Now showing 1 - 8 of 8
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    Anisotropic 3D confinement of MCF-7 cells induces directed cell-migration and viscoelastic anisotropy of cell-membrane
    (01-01-2023)
    Edwin, Privita Edwina Rayappan George
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    Kumar, Sumeet
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    Roy, Srestha
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    Tumor-associated collagen signature-3 (TACS-3) is a prognostic indicator for breast cancer survival. It is characterized by highly organized, parallel bundles of collagen fibers oriented perpendicular to the tumor boundary, serving as directional, confining channels for cancer cell invasion. Here we design a TACS-3-mimetic anisotropic, confined collagen I matrix and examine the relation between anisotropy of matrix, directed cellular migration, and anisotropy of cell membrane-the first direct contact between TACS-3 and cell-using Michigan Cancer Foundation-7 (MCF-7) cells as cancer-model. Using unidirectional freezing, we generated ∼50 μm-wide channels filled with collagen I. Optical tweezer (OT) microrheology shows that anisotropic confinement increases collagen viscoelasticity by two orders of magnitude, and the elastic modulus is significantly greater along the direction of anisotropic confinement compared to that along the orthogonal direction, thus establishing matrix anisotropy. Furthermore, MCF-7 cells embedded in anisotropic collagen I, exhibit directionality in cellular morphology and migration. Finally, using customized OT to trap polystyrene probes bound to cell-membrane (and not to ECM) of either free cells or cells under anisotropic confinement, we quantified the effect of matrix anisotropy on membrane viscoelasticity, both in-plane and out-of-plane, vis-à-vis the membrane. Both bulk and viscous modulus of cell-membrane of MCF-7 cells exhibit significant anisotropy under anisotropic confinement. Moreover, the cell membrane of MCF-7 cells under anisotropic confinement is significantly softer (both in-plane and out-of-plane moduli) despite their local environment being five times stiffer than free cells. In order to test if the coupling between anisotropy of extracellular matrix and anisotropy of cell-membrane is regulated by cell-cytoskeleton, actin cytoskeleton was depolymerized for both free and confined cells. Results show that cell membrane viscoelasticity of confined MCF-7 cells is unaffected by actin de-polymerization, in contrast to free cells. Together, these findings suggest that anisotropy of ECM induces directed migration and correlates with anisotropy of cell-membrane viscoelasticity of the MCF-7 cells in an actin-independent manner.
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    Detection of sub-degree angular fluctuations of the local cell membrane slope using optical tweezers
    (28-08-2020)
    Vaippully, Rahul
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    Ramanujan, Vaibavi
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    Normal thermal fluctuations of the cell membrane have been studied extensively using high resolution microscopy and focused light, particularly at the peripheral regions of a cell. We use a single probe particle attached non-specifically to the cell-membrane to determine that the power spectral density is proportional to (frequency)-5/3 in the range of 5 Hz to 1 kHz. We also use a new technique to simultaneously ascertain the slope fluctuations of the membrane by relying upon the determination of pitch motion of the birefringent probe particle trapped in linearly polarized optical tweezers. In the process, we also develop the technique to identify pitch rotation to a high resolution using optical tweezers. We find that the power spectrum of slope fluctuations is proportional to (frequency)-1, which we also explain theoretically. We find that we can extract parameters like bending rigidity directly from the coefficient of the power spectrum particularly at high frequencies, instead of being convoluted with other parameters, thereby improving the accuracy of estimation. We anticipate this technique for determination of the pitch angle in spherical particles to high resolution as a starting point for many interesting studies using the optical tweezers. This journal is
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    Direct detection of cell membrane slope fluctuations upon adding Latrunculin B using optical tweezers and single probe particle
    (01-01-2022)
    Roy, Srestha
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    Chakraborty, Snigdhadev
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    Muruga, Lokesh
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    Vaippuly, Rahul
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    Yadav, Vandana
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    Edwina, Privitha
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    The cell membrane has fluctuations due to thermal and athermal sources. That causes the membrane to flicker. Conventionally, only the normal (perpendicular to the membrane) fluctuations are studied and then used to ascertain the membrane properties like the bending rigidity. It is here that we introduce a different concept, namely the slope fluctuations of the cell membrane which can be modelled as a gradient of the normal fluctuations. This can be studied using a new technique where a birefringent particle placed on the membrane turns in the out of plane sense, called the pitch sense. We introduce the pitch detection technique in optical tweezers relying upon asymmetric scattering from a birefringent particle under crossed polarizers. We then go on to use this pitch detection technique to ascertain the power spectral density of membrane slope fluctuations and find it to be (frequency)-1 while the normal fluctuations yields (frequency)-5/3. We also explore a different regime where the cell is applied with the drug Latrunculin-B which inhibits actin polymerization and find the effect on membrane fluctuations. We find that even as the normal fluctuations now become (frequency)-4/3, the slope fluctuations spectrum still remains (frequency)-1, with exactly the same coefficient as the case when the drug was not applied. Thus, this presents a convenient opportunity to study the membrane parameters like bending rigidity as a function of time after applying the drug. This would be the first time the membrane bending rigidity could be studied as a function of time upon the application of Lat-B without reverting to AFM.
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    Determination of local cell membrane slope fluctuations using the pitch rotational mode of optical tweezers
    (01-01-2020)
    Vaippully, Rahul
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    Ramanujam, Vaibavi
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    We study the normal fluctuations of an MCF-7 cell membrane and calibrate the optical trap to detect pitch motion to get information about the rocking motion of a birefringent particle. We could show both theoretically and experimentally that the Z power spectrum has a power-law behavior of (frequency)-5/3, and We find that the power spectrum of slope fluctuations is proportional to (frequency)-1. We could extract parameters like bending rigidity directly from the power spectrum fitting parameters in 5 Hz to 1 kHz range. Our method was powerful enough to identify pitch rotation for a spherical birefringent particle to a high resolution using optical tweezers.
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    Comparison of translational and rotational modes towards passive rheology of the cytoplasm of MCF-7 cells using optical tweezers
    (09-01-2023)
    Roy, Srestha
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    Vaippully, Rahul
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    Lokesh, Muruga
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    Nalupurackal, Gokul
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    Edwina, Privita
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    A colloidal particle placed inside the cell cytoplasm is enmeshed within a network of cytoskeletal fibres immersed in the cytosolic fluid. The translational mode is believed to yield different rheological parameters than the rotational mode, given that these modes stretch the fibers differently. We compare the parameters for Michigan Cancer Foundation-7 (MCF-7) cells in this manuscript and find that the results are well comparable to each other. At low values of 0 Hz viscosity, the rotational and translational viscoelasticity matches well. However, discrepancies appear at higher values which may indicate that the cytoskeletal modes involved in rotation and translation of the particle are getting invoked. We also show that the 0 Hz viscosity increases as the cell ages under the conditions of constant room temperature of 25°C on the sample chamber.
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    Measurement of viscoelastic properties of the cellular cytoplasm using optically trapped Brownian probes
    (12-03-2020)
    Vaippully, Rahul
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    Ramanujan, Vaibavi
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    Measurement of the viscoelastic properties of a cell using microscopic tracer particles has been complicated given that the medium viscosity is dependent upon the size of the measurement probe leading to reliability issues. Further, a technique for direct calibration of optically trapped particles in vivo has been elusive due to the frequency dependence and spatial inhomogeneity of the cytoplasmic viscosity, and the requirement of accurate knowledge of the medium refractive index. Here, we employ a recent extension of Jeffery's model of viscoelasticity in the microscopic domain to fit the passive motional power spectra of micrometer-sized optically trapped particles embedded in a viscoelastic medium. We find excellent agreement between the 0 Hz viscosity in MCF7 cells and the typical values of viscosity in literature, between 2 to 16 mPa sec expected for the typical concentration of proteins inside the cytoplasmic solvent. This bypasses the dependence on probe size by relying upon small thermal displacements. Our measurements of the relaxation time also match values reported with magnetic tweezers, at about 0.1 s. Finally, we calibrate the optical tweezers and demonstrate the efficacy of the technique to the study of in vivo translational motion.
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    Anti-hypertensive Calcium-Blocking Drugs Induce a Change in Viscoelasticity of Mcf-7 Cancer Cells
    (15-09-2020)
    Vaibavi, S. R.
