Krishnan Raghunathan

Membrane Transition Temperature Determines Cisplatin Response

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients.

Two-color DNA nanoprobe of intracellular dynamics.

We have developed a correlation microscopy technique to follow the dynamics of quantum dot labeled DNA within living cells. The temporal correlation functions of the labels reflect the fluctuations of the DNA nanoprobe as a result of its interactions with the cellular environment. They provide a sensitive measure for the length of the probe on the scale of a persistence length (∼50 nm) and reveal strong nonthermal dynamics of the cell. These results pave the way for dynamic observations of DNA conformational changes in vivo.

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs. The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension. Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

Using DNA looping to measure sequence dependent DNA elasticity

We are using tethered particle motion (TPM) microscopy to observe protein-mediated DNA looping in the lactose repressor system in DNA constructs with varying AT / CG content. We use these data to determine the persistence length of the DNA as a function of its sequence content and compare the data to direct micromechanical measurements with constant-force axial optical tweezers. The data from the TPM experiments show a much smaller sequence effect on the persistence length than the optical tweezers experiments.

Mechanics of DNA : Sequence Dependent Elasticity

We have used constant force axial optical tweezers to understand the subtle eects of sequence variations on the mechanical properties of DNA. Using designed sequences of DNA with nearly identical curvatures, but varied AT content, we have shown the persistence length to be highly dependent on the elasticity of DNA. The persistence length varies by almost thirty percent between sequences containing 61% AT and 45% AT. The biological implications of this can be substantial, as the need to bend DNA is involved in a host of regulatory schemes, ranging from nucleosome positioning to the formation of protein-mediated repressor and enhancer loops.

DNA Nanoprobe for Intracellular Dynamics

We have developed a novel method to follow the dynamics of DNA interacting with the cellular environment in vivo using two-color correlation microscopy. A DNA probe is end-labeled with two quantum dots and transfected into an axenic strain of Dictyostelium discoideum. The motion of the quantum dots is observed with two-color fluorescence video microscopy. The computed time correlation functions of this two-particle motion reflect the fluctuations of the DNA probe as a result of its interactions with the cellular environment. Substantial differences between live cells and dead yet structurally intact cells point to a strong coupling of active, motor-driven fluctuations in the cell to the DNA probe. This suggests that the motion of native cellular DNA may similarly be driven by active processes instead of relying on purely thermal passive fluctuations. We also note that the difference between the autocorrelations of the center of mass motion and the relative motion of the two quantum dots is a sensitive measure for the effective length of the DNA probe on a length scale around one persistence length (∼ 50 nm). This paves the way for experiments with more complex DNA probes that can bind to intracellular proteins, and report single-molecule binding events through apparent length changes and consequently changes in this correlation measure.

Sequence-Dependent Mechanics of DNA

Double-stranded DNA is a semiflexible polymer that can naturally bend on length scales comparable to the size of large DNA-protein complexes like nucleosomes or protein-mediated DNA loops. The sequence of the substrate DNA does not only provide biochemical binding sites for the proteins, but also affects the local mechanical properties of the DNA. Notably, sequence can affect the intrinsic curvature of the DNA, as well as its bendability, or elasticity. While intrinsic bends in DNA and their role in protein-DNA complex formation are well studied, sequence-dependent elasticity still remains only vaguely explored. In order to separate sequence effects on elasticity from those on intrinsic curvature, we have designed sequences of DNA which have nearly identical curvatures but varied AT content and directly measured their mechanical elasticity using constant force axial optical tweezers. We found the persistence length to be highly dependent on the AT content of the DNA, differing almost thirty percent between sequences with nearly identical curvature but different sequence composition. This is a departure from conventional dinucleotide and trinucleotide models, which predict a much smaller difference between the two sequences, but consistent with estimates obtained from the crystallographic structures of protein-DNA complexes.

Insights Into Sequence Dependent Effects on DNA Elasticity Using Single Molecule Techniques

The mechanical properties of DNA play an important role in regulating gene expression within a cell. Sequence does not only code, but also affects structural properties of the DNA. These variations can change the intrinsic curvature and the stiffness of the DNA. To study the effect of sequence on elasticity, it must be decoupled from curvature effects. For this aim, we designed two DNA constructs with similar curvature but different sequences and measured their elasticity in single-molecule stretching experiments with optical tweezers. We report substantial differences in their persistence length. We complement these experiments with studies on the effect of these differences in elasticity on protein-mediated DNA looping as a means of transcriptional control, using the Lac repressor as a model system.

Boundary-condition dependent elasticity of short double-stranded DNA molecules

We have measured the entropic elasticity of ds-DNA molecules ranging from 247 to 1298 base pairs in length, using axial optical force-clamp tweezers. We show that entropic end effects and excluded-volume forces become significant for such short molecules. In this geometry, the effective persistence length of the shortest molecules decreases by a factor of two compared to the established value for long molecules, and excluded-volume forces extend the molecules to about one third of their nominal contour lengths in the absence of any external forces. We interpret these results in the framework of a modified wormlike chain model.