Yongdae Shin

Experimental characterization of electrospinning: the electrically forced jet and instabilities

In the electrospinning process, polymer fibers with submicron-scale diameters are formed by subjecting a fluid jet to a high electric field. We report an experimental investigation of the electrically forced jet and its instabilities. The results are interpreted within the framework of a recently developed theory for electrified fluid jets. We find that the process can be described by a small set of operating parameters and summarized through the use of operating diagrams of electric field versus flow rate. In addition, the jet current is related to the net charge density and found to depend on the fluid properties, the applied electric field and the equipment configuration. The net charge density appears to be relatively insensitive to the flow rate, at least for high flow rates. The experiments reveal that a key process in the formation of submicron-scale solid fibers is a convective instability, the rapidly whipping jet. The dependence of this instability on electric field and flow rate, and the exponential nature of its growth rate are in accord with the theory.

Electrospinning : A Whipping Fluid Jet Generates Submicron Polymer Fibers

Polymeric fibers with diameters in the range from 50 nm to 5 μm are produced by accelerating a fluid jet in an electric field, in a process known as “electrospinning.” Here we show that an essential element of the process is a fluid instability, the rapidly whipping jet. The phenomena responsible for the onset of whipping are revealed by a linear instability analysis that describes the jet behavior in terms of known fluid properties and operating conditions. The behavior of two competing instabilities, the Rayleigh mode and the axisymmetric conducting mode, is also described. The results are summarized using operating diagrams, delineating regimes of operation in electrospinning, which are in good agreement with experimental observations.

TCR Mechanobiology: Torques and Tunable Structures Linked to Early T Cell Signaling

Mechanotransduction is a basis for receptor signaling in many biological systems. Recent data based upon optical tweezer experiments suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into biochemical signals upon specific peptide-MHC complex (pMHC) ligation. Tangential force applied along the pseudo-twofold symmetry axis of the TCR complex post-ligation results in the αβ heterodimer exerting torque on the CD3 heterodimers as a consequence of molecular movement at the T cell–APC interface. Accompanying TCR quaternary change likely fosters signaling via the lipid bilayer predicated on the magnitude and direction of the TCR–pMHC force. TCR glycans may modulate quaternary change, thereby altering signaling outcome as might the redox state of the CxxC motifs located proximal to the TM segments in the heterodimeric CD3 subunits. Predicted alterations in TCR TM segments and surrounding lipid will convert ectodomain ligation into the earliest intracellular signaling events.

Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease

ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5′-[γ-thio]triphosphate (ATPγS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function.

Kinesin-12 Kif15 targets kinetochore fibers through an intrinsic two-step mechanism.

Proteins that recognize and act on specific subsets of microtubules (MTs) enable the varied functions of the MT cytoskeleton. We recently discovered that Kif15 localizes exclusively to kinetochore fibers (K-fibers) or bundles of kinetochore-MTs within the mitotic spindle. It is currently speculated that the MT-associated protein TPX2 loads Kif15 onto spindle MTs, but this model has not been rigorously tested. Here, we show that Kif15 accumulates on MT bundles as a consequence of two inherent biochemical properties. First, Kif15 is self-repressed by its C terminus. Second, Kif15 harbors a nonmotor MT-binding site, enabling dimeric Kif15 to crosslink and slide MTs. Two-MT binding activates Kif15, resulting in its accumulation on and motility within MT bundles but not on individual MTs. We propose that Kif15 targets K-fibers via an intrinsic two-step mechanism involving molecular unfolding and two-MT binding. This work challenges the current model of Kif15 regulation and provides the first account of a kinesin that specifically recognizes a higher-order MT array.

Stochastic optical active rheology

We demonstrate a stochastic based method for performing active rheology using optical tweezers. By monitoring the displacement of an embedded particle in response to stochastic optical forces, a rapid estimate of the frequency dependent shear moduli of a sample is achieved in the range of 10(-1)-10(3)u2009Hz. We utilize the method to probe linear viscoelastic properties of hydrogels at varied cross-linker concentrations. Combined with fluorescence imaging, our method demonstrates non-linear changes of bond strength between T cell receptors and an antigenic peptide due to force-induced cell activation.