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    Vaippully, Rahul
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    In this work, we studied the effect of common calcium channel blockers (Verapamil Hydrochloride and Nifedipine, usually prescribed for cardiovascular diseases) on the viscoelastic property of both tumorigenic (MCF-7) and vascular endothelial (HUVEC) cell-line. Specifically, an optical-tweezer was used to locally "press-down" on the cell membrane. Fluctuations of optically-trapped dielectric beads attached to the cell membrane were measured to study the effect of drugs on the mechanical properties of the membrane and the cytoskeleton. We observed that MCF-7 cells exhibit a significantly higher mean squared displacement (MSD) in response to these drugs as compared to HUVEC cells, under no-compression condition. Furthermore, optically induced compression of cell-membrane induced a significant drop in MSD of MCF-7 cells, whereas HUVEC cells showed a significant rise in MSD. To further test these results, we depolymerized actin-cytoskeleton using latrunculin-B and measured fluctuations of beads on the surface of cells. Immunofluorescence imaging of the actin cytoskeleton correlates with the variation in the viscoelastic response of MCF-7 and HUVEC under the influence of these drugs. Our power-spectrum analysis at 10 Hz shows that actin-depolymerization decreased the activity of both cell types; however, cells treated with nifedipine or verapamil showed contrasting effects. Together, these results show that clinically approved calcium-ion blocking cardiovascular drugs have a significant effect on the viscoelastic properties of circulating tumor cells in the blood.
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    Calcium-channel-blockers exhibit divergent regulation of cancer extravasation through the mechanical properties of cancer cells and underlying vascular endothelial cells
    (01-03-2022)
    Vaibavi, S. R.
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    Sivasubramaniapandian, Manoj
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    Vaippully, Rahul
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    Edwina, Privita
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    Cardiovascular and cancer illnesses often co-exist, share pathological pathways, and complicate therapy. In the context of the potential oncological role of cardiovascular-antihypertensive drugs (AHD), here we examine the role of calcium-channel blocking drugs on mechanics of extravasating cancer cells, choosing two clinically-approved calcium-channel blockers (CCB): Verapamil-hydrochloride and Nifedipine, as model AHD to simultaneously target cancer cells (MCF7 and or MDA231) and an underlying monolayer of endothelial cells (HUVEC). First, live-cell microscopy shows that exposure to Nifedipine increases the spreading-area, migration-distance, and frequency of transmigration of MCF-7 cells through the HUVEC monolayer, whereas Verapamil has the opposite effect. Next, impedance-spectroscopy shows that for monolayers of either endothelial or cancer cells, Nifedipine-treatment alone decreases the impedance of both cases, suggesting compromised cell-cell integrity. Furthermore, upon co-culturing MCF-7 on the HUVEC monolayers, Nifedipine-treated MCF-7 cells exhibit weaker impedance than Verapamil-treated MCF-7 cells. Following, fluorescent staining of CCB-treated cytoskeleton, focal adhesions, and cell-cell junction also indicated that Nifedipine treatment diminished the cell-cell integrity, whereas verapamil treatment preserved the integrity. Since CCBs regulate intracellular Ca2+, we next investigated if cancer cell’s exposure to CCBs regulates calcium-dependent processes critical to extravasation, specifically traction and mechanics of plasma membrane. Towards this end, first, we quantified the 2D-cellular traction of cells in response to CCBs. Results show that exposure to F-actin depolymerizing drug decreases traction stress significantly only for Nifedipine-treated cells, suggesting an actin-independent mechanism of Verapamil activity. Next, using an optical tweezer to quantify the mechanics of plasma membrane (PM), we observe that under constant, externally-applied tensile strain, PM of Nifedipine-treated cells exhibits smaller relaxation-time than Verapamil and untreated cells. Finally, actin depolymerization significantly decreases MSD only for Verapamil treated cancer-cells and endothelial cells and not for Nifedipine-treated cells. Together, our results show that CCBs can have varied, mechanics-regulating effects on cancer-cell transmigration across endothelial monolayers. A judicious choice of CCBs is critical to minimizing the pro-metastatic effects of antihypertension therapy.