Single-Molecule Imaging: Capturing Nanoscale Cellular Machines in Action

Readings

Week #	Topics	readings
1	Introduction to Single Molecule Imaging Techniques See the Lecture Summary	No Readings
2	Single Optical Traps See the Lecture Summary	Block, S.M., C.L. Asbury, J.W. Shaevitz, et al., 2003. Probing the Kinesin Reaction Cycle with a 2D Optical Force Clamp. Proceedings of the National Academy of Sciences 100 (5), 2351–2356. Sheinin, M., M. Li, M. Soltani, et al., 2013. Torque Modulates Nucleosome Stability and Facilitates H2A/H2B Dimer Loss. Nature Communications 4 (1), 2579.
3	Magnetic Tweezers See the Lecture Summary	Dulin, D., T. Cui, J. Cnossen, et al., 2015. High Spatiotemporal Resolution Magnetic Tweezers for Single-Molecule Force Spectroscopy: Calibration and Applications for DNA Dynamics. Biophysical Journal 109 (10), 2113–2125. van der Heijden, T., J. van Noort, H. van Leest, et al., 2005. Torque-Limited RecA Polymerization on dsDNA. Nucleic Acids Research 33 (7), 2099–2105.
4	Double Optical Traps See the Lecture Summary	Lisica, A., C. Engel, M. Jahnel, et al., 2016. Mechanisms of Backtrack Recovery by RNA Polymerases I and II. Proceedings of the National Academy of Sciences 113 (11), 2946–2951. Gardini, L., S.M. Heissler, C. Arbore, et al., 2018. Dissecting Myosin-5B Mechanosensitivity and Calcium Regulation at the Single Molecule Level. Nature Communications 9 (1), 2844–2844.
5	Total Internal Reflection Fluorescence Microscopy (TIRFM) See the Lecture Summary	Yardimci, H., A.B. Loveland, S. Habuchi, et al., 2010. Uncoupling of Sister Replisomes During Eukaryotic DNA Replication. Molecular Cell 40 (5), 834–840. Thawani, A., M.J. Rale, N. Coudray, et al., 2020. The Transition State and Regulation of γ-TuRC-Mediated Microtubule Nucleation Revealed by Single Molecule Microscopy. eLife 9, e54253.
6	Single-Molecule FRET (smFRET) See the Lecture Summary	Duchi, D., D.L.V. Bauer, L. Fernandez, et al., 2016. RNA Polymerase Pausing during Initial Transcription. Molecular Cell 63 (6), 939–950. Ticau, S., L.J. Friedman, N.A. Ivica, et al., 2015. Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading. Cell 161 (3), 513–525.
7	DNA Curtains and Extensions by Flow See the Lecture Summary	Kim, J.H., and R.G. Larson, 2007. Single-Molecule Analysis of 1D Diffusion and Transcription Elongation of T7 RNA Polymerase along Individual Stretched DNA Molecules. Nucleic Acids Research 35 (11) 3848–3858. Kong, M., E.E. Cutts, D. Pan, et al., 2020. Human Condensin I and II Drive Extensive ATP-Dependent Compaction of Nucleosome-Bound DNA. Molecular Cell 79 (1), 99–114.
8	Fleezers See the Lecture Summary	Hilario, J., I. Amitani, R.J. Baskin, et al., 2009. Direct Imaging of Human Rad51 Nucleoprotein Dynamics on Individual DNA Molecules. Proceedings of the National Academy of Sciences 106 (2), 361–368. van Mameren, J., M. Modesti, R. Kanaar, et al., 2009. Counting RAD51 Proteins Disassembling from Nucleoprotein Filaments under Tension. Nature 457 (7230), 745–748.
9	Fleezers 2.0: Dual traps combined with confocal microscopy See the Lecture Summary	Desai, V. P., F. Frank, A. Lee, et al., 2019. Co-temporal Force and Fluorescence Measurements Reveal a Ribosomal Gear Shift Mechanism of Translation Regulation by Structured mRNAs. Molecular Cell 75 (5), 1007–1019. Wasserman, M.R., G.D. Schauer, M.E. O’Donnell, et al., 2019. Replication Fork Activation is Enabled by a Single-Stranded DNA Gate in CMG Helicase. Cell 178 (3), 600–611.
10	Optical Traps Meet Both Confocal and FRET See the Lecture Summary	Ngo, T. T., Q. Zhang, R. Zhou, et al., 2015. Asymmetric Unwrapping of Nucleosomes under Tension Directed by DNA Local Flexibility. Cell 160 (6), 1135–1144. Suksombat, S., R. Khafizov, A.G. Kozlov, et al., 2015. Structural Dynamics of E. coli Single-Stranded DNA Binding Protein Reveal DNA Wrapping and Unwrapping Pathways. eLife 4, e08193.
11	STORM See the Lecture Summary	Xu, K., G. Zhong, and X. Zhuang, 2012. Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons. Science 339 (6118), 452–456. Bálint, Š., V.I. Vilanova, A.S. Alvarez, et al., 2013. Correlative Live-Cell and Superresolution Microscopy Reveals Cargo Transport Dynamics at Microtubule Intersections. Proceedings of the National Academy of Sciences 110 (9), 3375–3380.
12	PALM and STED See the Lecture Summary	Rossier, O., V. Octeau, J.B. Sibarita, et al., 2012. Integrins β1 and β3 Exhibit Distinct Dynamic Nanoscale Organizations inside Focal Adhesions. Nature Cell Biology 14 (10), 1057–1067. Hein, B. K.I. Willig, and S.W. Hell, 2008. Stimulated Emission Depletion (STED) Nanoscopy of a Fluorescent Protein-Labeled Organelle inside a Living Cell. Proceedings of the National Academy of Sciences 105 (38), 14271–14276.
13	SIM and SMLM See the Lecture Summary	Fiolka, R., L. Shao, E.H. Rego, et al., 2012. Time-Lapse Two-Color 3D Imaging of Live Cells with Doubled Resolution Using Structured Illumination. Proceedings of the National Academy of Sciences 109 (14), 5311–5315.
14	Student Presentations See the Lecture Summary	No Readings