Salt-dependent rheology and surface tension of protein condensates using optical traps

An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquid-like phases. These protein condensates serve as simple models for the investigation of the more complex membrane-less organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties.Comment: 5 pages, 3 figures, 1 supplemen

Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development.

Ribonucleoprotein condensates can exhibit diverse physical states in vitro and in vivo. Despite considerable progress, the relevance of condensate physical states for in vivo biological function remains limited. Here, we investigated the physical properties of processing bodies (P bodies) and their impact on mRNA storage in mature Drosophila oocytes. We show that the conserved DEAD-box RNA helicase Me31B forms viscous P body condensates, which adopt an arrested physical state. We demonstrate that structurally distinct proteins and protein-protein interactions, together with RNA, regulate the physical properties of P bodies. Using live imaging and in situ hybridization, we show that the arrested state and integrity of P bodies support the storage of bicoid (bcd) mRNA and that egg activation modulates P body properties, leading to the release of bcd for translation in the early embryo. Together, this work provides an example of how physical states of condensates regulate cellular function in development

Force Spectroscopy with Dual-Trap Optical Tweezers: Molecular Stiffness Measurements and Coupled Fluctuations Analysis

ABSTRACT Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any optical tweezers setup. In this article, we analyze, both from the theoretical and the experimental points of view, the most common source for these couplings in dual-trap optical-tweezers setups: the misalignment of traps and tether. We give criteria to distinguish different kinds of misalignment, to estimate their quantitative relevance and to include them in the data analysis. The experimental data is obtained in a, to our knowledge, novel dual-trap optical-tweezers setup that directly measures forces. In the case in which misalignment is negligible, we provide a method to measure the stiffness of traps and tether based on variance analysis. This method can be seen as a calibration technique valid beyond the linear trap region. Our analysis is then employed to measure the persistence length of dsDNA tethers of three different lengths spanning two orders of magnitude. The effective persistence length of such tethers is shown to decrease with the contour length, in accordance with previous studies

RNA buffers the phase separation behavior of prion-like RNA binding proteins

Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNAcritically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.1125sciescopu

Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

[EN] During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (~50 pN).We thank Stephan Grill laboratory (MPI-CBG, Dresden) for help with data collection and E. Galburt, M. Manosas and M. De Vega for critical reading of the manuscript. Spanish Ministry of Economy and Competitiveness [BFU2011-29038 to J.L.C., BFU2013-44202 to J.M.V., BFU2011-23645 to M.S., FIS2010-17440, GR35/10-A920GR35/10-A-911 to F.J.C., MAT2013-49455-EXP to J.R.A.-G. and BFU2012-31825 to B.I.]; Regional Government of Madrid [S2009/MAT 1507 to J.L.C. and CDS2007-0015 to M.S.]; European Molecular Biology Organization [ASTF 276-2012 to J.M.L.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2012-31825 to B.I.].Morin, J.; Cao, F.; Lázaro, J.; Arias-Gonzalez, JR.; Valpuesta, J.; Carrascosa, J.; Salas, M.... (2015). Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase. Nucleic Acids Research. 43(7):3643-3652. https://doi.org/10.1093/nar/gkv204S36433652437Steitz, T. A., & Steitz, J. A. (1993). A general two-metal-ion mechanism for catalytic RNA. Proceedings of the National Academy of Sciences, 90(14), 6498-6502. doi:10.1073/pnas.90.14.6498Nakamura, T., Zhao, Y., Yamagata, Y., Hua, Y., & Yang, W. (2012). Watching DNA polymerase η make a phosphodiester bond. Nature, 487(7406), 196-201. doi:10.1038/nature11181Kohlstaedt, L., Wang, J., Friedman, J., Rice, P., & Steitz, T. (1992). Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science, 256(5065), 1783-1790. doi:10.1126/science.1377403Steitz, T. A. (2006). Visualizing polynucleotide polymerase machines at work. The EMBO Journal, 25(15), 3458-3468. doi:10.1038/sj.emboj.7601211Zhang, H., Cao, W., Zakharova, E., Konigsberg, W., & De La Cruz, E. M. (2007). Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates. Nucleic Acids Research, 35(18), 6052-6062. doi:10.1093/nar/gkm587Wang, W., Wu, E. Y., Hellinga, H. W., & Beese, L. S. (2012). Structural Factors That Determine Selectivity of a High Fidelity DNA Polymerase for Deoxy-, Dideoxy-, and Ribonucleotides. Journal of Biological Chemistry, 287(34), 28215-28226. doi:10.1074/jbc.m112.366609Berezhna, S. Y., Gill, J. P., Lamichhane, R., & Millar, D. P. (2012). Single-Molecule Förster Resonance Energy Transfer Reveals an Innate Fidelity Checkpoint in DNA Polymerase I. Journal of the American Chemical Society, 134(27), 11261-11268. doi:10.1021/ja3038273Hariharan, C., Bloom, L. B., Helquist, S. A., Kool, E. T., & Reha-Krantz, L. J. (2006). Dynamics of Nucleotide Incorporation:  Snapshots Revealed by 2-Aminopurine Fluorescence Studies†. Biochemistry, 45(9), 2836-2844. doi:10.1021/bi051644sJoyce, C. M., Potapova, O., DeLucia, A. M., Huang, X., Basu, V. P., & Grindley, N. D. F. (2008). Fingers-Closing and Other Rapid Conformational Changes in DNA Polymerase I (Klenow Fragment) and Their Role in Nucleotide Selectivity†. Biochemistry, 47(23), 6103-6116. doi:10.1021/bi7021848Vande Berg, B. J., Beard, W. A., & Wilson, S. H. (2000). DNA Structure and Aspartate 276 Influence Nucleotide Binding to Human DNA Polymerase β. Journal of Biological Chemistry, 276(5), 3408-3416. doi:10.1074/jbc.m002884200Showalter, A. K., & Tsai, M.-D. (2002). A Reexamination of the Nucleotide Incorporation Fidelity of DNA Polymerases†. Biochemistry, 41(34), 10571-10576. doi:10.1021/bi026021iShah, A. M., Li, S.-X., Anderson, K. S., & Sweasy, J. B. (2001). Y265H Mutator Mutant of DNA Polymerase β. Journal of Biological Chemistry, 276(14), 10824-10831. doi:10.1074/jbc.m008680200Rothwell, P. J., Mitaksov, V., & Waksman, G. (2005). Motions of the Fingers Subdomain of Klentaq1 Are Fast and Not Rate Limiting: Implications for the Molecular Basis of Fidelity in DNA Polymerases. Molecular Cell, 19(3), 345-355. doi:10.1016/j.molcel.2005.06.032Patel, S. S., Wong, I., & Johnson, K. A. (1991). Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry, 30(2), 511-525. doi:10.1021/bi00216a029Luo, G., Wang, M., Konigsberg, W. H., & Xie, X. S. (2007). Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase. Proceedings of the National Academy of Sciences, 104(31), 12610-12615. doi:10.1073/pnas.0700920104Joyce, C. M., & Benkovic, S. J. (2004). DNA Polymerase Fidelity:  Kinetics, Structure, and Checkpoints†. Biochemistry, 43(45), 14317-14324. doi:10.1021/bi048422zFiala, K. A., & Suo, Z. (2004). Mechanism of DNA Polymerization Catalyzed bySulfolobus solfataricusP2 DNA Polymerase IV†. Biochemistry, 43(7), 2116-2125. doi:10.1021/bi035746zCramer, J., & Restle, T. (2005). Pre-steady-state Kinetic Characterization of the DinB Homologue DNA Polymerase ofSulfolobus solfataricus. Journal of Biological Chemistry, 280(49), 40552-40558. doi:10.1074/jbc.m504481200Choi, J.-Y., & Guengerich, F. P. (2005). Adduct Size Limits Efficient and Error-free Bypass Across Bulky N2-Guanine DNA Lesions by Human DNA Polymerase η. Journal of Molecular Biology, 352(1), 72-90. doi:10.1016/j.jmb.2005.06.079Olsen, T. J., Choi, Y., Sims, P. C., Gul, O. T., Corso, B. L., Dong, C., … Weiss, G. A. (2013). Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment). Journal of the American Chemical Society, 135(21), 7855-7860. doi:10.1021/ja311603rAllen, W. J., Rothwell, P. J., & Waksman, G. (2008). An intramolecular FRET system monitors fingers subdomain opening in Klentaq1. Protein Science, 17(3), 401-408. doi:10.1110/ps.073309208Johnson, S. J., & Beese, L. S. (2004). Structures of Mismatch Replication Errors Observed in a DNA Polymerase. Cell, 116(6), 803-816. doi:10.1016/s0092-8674(04)00252-1Yin, Y. W., & Steitz, T. A. (2004). The Structural Mechanism of Translocation and Helicase Activity in T7 RNA Polymerase. Cell, 116(3), 393-404. doi:10.1016/s0092-8674(04)00120-5Golosov, A. A., Warren, J. J., Beese, L. S., & Karplus, M. (2010). The Mechanism of the Translocation Step in DNA Replication by DNA Polymerase I: A Computer Simulation Analysis. Structure, 18(1), 83-93. doi:10.1016/j.str.2009.10.014Zhang, C., & Burton, Z. F. (2004). Transcription Factors IIF and IIS and Nucleoside Triphosphate Substrates as Dynamic Probes of the Human RNA Polymerase II Mechanism. Journal of Molecular Biology, 342(4), 1085-1099. doi:10.1016/j.jmb.2004.07.070Nedialkov, Y. A., Gong, X. Q., Hovde, S. L., Yamaguchi, Y., Handa, H., Geiger, J. H., … Burton, Z. F. (2003). NTP-driven Translocation by Human RNA Polymerase II. Journal of Biological Chemistry, 278(20), 18303-18312. doi:10.1074/jbc.m301103200Gong, X. Q., Zhang, C., Feig, M., & Burton, Z. F. (2005). Dynamic Error Correction and Regulation of Downstream Bubble Opening by Human RNA Polymerase II. Molecular Cell, 18(4), 461-470. doi:10.1016/j.molcel.2005.04.011Guajardo, R., & Sousa, R. (1997). A model for the mechanism of polymerase translocation 1 1Edited by A. R. Fersht. Journal of Molecular Biology, 265(1), 8-19. doi:10.1006/jmbi.1996.0707Thomen, P., Lopez, P. J., & Heslot, F. (2005). Unravelling the Mechanism of RNA-Polymerase Forward Motion by Using Mechanical Force. Physical Review Letters, 94(12). doi:10.1103/physrevlett.94.128102Larson, M. H., Zhou, J., Kaplan, C. D., Palangat, M., Kornberg, R. D., Landick, R., & Block, S. M. (2012). Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II. Proceedings of the National Academy of Sciences, 109(17), 6555-6560. doi:10.1073/pnas.1200939109Bar-Nahum, G., Epshtein, V., Ruckenstein, A. E., Rafikov, R., Mustaev, A., & Nudler, E. (2005). A Ratchet Mechanism of Transcription Elongation and Its Control. Cell, 120(2), 183-193. doi:10.1016/j.cell.2004.11.045Bai, L., Fulbright, R. M., & Wang, M. D. (2007). Mechanochemical Kinetics of Transcription Elongation. Physical Review Letters, 98(6). doi:10.1103/physrevlett.98.068103Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R., & Block, S. M. (2005). Direct observation of base-pair stepping by RNA polymerase. Nature, 438(7067), 460-465. doi:10.1038/nature04268Dangkulwanich, M., Ishibashi, T., Liu, S., Kireeva, M. L., Lubkowska, L., Kashlev, M., & Bustamante, C. J. (2013). Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. eLife, 2. doi:10.7554/elife.00971Lieberman, K. R., Dahl, J. M., Mai, A. H., Cox, A., Akeson, M., & Wang, H. (2013). Kinetic Mechanism of Translocation and dNTP Binding in Individual DNA Polymerase Complexes. Journal of the American Chemical Society, 135(24), 9149-9155. doi:10.1021/ja403640bLieberman, K. R., Dahl, J. M., Mai, A. H., Akeson, M., & Wang, H. (2012). Dynamics of the Translocation Step Measured in Individual DNA Polymerase Complexes. Journal of the American Chemical Society, 134(45), 18816-18823. doi:10.1021/ja3090302Dahl, J. M., Mai, A. H., Cherf, G. M., Jetha, N. N., Garalde, D. R., Marziali, A., … Lieberman, K. R. (2012). Direct Observation of Translocation