A Microwave Ring Resonator Based Glucose Sensor

A microwave ring resonator based glucose detecting biosensor incorporating glucose oxidase enzyme is presented. Sensor uses a split ring resonator as a transducer, where the sensing operation is done by the observation of shifts in its resonant frequency. Resonator was fabricated with basic fabrication techniques and the enzyme was immobilized via conductive polymer agent PEDOT:PSS. Experimentally observed redshift of resonant frequency of the sensor in response to different loading conditions are in agreement with simulation results and theoretical expectations. Sensor selectivity is confirmed with control experiments conducted with NaCl solutions. Experiments done with different glucose solution concentrations yielded a sensor sensitivity of 0.174MHz/mgml-1

Regulation of Dynein Motility and Force Generation by Lissencephaly-1

Molecular motors hydrolyze ATP to produce mechanical work by stepping along the cytoskeleton network and carrying cargos. Dynein has many cellular roles which require its minus end-directed motility and force generation along the microtubules (MTs). All dynein activity needs to be tightly regulated by its many associated factors. Lis1 is the only associated factor that directly binds to dynein’s ATP hydrolyzing AAA ring, and it is involved in most, if not all, cellular processes that require dynein activity. In my thesis work, working with both mammalian and yeast proteins, I showed how dynein motility and force generation is regulated by Lis1 and its yeast homolog Pac1 (both Lis1 from here on). Mammalian dynein is mostly autoinhibited, that is, it cannot take many steps before detaching from the microtubules, a property that is essential for dynein-mediated cargo transportation. For processive motility, dynein needs to be relieved from this autoinhibition and needs to bind to dynactin and a cargo adapter. Using protein engineering, single molecule motility, optical trapping, and biophysical characterization assays, we have shown that Lis1 relieves dynein from autoinhibition, thus allowing the formation of dynein dynactin cargo adapter complex (DDX). Through the same mechanism, Lis1 increases the copy number of dynein in DDX complexes which enables faster motility and higher force generation. However, even after the formation of these complexes Lis1 can remain bound to dynein. In that case, we see an inhibitory effect of Lis1. In my thesis, I have shown the mechanism by which Lis1 binding affects dynein motility I switched my research to S. cerevisiae cytoplasmic dynein which has inherently processive motility without needing any cofactors, unlike mammalian dynein. I showed that Lis1 binding to the motor domain slows down dynein motility thus confirming previous studies done on yeast dynein and Lis1. Through multicolor TIRF colocalization assays, I have demonstrated that binding of individual Lis1 molecules causes dynein to pause or stop, and its unbinding restores dynein velocity. I have made three discoveries: 1. Lis1 binding to dynein has been proposed to inhibit or slow dynein motility by tethering dynein to the microtubule. I ruled out this model by showing that Lis1 only weakly interacts with the microtubule lattice, and this interaction does not slow dynein motility. 2. Lis1 binding has been proposed to block the force-generating conformational changes of the dynein linker domain. Using optical trapping, we ruled out this model by showing that Lis1 does not reduce the dynein stall force. 3. I observed that Lis1 binding decreases the asymmetry in detachment kinetics of force-induced detachment of dynein from the microtubule. Mutations that disrupt Lis1’s interactions with dynein’s stalk (an anti-parallel coiled-coil that leads to dynein’s microtubule-binding domain) partially restore the asymmetry. Because dynein’s stalk “slides” or changes its coiled-coil registry in a nucleotide-dependent manner, my data suggest that Lis1’s interaction with the dynein stalk interferes with the stalk sliding mechanism. I propose that this is what leads to slowing the detachment of dynein from the microtubule under force. These results are compatible with studies of Lis1 in live cells and provide a mechanistic explanation for why Lis1 needs to dissociate from dynein for efficient minus-end-directed motility. They also suggest an additional regulatory role for Lis1, such as anchoring dynein to the microtubule in order to facilitate the proper assembly of dynein with dynactin. I believe that the studies presented in this thesis will be broadly interesting to biophysicists studying the mechanics of motor proteins in vitro, cell biologists interested in the mechanism and regulation of intracellular transport, and neurobiologists who study the molecular basis of neurodevelopmental disorders

Cost-Effective, Microstrip Antenna Driven Ring Resonator Microwave Biosensor for Biospecific Detection of Glucose

We present a biosensor based on electromagnetic ring resonator for label-free detection of glucose. The sensing mechanism is based on the principle that the resonant frequencies of such structures depend on the structure geometry and the physical properties of the medium they are in, such as electrical permittivity. The sensor in this paper uses a split-ring resonator fabricated on a flame retardant four substrate via simple printed circuit board fabrication techniques. Glucose oxidase enzyme was incorporated in order to provide biospecificity for glucose. Conductive polymer poly(3,4-ethylenedioxythiophene)poly(styrenesulfonate), also known as PEDOT:PSS, was used for the immobilization of the enzyme on sensor surface. The redshift of the resonant frequency of the sensor in response to DI water, glucose, and NaCl solutions are shown to be in agreement with simulation results and theoretical expectations. In the presence of the enzyme, the sensor loaded with a glucose solution was observed to experience a resonant frequency shift of 17.5 MHz in 15 min, whereas other reagents such as fructose, sucrose, and NaCl did not respond significantly, confirming the biospecificity. The sensor was measured to have a sensitivity of 0.107 MHz/mgml-1.

Structure and Mechanics of Dynein Motors

Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia

Lis1 activates dynein motility by modulating its pairing with dynactin.

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favours the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells

Lis1 activates dynein motility by modulating its pairing with dynactin.

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favours the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells

Integrated Silicon Photovoltaics on CMOS With MEMS Module for Catheter Tracking

This paper presents an electromagnetic actuation-based optoelectronic active catheter tracking system for magnetic resonance imaging (MRI). The system incorporates a radio frequency (RF) microelectromechanical system (MEMS) resonator array actuated by the Lorentz force induced due to the strong dc magnetic field available in MRI environment. Power transfer to the system and the actuation detection are done optically via fiber optic cables that replace conventional conductive transmission lines; thereby, enabling the tracking system to function safely under MRI. The complementary metal-oxide-semiconductor (CMOS) receiver, optically powered by a supply unit housing an on-chip silicon photovoltaic cell, detects the location of the catheter tip. The RF MEMS resonator array transmits the position data by transducing the electrical signal into a resonant mechanical vibration linearly. The optical reading of this actuation can be done by diffraction grating interferometry or laser doppler vibrometry. The fabricated resonator array is tested with the optically powered CMOS chip (0.18-μm UMC technology) in laboratory conditions. The driving electrical current supplied by the chip for resonator actuation is 25-μA rms, where the magnetic field provided by the experimental setup is 0.62 T. The resonator array is observed to be functional with real-world application by showing a frequency response of 10 dB, which will be enhanced further under the stronger magnetic field available in 3-T MRI