Single-Walled Carbon Nanotubes Probed with Insulator-Based Dielectrophoresis

Single-walled carbon nanotubes (SWNTs) offer unique electrical and optical properties. Common synthesis processes yield SWNTs with large length polydispersity (several tens of nanometers up to centimeters) and heterogeneous electrical and optical properties. Applications often require suitable selection and purification. Dielectrophoresis is one manipulation method for separating SWNTs based on dielectric properties and geometry. Here, we present a study of surfactant and single-stranded DNA-wrapped SWNTs suspended in aqueous solutions manipulated by insulator-based dielectrophoresis (iDEP). This method allows us to manipulate SWNTs with the help of arrays of insulating posts in a microfluidic device around which electric field gradients are created by the application of an electric potential to the extremities of the device. Semiconducting SWNTs were imaged during dielectrophoretic manipulation with fluorescence microscopy making use of their fluorescence emission in the near IR. We demonstrate SWNT trapping at low-frequency alternating current (AC) electric fields with applied potentials not exceeding 1000 V. Interestingly, suspended SWNTs showed both positive and negative dielectrophoresis, which we attribute to their ζ potential and the suspension properties. Such behavior agrees with common theoretical models for nanoparticle dielectrophoresis. We further show that the measured ζ potentials and suspension properties are in excellent agreement with a numerical model predicting the trapping locations in the iDEP device. This study is fundamental for the future application of low-frequency AC iDEP for technological applications of SWNTs

Lengths of untreated and sulfo-SMCC treated MTs at different time points.

<p>The table shows the total number of microtubules (from three independent sets of experiments), average calculated mean length (<i>L</i>_<i>mean</i>) and average mean length from the fitting curve (<i>L</i>_{<i>meanfit</i>}) for sulfo-SMCC treated and untreated MTs, measured after 0 h, 6 h, and 24 h of incubation.</p

Chemically treated and untreated MTs.

<p>The table shows the total numbers of microtubules (from one set of experiments), average calculated mean lengths (<i>L</i>_<i>mean</i>) and average mean lengths from the fitting curve (<i>L</i>_{<i>meanfit</i>}) for untreated MTs, and MTs treated with sulfo-SMCC, maleimide dye, and NHS ester dye for 24 h.</p

Length distributions of chemically treated and untreated MTs after 24 h of incubation.

<p>Length distributions of 2 mg/ml (tubulin) solution of untreated MTs, MTs treated with 250 <i>μ</i>M sulfo-SMCC, 250 <i>μ</i>M maleimide dye and 250 <i>μ</i>M NHS ester dye are shown. Lengths of MTs are distributed exponentially in all cases; single-exponential fits shown as dashed lines. Treatment with sulfo-SMCC resulted in drastically shorter MTs in comparison to untreated MTs (mean length = 2 <i>μ</i>m and 17 <i>μ</i>m, respectively). Maleimide dye treated MTs, however, showed a less drastic effect with an intermediate mean length of 9.2 <i>μ</i>m. Addition of NHS ester dye to MTs as a control did not affect the lengths of MTs.</p

Dual-colored microtubule experiments.

<p>The table shows the total number of microtubules (from three independent sets of experiments), average calculated mean length (<i>L</i>_<i>mean</i>) and average mean length from the fitting curve (<i>L</i>_{<i>meanfit</i>}) for different time points of sulfo-SMCC treated and untreated MTs.</p

Length distribution of sulfo-SMCC treated and untreated MTs after different incubation times.

