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Molecular and Cellular Biosciences at Wake Forest University


Wake Forest University Graduate School » Molecular and Cellular Biosciences

Jed Macosko, Ph.D.

Jed Macosko, Ph.D.
Research in the Macosko group is focused on understanding the mechanics of protein machines. We use techniques, including atomic force microscopy (AFM), single molecule fluorescence microscopy and motion-enhanced differential interference contrast light microscopy (MEDIC), to study how protein motors use chemical fuel to power their work cycle. One of our strategies in this effort is to use force and temperature to slow down or speed up the movements of the motors. This gives us information about the potential energy surface corresponding to the rate limiting mechanical transition in the molecular motor. Our long-term goal is the identification of precise mechanical-chemical couplings in molecular machines and the characterization of the overall pathways of their physical motion.

 

Grabbing the cat by the tail: manipulating molecules one by one. Bustamante, C., Macosko, J.C., & Wuite, G.J.L. Nature Reviews Molecular and Cell Biology 1, 130-136 (2000). Outside the Business School, Teaching Entrepreneurship through Teams and Projects: Three Case Studies. Macosko J.C., Johnson A.D., & Yocum S.M., Edward Elgar Publishing ISBN: 978 1 84720 455 4 (August, 2009) Kinesin velocity increases with the number of motors pulling a viscoelastic load. Gagliano JM, Walb MC, Blaker BD, Macosko JC & Holzwarth G. accepted in European Journal of Biophysics Selection of Bead-Displayed, PNA-encoded Chemicals. Gassman NR, Nelli JP, Dutta S, Kuhn AM, Bonin KD, Guthold M & Macosko JC accepted in Journal of Molecular Recognition In vivo Multimotor Force-Velocity Curves by Tracking and Sizing Sub-Diffraction Limited Vesicles. Shtridelman Y, Holzwarth GM, Bauer CT, Gassman NR, DeWitt DA & Macosko JC Cellular and Molecular Bioengineering, 2: 190-199 (2009) Structural and sequence analysis of fast evolving duplicated yeast genes indicates functional specialization with partially relaxed purifying selection. Turunen, O., Seelke, R., & Macosko, J.C. FEMS Yeast Res. 9: 16-31 (2009) The Genetic Code—more than just a table. Berleant D., White M., Pierce E., Tudoreanu E., Boeszoermenyi A., Shtridelman Y., and Macosko J.C. Cell Biochem Biophys, 55: 107-116(2009) Intraneuronal vesicular organelle transport changes with cell population density in vitro. Bauer CT, Shtridelman Y, Tome CM, Grim JQ, Turner CP, Tytell M & Macosko JC Neurosci Lett 441: 173-177 (2008) Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated. Hill DB, Macosko JC & Holzwarth G J Microsc 231: 433-439 (2008) Force-Velocity Curves of Motor Proteins Cooperating In Vivo. Shtridelman Y, Cahyuti T, Townsend B, Dewitt D & Macosko JC Cell Biochem Biophys 52:19-29 (2008) Fewer active motors per vesicle may explain slowed vesicle transport in chick motoneurons after three days in vitro. Macosko, J.C., Newbern, J.M., Rockford, J., Chisena, E.N., Brown, C.M., Holzwarth, G., and Milligan C.E. Brain Research, In Press, Accepted Manuscript, Available online 20 March 2008 Speckling Improves Tracking in Gliding Assays. Chisena, E.N. Wall, R.A., Macosko, J.C., and Holzwarth, G. Physical Biology, 4, 10-5 (2007) Stepping Statistics of Single HIV-1 Reverse Transcriptase Molecules during DNA Polymerization Ortiz, T.P., Marshall, J.A., Meyer, L.A., Davis, R.W., Macosko, J.C., Hatch, J., Keller, D.J. & Brozik, J.A. Journal of Physical Chemistry B 109,16127 – 16131 (2005) Closing of the Fingers Domain Generates Motor Forces in the HIV Reverse Transcriptase. Lu, H., Macosko, J.C., Habel-Rodriguez, D., Keller, R.W., Brozik, J.A. & Keller, D.J. Journal Biological Chemistry 24, 54529-54532 (2004) Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization. Poirier, M.A., Li H., Macosko, J.C., Cai, S., Amzel, M., Ross, C.A., Journal of Biological Chemistry. 43, 41032-41037. (2002) The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion. Leikina E., LeDuc D.L., Macosko J.C. Epand, R., Epand, R., Shin ,Y.K., Chernomordik, L.V., Biochemistry 40, 8378-8386 (2001). 15N NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions. Zhou Z., Macosko J.C., Hughes, D.W., Sayer, B.G., Hawes, J., Epand, R.M..Biophysical Journal 78, 2418-2425 (2000). A novel 5' displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA. Macosko, J.C., Pio, M.S., Tinoco, I. Jr, Shin, Y.K. RNA Journal 9, 1158-1166 (1999). The ectodomain of HA2 of influenza virus promotes rapid pH dependent membrane fusion. Epand, R.F., Macosko, J.C., Russell, C.J., Shin, Y.K., Epand, R.M., Journal of Molecular Biology 286, 489-503 (1999). The synaptic SNARE complex is a parallel four-stranded helical bundle. Poirier, M.A., Xiao, W., Macosko, J.C., Chan, C., Shin ,Y.K., Bennett, M.K., Nature Strucural. Biology 5, 765-769 (1998) The mechanism for low-pH-induced clustering of phospholipid vesicles carrying the HA2 ectodomain of influenza hemagglutinin. Kim, C.H., Macosko, J.C., & Shin, Y.-K. Biochemistry 37, 137-144 (1998) The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR. Macosko, J.C., Kim, C.H., Shin. Y.K. Journal of Molecular Biology 267, 1139-1148 (1997) On the dynamics and conformation of the HA2 domain of the influenza virus hemagglutinin. Kim, C.H., Macosko, Yu, Y.G., Shin, Y.K., Biochemistry 35, 5359- 5365 (1996).

