OPTICAL TRAPPING AND ITS MODELING**

In the so called optical trapping technique a bead is held suspended in an aqueous solution by the presence of a lasing system. The laser light provides a trapping potential, similar to a harmonic potential, with a stiffness constant that is related to the laser intensity. It is possible for the bead, therefore, to perform simple harmonic motion, albeit under the action of random forces due to thermal motion. Thus, we look at a simple model for the bead\u27s motion as a function of time through the application of Newton\u27s 2nd law in the presence of a spring force, a damping force, and a random force. Due to the fact that the suspended bead could actually be an organelle in a cell or be a cell, with very low mass, we look at what might the differences of a model with negligible mass be compared to the one when the mass is not ignored. We have experimental results of a 500 nm diameter carboxylate coated polystyrene bead trapped in a 980 nm laser beam with trap stiffness of 0.05 pN/nm. The bead position in the trap was determined by using a position detection diode having 10 nanometer spatial resolution and a time resolution of 0.5 milliseconds. We look at possible interpretation of this data with the above mentioned model

Water-Glycerol Mixture Viscosity through Optical trapping

A cellular medium is highly viscoelastic in nature. In order to understand the viscoelastic nature of a medium, we have developed a new simple approach to quantitatively measure the viscosity of water-glycerol mixture medium using an optical tweezers technique. In this approach, the position of a trapped bead of radius r in a glycerol-water mixture is measured using a position sensing diode with 40 kHz acquisition rate. By using a Lorentzian fit to the Power Spectrum of the position signal, the corner frequency is obtained. For a small change in volume concentration of glycerol in a water-glycerol medium, the change in trap stiffness can be ignored if incident laser power and bead radius remain unchanged. By following an iterative experimental approach, the viscosity of the water-glycerol mixture can be obtained by comparing the corner frequencies obtained experimentally in a medium of known viscosity. Using this approach, the viscosity of a water-glycerol mixture has been measured for glycerol volume concentration of 10%, 20%, 30%, and 40%. The experimental results are compared with the work of Segur and Oberstar [1] as well as with a simple theoretical approach that models the data behavior versus glycerol concentration. The comparison shows that with this experimental approach using an optical tweezers technique, the viscosity of water-glycerol mixture is determined to an accuracy of 0.0082 percent. [1] J. B. Segur and H. E. Oberstar, Industrial & Engineering Chemistry, V43, No. 9 (1951), p2117

Calibration of Optical Tweezers for In Vivo Force Measurements: How do Different Approaches Compare?

There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties. We evaluate the relative merits of these methods and directly compare two of them, a refractive index matching method, and a light-momentum-change method. Since in vivo forces are frequently relatively high (e.g., can exceed 15 pN for lipid droplets), a high-power laser is employed. We discover that this high-powered trap induces local temperature changes, and we develop an approach to compensate for uncertainties in the magnitude of applied force due to such temperature variations

Detachment Kinetics of Single Kinesin and Dynein

Intra-cellular transport via the microtubule motors kinesin and dynein plays animportant role in maintaining cell structure and function. Often, multiple kinesinor dynein motors move the same cargo. Their collective function dependscritically on the single motors’ detachment kinetics under load. Single Kinesin’sand Dynein’s super-force off rates have been measured using anoptical-trap based method. We rapidly increased the force on a moving beadand measured the time to detachment. From such events, detachment time distributionsfor specific super-force values have been measured. In contrast toa possible constant off-rate kinesin has an off-rate increasing with force. Atlow loads, dynein is sensitive to load; detaching easily but at higher load it exhibiteda catch-bond type behavior, with off rate decreasing with load. Thesuper-force experiments also allowed us to determine the probability of backwardstepping for the motors. Kinesin and dynein can back-step under load, butthis was relatively rare in both directions (<20%), and the typical backwardtravel distance was short

Detachment Kinetics of Single Kinesin and Dynein

Intra-cellular transport via the microtubule motors kinesin and dynein plays animportant role in maintaining cell structure and function. Often, multiple kinesinor dynein motors move the same cargo. Their collective function dependscritically on the single motors’ detachment kinetics under load. Single Kinesin’sand Dynein’s super-force off rates have been measured using anoptical-trap based method. We rapidly increased the force on a moving beadand measured the time to detachment. From such events, detachment time distributionsfor specific super-force values have been measured. In contrast toa possible constant off-rate kinesin has an off-rate increasing with force. Atlow loads, dynein is sensitive to load; detaching easily but at higher load it exhibiteda catch-bond type behavior, with off rate decreasing with load. Thesuper-force experiments also allowed us to determine the probability of backwardstepping for the motors. Kinesin and dynein can back-step under load, butthis was relatively rare in both directions (<20%), and the typical backwardtravel distance was short

Casein kinase 2 reverses tail-independent inactivation of kinesin-1

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signalling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (approximately fourfold) without altering kinesin\u27s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase upregulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin