Single filament behavior of microtubules in the presence of added divalent counterions.

Microtubules (MTs) are hollow biopolymeric filaments that function to define the shape of eukaryotic cells, serve as a platform for intracellular vesicular transport, and separate chromosomes during mitosis. One means of physiological regulation of MT mechanics and dynamics, critical to their adaptability in such processes, is through electrostatics due to the strong polyelectrolyte nature of MTs. Here, we show that in the presence of physiologically relevant amounts of divalent salts, MTs experience a dramatic increase in persistence length or stiffness, which is counter to theoretical expectations and experimental observations in similar systems (e.g., DNA). Divalent salt-dependent effects on MT dynamics were also observed with respect to suppressing depolymerization as well as reducing dispersion in kinesin-driven molecular motor transport assays. These results establish a novel mechanism by which MT dynamics, mechanics, and interaction with molecular motors may be regulated by physiologically relevant concentrations of divalent salts.

[1]  Arpita Mitra,et al.  Taxol allosterically alters the dynamics of the tubulin dimer and increases the flexibility of microtubules. , 2008, Biophysical journal.

[2]  M. Elowitz,et al.  Spiral defects in motility assays: A measure of motor protein force. , 1995, Physical review letters.

[3]  R. Williams,et al.  Taxol-induced flexibility of microtubules and its reversal by MAP-2 and Tau. , 1993, The Journal of biological chemistry.

[4]  J. Howard,et al.  Kinesin Takes One 8-nm Step for Each ATP That It Hydrolyzes* , 1999, The Journal of Biological Chemistry.

[5]  J. Tuszynski,et al.  The evolution of the structure of tubulin and its potential consequences for the role and function of microtubules in cells and embryos. , 2006, The International journal of developmental biology.

[6]  E. Muto,et al.  Dielectric measurement of individual microtubules using the electroorientation method. , 2006, Biophysical journal.

[7]  J. Skolnick,et al.  Electrostatic Persistence Length of a Wormlike Polyelectrolyte , 1977 .

[8]  Myung Chul Choi,et al.  Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules. , 2013, Faraday discussions.

[9]  E. Salmon,et al.  Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies , 1988, The Journal of cell biology.

[10]  H. Erickson,et al.  Polycation-induced assembly of purified tubulin. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Enrique M De La Cruz,et al.  Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness , 2012, Proceedings of the National Academy of Sciences.

[12]  A C Maggs,et al.  Analysis of microtubule rigidity using hydrodynamic flow and thermal fluctuations. , 1994, The Journal of biological chemistry.

[13]  M J Schilstra,et al.  The effect of solution composition on microtubule dynamic instability. , 1991, The Biochemical journal.

[14]  M. Yaffe,et al.  Microtubule assembly is dependent on a cluster of basic residues in alpha-tubulin. , 1986, Biochemistry.

[15]  A. Matus,et al.  Domains of Neuronal Microtubule-associated Proteins and Flexural Rigidity of Microtubules , 1997, The Journal of cell biology.

[16]  R. Lal,et al.  Microtubule-dependent Oligomerization of Tau , 2003, Journal of Biological Chemistry.

[17]  H. Larsson,et al.  Induction of a sheet polymer of tubulin by Zn2+. , 1976, Experimental cell research.

[18]  W. B. Derry,et al.  Substoichiometric binding of taxol suppresses microtubule dynamics. , 1995, Biochemistry.

[19]  Ronald D. Vale,et al.  Engineering the Processive Run Length of the Kinesin Motor , 2000, The Journal of cell biology.

[20]  N. Hirokawa,et al.  Kinesin and dynein superfamily proteins and the mechanism of organelle transport. , 1998, Science.

[21]  Tian Shen,et al.  Segmentation and tracking of cytoskeletal filaments using open active contours , 2010, Cytoskeleton.

[22]  Erwin Frey,et al.  Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length , 2005, Proceedings of the National Academy of Sciences.

[23]  Christoph F. Schmidt,et al.  Direct observation of kinesin stepping by optical trapping interferometry , 1993, Nature.

[24]  J. Tuszynski,et al.  Transitions in microtubule C-termini conformations as a possible dendritic signaling phenomenon , 2005, European Biophysics Journal.

[25]  B. Mickey,et al.  Rigidity of microtubules is increased by stabilizing agents , 1995, The Journal of cell biology.

[26]  J. Howard,et al.  Mechanics of Motor Proteins and the Cytoskeleton , 2001 .

[27]  Takahiro Nitta,et al.  Dispersion in active transport by kinesin-powered molecular shuttles. , 2005, Nano letters.

[28]  R. Maccioni,et al.  Controlled proteolysis of tubulin by subtilisin: localization of the site for MAP2 interaction. , 1984, Biochemistry.

[29]  D. Grier,et al.  Methods of Digital Video Microscopy for Colloidal Studies , 1996 .

[30]  R. Golestanian,et al.  Conformational instability of rodlike polyelectrolytes due to counterion fluctuations. , 2002, Physical review. E, Statistical, nonlinear, and soft matter physics.

[31]  J. Howard,et al.  Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape , 1993, The Journal of cell biology.

[32]  Yoshihito Osada,et al.  Ring-shaped assembly of microtubules shows preferential counterclockwise motion. , 2008, Biomacromolecules.