in Individual DNA Polymerase Complexes. Journal of Biological Chemistry, 287(16), 13407-13421. doi:10.1074/jbc.m111.338418Rodriguez, I., Lazaro, J. M., Blanco, L., Kamtekar, S., Berman, A. J., Wang, J., … de Vega, M. (2005). A specific subdomain in  29 DNA polymerase confers both processivity and strand-displacement capacity. Proceedings of the National Academy of Sciences, 102(18), 6407-6412. doi:10.1073/pnas.0500597102Morin, J. A., Cao, F. J., Valpuesta, J. M., Carrascosa, J. L., Salas, M., & Ibarra, B. (2012). Manipulation of single polymerase-DNA complexes: A mechanical view of DNA unwinding during replication. Cell Cycle, 11(16), 2967-2968. doi:10.4161/cc.21389Morin, J. A., Cao, F. J., Lazaro, J. M., Arias-Gonzalez, J. R., Valpuesta, J. M., Carrascosa, J. L., … Ibarra, B. (2012). Active DNA unwinding dynamics during processive DNA replication. Proceedings of the National Academy of Sciences, 109(21), 8115-8120. doi:10.1073/pnas.1204759109Ibarra, B., Chemla, Y. R., Plyasunov, S., Smith, S. B., Lázaro, J. M., Salas, M., & Bustamante, C. (2009). Proofreading dynamics of a processive DNA polymerase. The EMBO Journal, 28(18), 2794-2802. doi:10.1038/emboj.2009.219Bustamante, C., Chemla, Y. R., Forde, N. R., & Izhaky, D. (2004). Mechanical Processes in Biochemistry. Annual Review of Biochemistry, 73(1), 705-748. doi:10.1146/annurev.biochem.72.121801.161542Jahnel, M., Behrndt, M., Jannasch, A., Schäffer, E., & Grill, S. W. (2011). Measuring the complete force field of an optical trap. Optics Letters, 36(7), 1260. doi:10.1364/ol.36.001260Soengas, M. S., Esteban, J. A., Lázaro, J. M., Bernad, A., Blasco, M. A., Salas, M., & Blanco, L. (1992). Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3′-5′ exonuclease and strand-displacement activities. The EMBO Journal, 11(11), 4227-4237. doi:10.1002/j.1460-2075.1992.tb05517.xSoengas, M. S., Gutiérrez, C., & Salas, M. (1995). Helix-destabilizing Activity of φ29 Single-stranded DNA Binding Protein: Effect on the Elongation Rate During Strand Displacement DNA Replication. Journal of Molecular Biology, 253(4), 517-529. doi:10.1006/jmbi.1995.0570De Vega, M., Lazaro, J. M., Salas, M., & Blanco, L. (1996). Primer-terminus stabilization at the 3′-5′ exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. The EMBO Journal, 15(5), 1182-1192. doi:10.1002/j.1460-2075.1996.tb00457.xVisscher, K., Schnitzer, M. J., & Block, S. M. (1999). Single kinesin molecules studied with a molecular force clamp. Nature, 400(6740), 184-189. doi:10.1038/22146Pandey, M., & Patel, S. S. (2014). Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps. Cell Reports, 6(6), 1129-1138. doi:10.1016/j.celrep.2014.02.025Truniger, V. (2002). A positively charged residue of phi29 DNA polymerase, highly conserved in DNA polymerases from families A and B, is involved in binding the incoming nucleotide. Nucleic Acids Research, 30(7), 1483-1492. doi:10.1093/nar/30.7.1483Berman, A. J., Kamtekar, S., Goodman, J. L., Lázaro, J. M., de Vega, M., Blanco, L., … Steitz, T. A. (2007). Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases. The EMBO Journal, 26(14), 3494-3505. doi:10.1038/sj.emboj.7601780Thomen, P., Lopez, P. J., Bockelmann, U., Guillerez, J., Dreyfus, M., & Heslot, F. (2008). T7 RNA Polymerase Studied by Force Measurements Varying Cofactor Concentration. Biophysical Journal, 95(5), 2423-2433. doi:10.1529/biophysj.107.125096Keller, D., & Bustamante, C. (2000). The Mechanochemistry of Molecular Motors. Biophysical Journal, 78(2), 541-556. doi:10.1016/s0006-3495(00)76615-xHerbert, K. M., Greenleaf, W. J., & Block, S. M. (2008). Single-Molecule Studies of RNA Polymerase: Motoring Along. Annual Review of Biochemistry, 77(1), 149-176. doi:10.1146/annurev.biochem.77.073106.100741Wong, I., Patel, S. S., & Johnson, K. A. (1991). An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics. Biochemistry, 30(2), 526-537. doi:10.1021/bi00216a030Lowe, L. G., & Guengerich, F. P. (1996). Steady-State and Pre-Steady-State Kinetic Analysis of dNTP Insertion Opposite 8-Oxo-7,8-dihydroguanine byEscherichia coliPolymerases I exo-and II exo- †. Biochemistry, 35(30), 9840-9849. doi:10.1021/bi960485xKirmizialtin, S., Nguyen, V., Johnson, K. A., & Elber, R. (2012). How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations. Structure, 20(4), 618-627. doi:10.1016/j.str.2012.02.018Donlin, M. J., Patel, S. S., & Johnson, K. A. (1991). Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction. Biochemistry, 30(2), 538-546. doi:10.1021/bi00216a031Li, Y. (1998). Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. The EMBO Journal, 17(24), 7514-7525. doi:10.1093/emboj/17.24.7514Lieberman, K. R., Dahl, J. M., & Wang, H. (2014). Kinetic Mechanism at the Branchpoint between the DNA Synthesis and Editing Pathways in Individual DNA Polymerase Complexes. Journal of the American Chemical Society, 136(19), 7117-7131. doi:10.1021/ja5026408Subuddhi, U., Hogg, M., & Reha-Krantz, L. J. (2008). Use of 2-Aminopurine Fluorescence To Study the Role of the β Hairpin in the Proofreading Pathway Catalyzed by the Phage T4 and RB69 DNA Polymerases†. Biochemistry, 47(23), 6130-6137. doi:10.1021/bi800211fShamoo, Y., & Steitz, T. A. (1999). Building a Replisome from Interacting Pieces. Cell, 99(2), 155-166. doi:10.1016/s0092-8674(00)81647-5Lamichhane, R., Berezhna, S. Y., Gill, J. P., Van der Schans, E., & Millar, D. P. (2013). Dynamics of Site Switching in DNA Polymerase. Journal of the American Chemical Society, 135(12), 4735-4742. doi:10.1021/ja311641bKamtekar, S., Berman, A. J., Wang, J., Lázaro, J. M., de Vega, M., Blanco, L., … Steitz, T. A. (2004). Insights into Strand Displacement and Processivity from the Crystal Structure of the Protein-Primed DNA Polymerase of Bacteriophage φ29. Molecular Cell, 16(4), 609-618. doi:10.1016/j.molcel.2004.10.019Hogg, M., Wallace, S. S., & Doublié, S. (2004). Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site. The EMBO Journal, 23(7), 1483-1493. doi:10.1038/sj.emboj.7600150Seifert, U. (2012). Stochastic thermodynamics, fluctuation theorems and molecular machines. Reports on Progress in Physics, 75(12), 126001. doi:10.1088/0034-4885/75/12/126001Cao, F. J., & Feito, M. (2009). Thermodynamics of feedback controlled systems. Physical Review E, 79(4). doi:10.1103/physreve.79.041118Cao, F. J., Dinis, L., & Parrondo, J. M. R. (2004). Feedback Control in a Collective Flashing Ratchet. Physical Review Letters, 93(4). doi:10.1103/physrevlett.93.040603Bier, M. (2007). The stepping motor protein as a feedback control ratchet. Biosystems, 88(3), 301-307. doi:10.1016/j.biosystems.2006.07.013Astumian, R. D. (1997). Thermodynamics and Kinetics of a Brownian Motor. Science, 276(5314), 917-922. doi:10.1126/science.276.5314.917Komissarova, N., & Kashlev, M. (1997). Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3’ end of the RNA intact and extruded. Proceedings of the National Academy of Sciences, 94(5), 1755-1760. doi:10.1073/pnas.94.5.1755Brueckner, F., & Cramer, P. (2008). Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation. Nature Structural & Molecular Biology, 15(8), 811-818. doi:10.1038/nsmb.145

Nanoparticle Diffusion Measures Bulk Clot Permeability

A clot's function is to achieve hemostasis by resisting fluid flow. Permeability is the measurement of a clot's hemostatic potential. It is sensitive to a wide range of biochemical parameters and pathologies. In this work, we consider the hydrodynamic phenomenon that reduces the mobility of fluid near the fiber surfaces. This no-slip boundary condition both defines the gel's permeability and suppresses nanoparticle diffusion in gel interstices. Here we report that, unlike previous work where steric effects also hindered diffusion, our system—nanoparticles in fibrin gel—was subject exclusively to hydrodynamic diffusion suppression. This result enabled an automated, high-throughput permeability assay that used small clot volumes. Permeability was derived from nanoparticle diffusion using the effective medium theory, and showed one-to-one correlation with measured permeability. This technique measured permeability without quantifying gel structure, and may therefore prove useful for characterizing similar materials (e.g., extracellular matrix) where structure is uncontrolled during polymerization and difficult to measure subsequently. We also report that PEGylation reduced, but did not eliminate, the population of immobile particles. We studied the forces required to pull stuck PEG particles free to confirm that the attachment is a result of neither covalent nor strong electrostatic binding, and discuss the relevance of this force scale to particle transport through physiological clots