Detection and Quantification through a Lipid Membrane Using the Molecularly Controlled Semiconductor Resistor

The detection of covalent and noncovalent binding events between molecules and biomembranes is a fundamental goal of contemporary biochemistry and analytical chemistry. Currently, such studies are performed routinely using fluorescence methods, surface-plasmon resonance spectroscopy, and electrochemical methods. However, there is still a need for novel sensitive miniaturizable detection methods where the sample does not have to be transferred to the sensor, but the sensor can be brought into contact with the sample studied. We present a novel approach for detection and quantification of processes occurring on the surface of a lipid bilayer membrane, by monitoring the current change through the n-type GaAs-based molecularly controlled semiconductor resistor (MOCSER), on which the membrane is adsorbed. Since GaAs is susceptible to etching in an aqueous environment, a protective thin film of methoxysilane was deposited on the device. The system was found to be sensitive enough to allow monitoring changes in pH and in the concentration of amino acids in aqueous solution on top of the membrane. When biotinylated lipids were incorporated into the membrane, it was possible to monitor the binding of streptavidin or avidin. The device modified with biotin-streptavidin complex was capable of detecting the binding of streptavidin antibodies to immobilized streptavidin with high sensitivity and selectivity. The response depends on the charge on the analyte. These results open the way to facile electrical detection of protein–membrane interactions

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

<div><p><i>Candida albicans</i>, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to “trimeras,” three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in <i>C. albicans</i> is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related <i>Candida</i> species, and in an <i>in vivo</i> model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.</p></div

Trimera formation is not a general stress response.

<p>Cells exposed to triazoles ketoconazole, voriconazole, and itraconazole formed trimeras at frequencies similar to FLC. Cells that were exposed to caspofungin, an echinocandidn, also produced many trimera-like and multimera-like cells (upper and lower panels). Exposure to toxin 5-FOA as well as heat shock did not result in a significant number of trimeras. No trimeras were detectable following exposure to 2-DOG (unpublished data). Percentages in upper right corner of DAPI image denote frequency of trimera formation in 300–400 cells.</p

Bud emergence is delayed after 4 h of FLC exposure.

<p>(A) Individual frames from time-lapse images of Nop1-RFP/Tub1-GFP–expressing cells in the absence (top two rows) and presence (4 h, bottom two rows) of FLC. BE, bud emergence; SA, spindle assembly; AO, anaphase onset; SEP, sister separation; SEG, sister segregation across bud neck; SD, spindle disassembly. Illustrations in rows 3 and 4 show relative timing of events. Numbers in fluorescent images denote time (min) of FLC exposure. Scale bars, 5 µm. (B) Cartoon illustrating relative timing of cell cycle events in the absence (left) and presence (right) of FLC. Lower left panel, average time between bud emergence (BE, purple) and anaphase onset (AO, gray) events. Bar graph, average time between BE and AO (red), AO and SEG (green), SEG and SD (blue), and SD and BE (purple). Error bars are 1 standard deviation from mean based on <i>n</i> = 25 cells observed on 2 d. Asterisks denote statistically significant differences (<i>t</i> test, <i>p</i> value <0.05). (C) Proportion of cells with normal pre-START and pre-anaphase phenotypes (blue) and aberrant START (unbudded cells with two SPBs, yellow) phenotypes. Cells not represented here were in mitosis. “No drug” control cultures showed no significant deviation from t = 0 numbers (unpublished data).</p

Trimeras become prevalent after 8 h of FLC exposure and remain viable.

<p>(A) Time-lapse microscopy of control (top row) and FLC-exposed cells expressing Nop1-GFP. Numbers indicate time (min) of FLC exposure. (B) Relative proportion of cells with indicated morphologies (indicated in color-outlined images) at different times (t, time) of FLC exposure. “No drug” control cultures showed no significant deviation from t = 0 numbers (unpublished data). Colors in pie charts correspond to color of image outline. Scale bars, 5 µm. At least 300 cells from each of two different strains were analyzed for each time point. (C) Colony formation assay following cell micromanipulation. Following 12 h pre-exposure to 10 µg/ml FLC, single budded cells (red) and trimeras (purple) were transferred to plates with indicated FLC concentration. The number of cells analyzed for single budded cells on 0, 2, and 10 µg/ml FLC plates were 52, 53, and 48, respectively. The number of trimeras analyzed on 0, 2, and 10 µg/ml FLC plates were 78, 65, and 48, respectively. Error bars indicate standard error, and statistical significance was determined using a Fisher's exact test.</p

Nuclei within trimeras undergo mitotic collapse to form tetraploids.

<p>(A) Time-lapse images of trimera formation and mitotic dynamics. Numbers in fluorescent images denote time in minutes. Also indicated is the percent of trimeras (<i>n</i> = 19) that formed four individual nucleoli (top sequence) and that formed three nucleoli (lower sequence) Scale bars, 5 µm. (B) Violin plots of nuclear DNA content (Hhf1-GFP fluorescence) and SPB number (Tub4-mCherry foci). Colors correspond to measurements of individual nuclei within cells as illustrated below plots: trimeras with two nuclei (purple, <i>n</i> = 56 nuclei), trimeras with four nuclei (pink, <i>n</i> = 24), and trimeras with three nucleoli, one large (green, <i>n</i> = 11) and two small (cyan, <i>n</i> = 22). Gray regions define average fluorescence intensity of 2N and 4N nuclei ±1 standard deviation based on log-phase “no drug” control cells.</p

Cell size correlates with DNA content.

<p>(A) Flow cytometry data plotted as cell size (FSC) versus DNA content (FL1-A) for cultures grown in the absence (top row) or presence (bottom row) of FLC for times indicated. (B) Bar graphs represent percent of cells that fell into colored regions defined in scatterplots above as determined using Gaussian fitting (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001815#s4" target="_blank">Materials and Methods</a>).</p

A model for aneuploid formation in <i>C. albicans</i> cells exposed to FLC.

<p>Proposed model for aneuploid formation in <i>C. albicans</i> cells exposed to FLC. Nuclear membrane, thin black line; nucleolus, red; spindle, green.</p

Trimeras form <i>in vivo</i>.

<p>Percent of ConA-Texas Red/Eno1-GFP cells showing unbudded (blue), budded (red), trimera-like (purple), and hyphal (green) phenotypes within untreated (left, <i>n</i> = 195) and FLC-treated (right, <i>n</i> = 309) mouse host 48 h after injection. Error bars are 1 standard deviation. Scale bar, 5 µm.</p

Unequal segregation occurs in nuclei with more than one spindle.

<p>(A) Time-lapse microscopy of nuclear segregation patterns in tetraploid/diakaryotic cells. Type I segregation pattern (top two rows, 54% of events), both spindles elongated across the bud neck. Type II (bottom two rows, 46% of events), only one spindle elongated across the bud neck. Numbers denote time (min) of FLC exposure. Scale bars, 5 µm. Total of 13 cells analyzed. (B) Histone H4 (Hhf1)-GFP fluorescence intensity scatter plots. Sister nuclei are plotted relative to each other. Postanaphase cells containing a total of two SPBs (cyan) clustered around 1∶1, indicative of equal segregation (gray region, contains 95% of points from “no drug” cells). Postanaphase cells containing more than two SPBs (magenta) diverged significantly from 1∶1 at 12 h (two-tailed <i>t</i> test, <i>p</i> value <0.05).</p