 

Studies of vesicle transport by kinesin in living neurons Single-molecule fluorescence microscopy of actively transcribing T7 RNA polymerase Experiments to investigate gene expression and regulation at the single-molecule level Single-molecule fluorescence resonance energy transfer analysis of transcription initiation Integration of an atomic force microscope (AFM) with single molecule fluorescence microsopy Development of a centrifugal force scanning microscope based on CD/DVD technology -------------------------------------------------------------------------------- Studies of vesicle transport by kinesin in living neurons Our primary research project is an extension of Professor G. Holzwarth's work: the measurements of the drag force and mechanical work required for fast transport of vesicles and the relationship of this cellular task to the known limitations of motor proteins, especially kinesin. In buffer, against the force of an optical trap, the maximum of steady force which kinesin can exert is 6.5 pN. One ATP is hydrolyzed per 8 nm step, and each step takes 50 microseconds. About 100 such steps occur per second during processive movement. In a cell, the vesicle(load) is in cytoplasm, not buffer+trap, so the load on the motor is viscoelastic drag. The viscous part of this load differs by a factor of 10,000-100,000 from the viscous load in an optical trap, since the viscosity of water is .001 Pa*s and the zero-frequency viscosity of cytoplasm is roughly 50 Pa*s. Does kinesin develop the same force in these two environments? Our goal is to measure the forces and work required to move vesicles in live cells and to compare these to the limits established for kinesin in solution. One expects highly extended cells such as neurons to be particularly sensitive to the energy costs of vesicle transport. For this reason, we are measuring vesicle transport in differentiated PC12 cells, which are a good model system, for neurons, but easier to grow. -------------------------------------------------------------------------------- Single-molecule fluorescence microscopy of actively transcribing T7 RNA polymerase In the most basic T7 RNA polymerase (RNAP) experiment, a single fluorophore, covalently attached to GTP, will be incorporated at the 5' end of the RNA, thus marking the beginning of transcription (see figure). The signal from this incorporated fluorophore will persist until transcription terminates and the transcript diffuses away. By fitting histograms of the fluorescent persistence times, to kinetic models we can uncover essential information (average rate, processivity, abortive transcription percentage, etc.) regarding actively transcribing T7 RNAP. -------------------------------------------------------------------------------- Experiments to investigate gene expression and regulation at the single-molecule level One of the simplest known gene regulation systems is the autoregulation of lysozyme in T7 bacteriophage. T7 lysozyme is produced during a T7 infection to help lyse the bacterial cell wall in order to release the newly formed bacteriophage capsids. Additionally, T7 lysozyme autoregulates by inhibiting transcription by T7 RNAP. Using single-molecule fluorescence, we will observe the processive transcription rate of single T7 RNAP's in the presence of T7 lysozyme. Putative sequence dependence of the autoregulatory effect will also be examined. -------------------------------------------------------------------------------- Single-molecule fluorescence resonance energy transfer analysis of transcription initiation Single molecule FRET is a powerful tool to probe mechanical motion in protein machines. In the FRET experiment (see figure), donor-labeled GTP will associate with the template DNA at the first position of transcription. Meanwhile, a his-tag, genetically engineered at the N-terminus of recombinantly expressed T7 RNAP, will