cellular variability and it was mostly determined by the cytosol. Our shear modulus microscopy can thus map the spatial distribution of G of soft materials including gels, cells and tissues while allowing the visualization of microscopic structures such as the cytoskeleleton.Publication Corrigendum: Airway and Parenchymal Strains during Bronchoconstriction in the Precision Cut Lung Slice(Frontiers Media S.A., 2017) Hiorns, Jonathan E.; Bidan, Cécile M.; Jensen, Oliver E.; Gosens, Reinoud; Kistemaker, Loes E. M.; Fredberg, Jeffrey; Butler, Jim P.; Krishnan, Ramaswamy; Brook, Bindi S.Publication JunB Mediates Basal- and TGFβ1-Induced Smooth Muscle Cell Contractility(Public Library of Science, 2013) Ramachandran, Aruna; Gangopadhyay, Samudra Saurabh; Krishnan, Ramaswamy; Ranpura, Sandeep A.; Rajendran, Kavitha; Ram-Mohan, Sumati; Mulone, Michelle; Gong, Edward M.; Adam, RosalynSmooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFβ1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFβ1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFβ1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton.