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

Modulating transition temperature affects cisplatin mediated cellular response in UMSCC1 cells.

<p>(A) Transition temperature shifts measured for GPMVs isolated from UMSCC1 in the presence of 50mM isopropanol or 100 μM menthol or each of these treatments in combination with 10μM cisplatin. (B) Efficacy of 50mM isopropanol and 100μM menthol action on UMSCC1 cells calculated as the number of cells present after 24h of treatment divided by the number of cells present in an untreated control. (C) Efficacy of cisplatin action as a function of the transition temperature shift effected by the treatment in isolated GPMVs shown in A. Efficacy of cisplatin action on UMSCC1 cells as above was computed as above in (B) by dividing the number of cells present after 24h of treatment compared to the number of cells present in an untreated control. (D) Plots show relative cell counts as a function of cisplatin concentration either in the presence or absence of 50mM isopropanol or 100μM menthol. Each point represents the average and SEM of at least 4 independent measurements, and lines are fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a>. (E) Average IC₅₀ values as determined by fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a> to individual dose response curves. Values represent an average and SEM over at least 4 independent measurements.</p

Co-incubation of cisplatin with isopropanol leads to enhanced apoptosis without an increase in intercellular cisplatin concentration.

<p>(A) Expression levels of cleaved PARP, an apoptotic marker, as measured by western blot for cells incubated with 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone along with cisplatin free controls (B) Levels of intracellular cisplatin were measured using optical emission spectrometry for the three treatments, 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone. The differences between the three treatments are not statistically significant (n = 8 trials). Also shown are predicted levels of cisplatin obtained by assuming that intracellular cisplatin that is directly proportional to the external concentration determines the extent of cell death (as described in Methods). The solid line denotes the predicted mean theoretical value and corresponding dashed lines denote error bounds. (C) Levels of intracellular cisplatin for UMSCC1 and the more resistant cell line Me-180pt treated with 10μM cisplatin. The solid line as described previously denotes predicted levels given the assumptions stated in 4B and dashed lines indicate error bounds.</p

Changes in transition temperature in GPMVs correlate with the cell lines resistance to cisplatin.

<p>(A) GPMVs were isolated from four cell-lines as described in the Methods section. The transition temperature shifts are reported by comparing the transition temperatures of GPMVs probed in the presence of 10 μM cisplatin to untreated GPMVs. (B) Data points in panel A were plotted against a previously reported measure of surviving fraction to cisplatin obtained using clonogenic assays for the same four cell lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.ref008" target="_blank">8</a>]. Surviving fraction was measured using a clonogenic survival assay. Surviving fractions were measured 72 hours after treatment with 10uM cisplatin. The straight line is drawn to visually distinguish sensitive and resistant celllines. Transition temperature shifts upon incubation with 10 μM cisplatin or exposure to 10 Gy irradiation for GPMVs isolated from ME-180 pt cells (C) and RBL cells (D). In all cases, points represent the average of at least 3 independent measurements and error bounds represent the standard error of the mean. Significance between transition temperature shift measurements were evaluated using t-tests.</p

Helical Wheel Diagrams for Lid1 and Lid2

<p>Shown are the long helix in lid1 (A) and the helix in lid2 (B). Hydrophobic residues are shaded in grey; those in bold face the binding pocket and opposite lid. Letters stand for amino acids.</p

Simulation Results of Closed Form in Octane-Water Interface

<p>This model reiterates our hypothesis of the second lid in the lipase.</p> <p>(A) This shows the variation of the distance (black) and RMSD of the two lids (red for lid1 and blue for lid2) with the time course of simulation. As the lid opens, there is a corresponding increase in distance between the two lids. This movement stabilizes from 8 ns until the end of simulation.</p> <p>(B) Here the final structure of the simulation (after 20 ns; red) is superimposed on the crystal structure (blue) to a C-alpha RMSD of 0.08 nm.</p

Lid Movement in <i>P. aeruginosa</i> Lipase: Quantitative Measures

<p>Here we show the time variation of the distance between the lids and the RMSD of the two flaps as a function of time. The RMSD of lid1 (red) after least-square fit of the protein backbones is approximately 0.9 nm. The RMSD of the helix in lid2 (blue) is also shown. This is corroborated by a plot of distance between the two lids against time (black). There is an appreciable decrease in this distance after 6 ns. The lid1-lid2 distance stabilizes at about 8 ns, after which there is little relative movement between the two lids.</p

Lid Movement in <i>P. aeruginosa</i> Lipase: Qualitative Representation

<p>(A) This figure represents the simulation results. The structure on the left (i) is the open form of the lipase, which reveals the active site cavity. Binding pocket residues are coloured yellow, and lid residues red (lid1) and blue (lid2). There appears to be nearly a 100% decrease in the solvent-accessible area when the lids close (structure on right [ii]).</p> <p>(B) Time evolution of the structure backbone is shown. Blue corresponds to the open crystal structure of the lipase, while red indicates the position of the lids after 3 ns (left [i]), 7 ns (centre [ii]), and 10 ns (right [iii]).</p

Simulation with Lipase Double Mutants

<p>Both the alanine at 217 and the phenylalanine at 214 were mutated to serines, and the simulation was carried out in an aqueous medium. The lids do not close in an aqueous environment. Thus, by mutating the key contact residues to serines, the hydrophobicity of lid2 is lost, and it is unable to function as a trigger for the lids to converge and close.</p> <p>Red, lid1 RMSD; blue, lid2 RMSD; black, lid1-lid2 distance.</p

Lid1 Movement is Triggered by Lid2, but Lid2 Movement Is Independent of Lid1

<p>(A) Red represents the movement of lid1 when lid2 is restrained, while black is that in the unrestrained system. The movement of lid1 is significantly hindered when lid2 is restrained.</p> <p>(B) Blue represents the movement of lid2 when lid1 is restrained, and black represents the movement in the unrestrained system. The movement of lid2 is the same in both cases.</p