bind tightly to the acceptor fluorophore at the N-terminal end of each surface-immobilized polymerase. In this way fluorescent transfer between donor and acceptor will begin immediately upon transcription initiation. The fluorescent signal from the donor and the energy transfer to the acceptor will suddenly drop if RNA transcription is aborted. Since aborts are quite common, many of the data traces will last only a fraction of a second. These short traces will be useful in characterizing the transition from initiation to elongation. For example, in a single molecule study of the E. coli Rep helicase, researchers found that the FRET signal oscillated markedly when the helicase paused on the DNA. Accordingly, we will look for a characteristic FRET signal in the abortive data that will shed light on the failed transition to elongation. Longer traces will also be informative: their FRET signal intensity should abruptly decrease at the transition between initiation and elongation. We will correlate this abrupt change to the initiation and the elongation crystal structures and help clarify the mechanical motions of this important transition. -------------------------------------------------------------------------------- Integration of an atomic force microscope (AFM) with single molecule fluorescence microsopyIn collaboration with Professor M. Guthold (WFU, Physics) For AFM pulling experiments, we will "fish" for T7 RNAP using template DNA dangling from the AFM cantilever (see figure). Biotin-streptavidin links will fix T7 RNAP to the glass surface and also the template DNA to the cantilever. The AFM will be mounted over an inverted fluorescence microscope, and single-molecule fluorescence will be used to confirm the active transcription of T7 RNAP. Additionally, it is possible to reverse the orientation of the dangling template DNA and instead fish with a transcription-assisting pull rather than with an opposing force. -------------------------------------------------------------------------------- Development of a centrifugal force scanning microscope (CFSM) based on CD/DVD technology. A prototype CFSM will be further developed to facilitate single-molecule manipulation. A sealed gasket encloses the sample chamber, which will be mounted on a spinning compact disk (see figure). A ds-DNA library, containing important transcription initiation and termination sequences, will be spotted on the lower surface of the sample chamber using DNA microarray technology. Each ds-DNA, prepared with biotin at its distal end, will bind a strepavidin-coated fluorescent microsphere. The centrifugal force will push (when viewed in a rotating frame of reference) these microspheres radially and so cause the attached ds-DNA to stretch. The laser on a modified compact disk player will be used to track the bead displacement. The centrifugal force on each microsphere can be calculated based on their size, density and radial velocity. The centrifugal force plotted against microsphere displacement will provide information about the physical properties (persistence length, overstretching transition, etc) of different DNA sequences in the library. After analyzing the sequence dependence of the ds-DNA force-versus- displacement curves, the sample chamber will be filled with proteins that bind DNA: T7 RNAP binding its promoter sequences, for instance. In this example, the stretching properties of the promoter DNA will be examined; in the presence and absence of T7 RNAP (which has been shown to bend its promoter 80 degrees and unwind it by 8 base pairs). Furthermore, researchers have found that T7 RNAP responds differently to inhibition by T7 lysozyme depending on which type of promoter sequence is used to initiate transcription. CFM would be the ideal technique to quantify the interaction between T7 RNAP and all possible promoter sequences, in the presence or absence of T7 lysozyme.