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8

Significance The cellular distributions of kinesins are defined in part by their intrinsic biophysical properties. The well-characterized kinesin-8s, for example, translocate exceptionally long distances on a microtubule track, concentrating them at plus ends of long, stable microtubules. Kif18B, a little-studied kinesin-8, targets the plus ends of fast-growing, short-lived microtubules by binding the plus-end tracking protein EB1. Whether ultraprocessivity is conserved among kinesin-8s is thus unclear. Here, we show that Kif18B is not ultraprocessive and that the motor switches frequently between diffusive and directed modes of motility. Our work identifies properties of Kif18B that may have optimized the motor to explore the ∼1-μm domain of microtubule plus ends and show that biophysical motor properties cannot be generalized within any one kinesin subfamily. Kinesin-8s are plus-end–directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are “ultraprocessive,” a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end–directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.

Clpxp Degradation of Proteins Probed By Single-Molecule Fluorescence

ClpXP is an AAA+ protease that unfolds and degrades target proteins. ClpX, a hexameric ring-shaped ATPase, recognizes specific proteins and then powers their mechanical denaturation and translocation into the degradation chamber of ClpP where polypeptide bond cleavage occurs. Although ClpXP degradation activities have been widely studied at the bulk solution level, the operating principles and detailed mechanisms of this complex macromolecular machinery remain unanswered. Here, we probe the kinetics of substrate unfolding and degradation by ClpXP using a single-molecule fluorescence assay. These assays employ a covalently crosslinked ClpX hexamer, immobilized on PEG coated surface illuminated by total internal reflection fluorescence. A series of substrates are engineered to contain fusion of an N-terminal Cy3 and a C-terminal GFP-titin-ssrA module. In the presence of ATPγS, ClpX stalls at GFP after degradation of titin-ssrA domains. These stalled pre-engaged substrates are stably bound to ClpXP even in the absence of Mg++, but are released quickly upon the introduction of nucleotide-free solution. Exchange into ATP for pre-engaged substrate-ClpXP complexes allows synchronous resumption of unfolding and degradation of GFP and any following domains. The time required for complete degradation is measured by loss of the N-terminal Cy3 from the protease complex. GFP unfolding can also be monitored directly with quenching of intrinsic fluorescence by denaturation. Global fitting of single-molecule data for a set of related substrates yields time constants for ClpX unfolding, translocation, and a terminal step which may involve product release, and shows strong agreement with bulk solution measurements. It should be possible to extend these methods to allow single-molecule studies such as FRET for real-time assays of ATP-fueled conformational changes that drive the mechanical operations of the ClpXP protease. Support from the NSF Career Award (0643745) is gratefully acknowledged.

Active Stochastic Microrheology using Optical Tweezers

Cells are dynamic structures capable of generating and reacting to physical cues in their environment. Measuring mechanical properties is thus essential for elucidating cell or other material structure-function in particular during dynamic rearrangement of the cytoskeleton. Although a variety of rheological techniques have been developed using video microscopy, AFM, and magnetic traps, the measurable frequency range is limited by the time to obtain the measurement, and forcing conditions such as amplitude, direction, contact geometry, and probe location. Here, we developed active stochastic microrheology using optical tweezers to enhance the temporal resolution and precision of detection. A stochastic force is generated by moving the trap relative to the sample. Both bead displacement and trap position are monitored simultaneously by separate position sensitive devices. With this method, both storage and loss shear moduli of the extracellular matrices can be extracted over a wide frequency range of 10−2 – 103 Hz within a few minutes. Also, this method was used to probe the local mechanical environment of B-cell receptor using antigen specific interaction. We showed that the local mechanical properties are strengthened in response to antigen binding and repeated external excitation in a physiological range of 1–100pN. The mechanical responses can also be measured with respect to direction such as force applied normal and perpendicular to the cell membrane. This technique is useful in characterizing the mechanical properties at a user-defined location and magnitude, over a wide frequency spectrum, in a short time, and with a small deformation < 100nm. With these advantages, the method can also be applied to other cell processes, studies of complex fluids, fibril growth, and polymer solutions. Support from the NIGMS (GM-076689), an NSF Career Award (0643745), and the Singapore-MIT Alliance for Research and Technology (SMART-BioSyM) are gratefully acknowledged.