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Open Access Publications from the University of California

Scholarly Works (12 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2012)

The mechanical properties of the cell components, cell plasma membrane and cytoskeleton, as well as membrane-cytoskeleton associations, determine the mechanical properties of the whole cell which is important in cellular shape change behavior and mechanical signal transduction in living cells. Examples of biologically important processes involving cellular shape changes are deformation of erythrocytes in capillaries, cell division, phagocytosis, pseudopodium and dendritic spine formation, and electromotility of the outer hair cells.

This dissertation focuses on investigating the mechanical properties of the living cell plasma membrane and the local mechanical associations of the plasma membrane with the underlying cytoskeleton.

Mechanical properties of the living cell plasma membrane are investigated by forming membrane nanotubes (tethers) from human embryonic kidney cells using optical tweezers technique. In order to analyze the role of membrane composition on its nano-mechanical properties, the membrane cholesterol content, the major lipid component of the plasma membrane, is manipulated and the obtained membrane mechanical properties are correlated to the membrane cholesterol content. The results reveal significant effects of membrane cholesterol in specific, and membrane composition in general, on membrane nano-mechanical properties. Specifically, decreases in membrane cholesterol content were associated with increased plasma membrane equilibrium force, plasma membrane tether stiffness and plasma membrane-cytoskeleton adhesion energy per unit area. Elevation of the membrane cholesterol content was followed by lower membrane tether equilibrium force, lower stiffness values, and lower membrane-cytoskeleton adhesion energy. The membrane bending modulus was almost unchanged upon membrane cholesterol manipulations.

In order to discern the role of cell cytoskeleton on membrane mechanical properties the experiments were repeated after F-actin disruption and in control cell and cholesterol manipulated cells. The disruption of F-actin filaments showed a noticeable impact on the membrane mechanical properties and diminished the observed disparity in membrane mechanical properties upon cholesterol depletion and cholesterol enrichment.

This dissertation also focuses on local cell deformations (protrusions). These deformations occur at an intermediate region between deformations at cell plasma membrane level and whole cell deformation, and are biologically important in formation of pseudopodia and filopodia, deformation of macrophages to engulf particles, and the surface protrusions on the cells preceding formation of membrane tethers. We used a combined optical tweezers-fluorescent microscopy technique to study cellular protrusions in adherent living cells. The mechanical properties of the protrusions were analyzed by obtaining the associated force-length plots and a Maxwell viscoelastic model is used to fit the force-length plots a. The experiments are performed on adherent human embryonic kidney cells, under cholesterol depleted and cholesterol enriched conditions to examine the effects of membrane cholesterol on protrusions. The experiments indicated greater maximum protrusion forces and shorter protrusion length under cholesterol depleted conditions in addition to greater values of the protrusion stiffness. Cholesterol enrichment experiments were associated with lower values of maximum protrusion force and protrusion stiffness, and formation of longer protrusions The observations suggest a significant contribution of the cytoskeletal F-actin filaments on the observed mechanical properties of protrusion regardless of membrane cholesterol content.

    Cover page: Experimental Investigation of Cell Membrane Nano-mechanics and Plasma Membrane-Cytoskeletal Interactions Using Optical Tweezers
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2015)
    Whether or not an external force can make a trapped particle escape from optical tweezers can be used to measure optical forces. Combined with the linear dependence of optical forces on trapping power, a quantitative measurement of the force can be obtained. For this measurement, the particle is at the edge of the trap, away from the region near the equilbrium position where the trap can be described as a linear spring. This method provides the ability to measure higher forces for the same beam power, compared with using the linear region of the trap, with lower risk of optical damage to trapped specimens. Calibration is typically performed by using an increasing fluid flow to exert an increasing force on a trapped particle until it escapes. In this calibration technique, the particle is usually assumed to escape along a straight line in the direction of fluid-flow. Here, we show that the particle instead follows a curved trajectory, which depends on the rate of application of the force (i.e., the acceleration of the fluid flow). In the limit of very low acceleration, the particle follows the surface of zero axial optical force during the escape. The force required to produce escape depends on the trajectory, and hence the acceleration. This can result in variations in the escape force of a factor of two. This can have a major impact on calibration to determine the escape force efficiency. Even when calibration measurements are all performed in the low acceleration regime, variations in the escape force efficiency of 20% or more can still occur. We present computational simulations using generalized Lorenz-Mie theory and experimental measurements to show how the escape force efficiency depends on rate of increase of force and trapping power, and discuss the impact on calibration.
      Cover page: Escape forces and trajectories in optical tweezers and their effect on calibrationCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2018)
      This study aims to understand the phagocytic response of astrocytes to the injury of neurons or other astrocytes at the single cell level. Laser nanosurgery was used to damage individual cells in both primary mouse cortical astrocytes and an established astrocyte cell line. In both cases, the release of material/substances from laser-irradiated astrocytes or neurons induced a phagocytic response in near-by astrocytes. Propidium iodide stained DNA originating from irradiated cells was visible in vesicles of neighboring cells, confirming phagocytosis of material from damaged cortical cells. In the presence of an intracellular pH indicator dye, newly formed vesicles correspond to acidic pH fluorescence, thus suggesting lysosome bound degradation of cellular debris. Cells with shared membrane connections prior to laser damage had a significantly higher frequency of induced phagocytosis compared to isolated cells with no shared membrane. The increase in phagocytic response of cells with a shared membrane occurred regardless of the extent of shared membrane (a thin filopodial connection vs. a cell cluster with significant shared membrane). In addition to the presence (or lack) of a membrane connection, variation in phagocytic ability was also observed with differences in injury location within the cell and distance separating isolated astrocytes. These results demonstrate the ability of an astrocyte to respond to the damage of a single cell, be it another astrocyte, or a neuron. This single-cell level of analysis results in a better understanding of the role of astrocytes to maintain homeostasis in the CNS, particularly in the sensing and removal of debris in damaged or pathologic nervous tissue.
        Cover page: Phagocytic response of astrocytes to damaged neighboring cells.
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2013)
        Protrusions are deformations that form at the surface of living cells during biological activities such as cell migration. Using combined optical tweezers and fluorescent microscopy, we quantified the mechanical properties of protrusions in adherent human embryonic kidney cells in response to application of an external force at the cell surface. The mechanical properties of protrusions were analyzed by obtaining the associated force-length plots during protrusion formation, and force relaxation at constant length. Protrusion mechanics were interpretable by a standard linear solid (Kelvin) model, consisting of two stiffness parameters, k0 and k1 (with k0>k1), and a viscous coefficient. While both stiffness parameters contribute to the time-dependant mechanical behavior of the protrusions, k0 and k1 in particular dominated the early and late stages of the protrusion formation and elongation process, respectively. Lowering the membrane cholesterol content by 25% increased the k0 stiffness by 74%, and shortened the protrusion length by almost half. Enhancement of membrane cholesterol content by nearly two-fold increased the protrusion length by 30%, and decreased the k0 stiffness by nearly two-and-half-fold as compared with control cells. Cytoskeleton integrity was found to make a major contribution to protrusion mechanics as evidenced by the effects of F-actin disruption on the resulting mechanical parameters. Viscoelastic behavior of protrusions was further characterized by hysteresis and force relaxation after formation. The results of this study elucidate the coordination of plasma membrane composition and cytoskeleton during protrusion formation.
          Cover page: Effects of Plasma Membrane Cholesterol Level and Cytoskeleton F-Actin on Cell Protrusion Mechanics
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2014)
          In this study, we investigated the effects of size and surrounding media viscosity on trapping of microspheres. A continuous wave ytterbium fiber laser with a 1064 nm wavelength was used to create an optical tweezers system for optical manipulation experiments. Briefly, the system consisted of an inverted microscope, and a 100X 1.4 NA oil immersion objective through which the laser beam converged to form the optical trap. The laser beam was collimated, steered, and coupled to the microscope through the epifluorescence microscope port. The laser power at the trap focal spot was determined by measuring the input power at the back aperture of the objective multiplied by the objective transmission factor at 1064 nm measured by a modified dual objective method. Polystyrene microspheres varying in diameter from 5 to 15 microns were suspended in liquid media in glass bottom petri dishes prior to trapping experiments. The microspheres were trapped at different trapping powers, and fluidic viscous drag forces where applied to the optically trapped microspheres by driving a computer controlled 2D motorized microscope stage at known velocities. The drag forces were calculated at the point that the microspheres fell out of the trap, based on the Stokes equation for flow around spheres. The data show a linear relationship between trapping force and trap power within the range of the microsphere diameters and media viscosity values used. The work includes calculation of the dimensionless trap efficiency coefficient (Q) at 1064 nm wavelength and the corresponding effects of media viscosity and microsphere size on (Q). © 2014 SPIE.
            Cover page: Effects of media viscosity and particle size on optical trapping of microspheresCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2016)
            Laser microirradiation is a powerful tool for real-time single-cell analysis of the DNA damage response (DDR). It is often found, however, that factor recruitment or modification profiles vary depending on the laser system employed. This is likely due to an incomplete understanding of how laser conditions/dosages affect the amounts and types of damage and the DDR. We compared different irradiation conditions using a femtosecond near-infrared laser and found distinct damage site recruitment thresholds for 53BP1 and TRF2 correlating with the dose-dependent increase of strand breaks and damage complexity. Low input-power microirradiation that induces relatively simple strand breaks led to robust recruitment of 53BP1 but not TRF2. In contrast, increased strand breaks with complex damage including crosslinking and base damage generated by high input-power microirradiation resulted in TRF2 recruitment to damage sites with no 53BP1 clustering. We found that poly(ADP-ribose) polymerase (PARP) activation distinguishes between the two damage states and that PARP activation is essential for rapid TRF2 recruitment while suppressing 53BP1 accumulation at damage sites. Thus, our results reveal that careful titration of laser irradiation conditions allows induction of varying amounts and complexities of DNA damage that are gauged by differential PARP activation regulating protein assembly at the damage site.
              Cover page: Femtosecond near-infrared laser microirradiation reveals a crucial role for PARP signaling on factor assemblies at DNA damage sites
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2018)
              DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2015)
                Cells within the body are subject to various forces; however, the details concerning the way in which cells respond to mechanical stimuli are not well understood. We demonstrate that laser-induced shockwaves (LIS) combined with biosensors based on fluorescence resonance energy transfer (FRET) is a promising new approach to study biological processes in single live cells. As "proof-of-concept," using a FRET biosensor, we show that in response to LIS, cells release intracellular calcium. With the parameters used, cells retain their morphology and remain viable. LIS combined with FRET permits observation of the cells immediate response to a sudden shear force.
                  Cover page: Laser‐induced shockwave paired with FRET: A method to study cell signaling
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2014)
                  With suitable calibration, optical tweezers can be used to measure forces. If the maximum force that can be exerted is of interest, calibration can be performed using viscous drag to remove a particle from the trap, typically by moving the stage. The stage velocity required to remove the particle then gives the escape force. However, the escape force can vary by up to 30% or more, depending on the particle trajectory. This can have significant quantitative impact on measurements. We describe the variation of escape force and escape trajectory, using both experimental measurements and simulations, and discuss implications for experimental measurement of forces.
                    Cover page: Optical tweezers escape forcesCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2014)
                    We have estimated the mitotic forces exerted on individual isolated mammalian chromosomes using optical trapping. The chromosomes were trapped by an optical tweezers system created by a continuous wave ytterbium laser at 1064 nm. Individual chromosomes were trapped at different in situ powers in the range of 20-50 mW. The corresponding trapping forces were determined by a viscous drag method. In the range of laser powers used, the preliminary data show a linear relationship between the chromosome trapping forces and in situ powers. We have calculated the dimensionless trapping efficiency coefficient (Q) of the chromosomes at 1064 nm and the corresponding effects of trapping power on Q. The value of Q in our experiments was determined to be ≈ 0.01. The results of this study validate optical tweezers as a non-invasive and precise technique to determine intracellular forces in general, and specifically, the spindle forces exerted on the chromosomes during cell division.
                      Cover page: Optical trapping of isolated mammalian chromosomesCreative Commons 'BY' version 4.0 license
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