<p>Length distributions of 2 mg/ml (tubulin) solution of (A) untreated and (B) sulfo-SMCC treated (250 <i>μ</i>M) MTs after 0 h, 6 h, and 24 h are shown. Untreated MTs show a significant increase in their mean lengths after 6 h (p < 0.001) and 24 h (p < 0.001) of incubation, in comparison with those measured at 0 h. Sulfo-SMCC treated MTs show a constant mean length, independent of incubation time. Lengths of all samples are distributed exponentially; exponential fits with the normalized probability function <i>a</i>² <i>Lexp</i>(−<i>aL</i>) are shown as dashed lines. This functional form takes undersampling of short MTs due to the resolution limit into account.</p

(A) Kymograph of the displacement of 230 pM Eg5-513-GFP dimers versus time in 80 mM Pipes buffer with 0

2 mM ATP. A large majority of binding events last two frames or less (<p><b>Copyright information:</b></p><p>Taken from "Microtubule cross-linking triggers the directional motility of kinesin-5"</p><p></p><p>The Journal of Cell Biology 2008;182(3):421-428.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500128.</p><p></p

Data collected as described in , but in the presence of a mixture of 1 nM Eg5-GFP and 2 nM of unlabeled tetrameric Eg5

(A) Top kymograph shows sliding of a microtubule (MT) relative to a surface-attached microtubule. Below, the corresponding kymograph of Eg5-GFP shows directional runs between the overlapping microtubules (region marked with two red dotted lines). (B–F) Analysis of Eg5 motility during relative sliding. (B) Scatter plot of all pairs of short-term velocity and diffusion constant determined for a window of 15 s moving over the composite position-time trace of 94 Eg5 motors traced in the overlap zone of 11 microtubule pairs (2,335 points obtained from 2,349 s of total time). The horizontal dotted line indicates the average velocity of sliding microtubules (33 nm/s), and the vertical dotted line indicates the threshold used to discriminate slow and fast diffusion. (C and D) Similar analyses for Eg5 moving on individual microtubules at low ionic strength (C, 70 mM Pipes; ; 4,266 points) and high ionic strength (D, data pooled from 70 mM Pipes + 60 mM KCl and 70 mM Pipes + 80 mM KCl; 2,478 points). (E) Position-time traces. Black, fraction of the composite trace used for B. Green and red, sorted time points with a short-term diffusion constant; < 1,500 nm/s (green) and >1,500 nm/s (red). (F) Histograms of the short-term velocities as obtained from the time points in the green and red trace in E. The arrow indicates the average microtubule sliding velocity. (G) Graph summarizing Eg5 behavior under various conditions. Bar, 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Microtubule cross-linking triggers the directional motility of kinesin-5"</p><p></p><p>The Journal of Cell Biology 2008;182(3):421-428.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500128.</p><p></p

(A and B) Frames (A) and kymograph (B) from a time-lapse recording showing single molecules of Eg5-GFP (green) moving directionally along a microtubule (red) in the presence of 70 mM Pipes

(C and D) Frames (C) and kymograph (D) from a time-lapse recording showing single molecules of Eg5-GFP (green) diffusing along a microtubule (red) in the presence of 70 mM Pipes plus 40 mM KCl. (E–H) MD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) in 70 mM Pipes plus 0, 20, 40, or 60 mM KCl. Fits represent MD = . (I–M) MSD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) and at the indicated ionic strengths. Fits represent MD = and MD 2 + offset for the ATP data and MD 2 + offset for the ADP data. All numerical results are listed in . (N) Histogram of the duration of binding events for 0 mM KCl and for 60 mM KCl added. Lines are single exponential fits (exp[−t/t]) to the data (0 mM: t = 34 ± 3, = 212; 60 mM: t = 16 ± 2, = 119). Error bars represent SD. Bars, 1 μm.<p><b>Copyright information:</b></p><p>Taken from "Microtubule cross-linking triggers the directional motility of kinesin-5"</p><p></p><p>The Journal of Cell Biology 2008;182(3):421-428.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500128.</p><p></p

Endoplasmic Reticulum Sorting and Kinesin-1 Command the Targeting of Axonal GABA_B Receptors

<div><p>In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABA_BRs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABA_BRs are obligatory heteromers constituted by two subunits, GABA_BR1 and GABA_BR2. GABA_BR1a and GABA_BR1b are the most abundant subunit variants. GABA_BR1b is located in the somatodendritic domain whereas GABA_BR1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABA_BR1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABA_BRs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABA_BR1a operate in a pre-Golgi compartment. In the axon GABA_BR1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABA_BR1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABA_BRs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.</p> </div