[33]  J. Andrew McCammon,et al.  Electrostatically Biased Binding of Kinesin to Microtubules , 2011, PLoS biology.

[34]  S. Halpain,et al.  MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments , 2002, The Journal of cell biology.

[35]  G. S. Manning Limiting laws and counterion condensation in polyelectrolyte solutions. IV. The approach to the limit and the extraordinary stability of the charge fraction. , 1977, Biophysical chemistry.

[36]  Kazuhiro Oiwa,et al.  Single‐molecule investigation of the interference between kinesin, tau and MAP2c , 2002, The EMBO journal.

[37]  Marileen Dogterom,et al.  A bending mode analysis for growing microtubules: evidence for a velocity-dependent rigidity. , 2004, Biophysical journal.

[38]  P. Cremer,et al.  Role of carboxylate side chains in the cation Hofmeister series. , 2012, The journal of physical chemistry. B.

[39]  Gerald S. Manning,et al.  Limiting Laws and Counterion Condensation in Polyelectrolyte Solutions I. Colligative Properties , 1969 .

[40]  Erik David Spoerke,et al.  Biomolecular Motor‐Powered Self‐Assembly of Dissipative Nanocomposite Rings , 2008 .

[41]  M. Schliwa,et al.  Flexural rigidity of microtubules measured with the use of optical tweezers. , 1996, Journal of cell science.

[42]  C. Dekker,et al.  Microtubule curvatures under perpendicular electric forces reveal a low persistence length , 2008, Proceedings of the National Academy of Sciences.

[43]  Holy,et al.  "Gliding assays" for motor proteins: A theoretical analysis. , 1995, Physical review letters.

[44]  Jack A. Tuszynski,et al.  Molecular dynamics simulations of tubulin structure and calculations of electrostatic properties of microtubules , 2005, Math. Comput. Model..

[45]  J. V. José,et al.  A dynamical model of kinesin-microtubule motility assays. , 2001, Biophysical journal.

[46]  T. Odijk Polyelectrolytes near the rod limit , 1977 .

[47]  A. Hudspeth,et al.  Movement of microtubules by single kinesin molecules , 1989, Nature.

[48]  Jay X. Tang,et al.  Counterion induced bundle formation of rodlike polyelectrolytes , 1996 .

[49]  Polyelectrolyte persistence length: Attractive effect of counterion correlations and fluctuations , 2001, cond-mat/0112337.

[50]  D. Babcock,et al.  Adenylyl imidodiphosphate, an adenosine triphosphate analog containing a P--N--P linkage. , 1971, Biochemistry.

[51]  H. Gaub,et al.  Elasticity of Single Polyelectrolyte Chains and Their Desorption from Solid Supports Studied by AFM Based Single Molecule Force Spectroscopy , 2001 .

[52]  Kenneth H. Downing,et al.  Structure of the αβ tubulin dimer by electron crystallography , 1998, Nature.

[53]  S. Smith,et al.  Ionic effects on the elasticity of single DNA molecules. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[54]  Nathan F. Bouxsein,et al.  Biomolecular motors in nanoscale materials, devices, and systems. , 2014, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.

[55]  H. Isambert,et al.  Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins , 1995, The Journal of Biological Chemistry.

[56]  E. Salmon,et al.  How tubulin subunits are lost from the shortening ends of microtubules. , 1997, Journal of structural biology.

[57]  D. Sackett,et al.  Proteolysis of tubulin and the substructure of the tubulin dimer. , 1986, The Journal of biological chemistry.

[58]  E. Nogales,et al.  High-Resolution Model of the Microtubule , 1999, Cell.

[59]  E. Meyhöfer,et al.  The E-hook of tubulin interacts with kinesin's head to increase processivity and speed. , 2005, Biophysical journal.

[60]  M. Deserno,et al.  Theory and simulations of rigid polyelectrolytes , 2002, cond-mat/0203599.

[61]  G. C. Rogers,et al.  Microtubule motors in mitosis , 2000, Nature.

[62]  R. Maccioni,et al.  Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Hideo Tashiro,et al.  Flexural rigidity of individual microtubules measured by a buckling force with optical traps. , 2006, Biophysical journal.

[64]  Henry Hess,et al.  Biomolecular motors at the intersection of nanotechnology and polymer science , 2010 .

[65]  Jay X. Tang,et al.  The Polyelectrolyte Nature of F-actin and the Mechanism of Actin Bundle Formation (*) , 1996, The Journal of Biological Chemistry.

[66]  D. Haar,et al.  Statistical Physics , 1971, Nature.

[67]  David A Weitz,et al.  Bending dynamics of fluctuating biopolymers probed by automated high-resolution filament tracking. , 2007, Biophysical journal.

[68]  R. W. Wilson,et al.  Counterion-induced condesation of deoxyribonucleic acid. a light-scattering study. , 1979, Biochemistry.

[69]  R. Lasek,et al.  Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP-PNP , 1985, Nature.

[70]  Persistence length of a strongly charged rodlike polyelectrolyte in the presence of salt. , 2002, Physical review. E, Statistical, nonlinear, and soft matter physics.

[71]  J. Wolff Promotion of microtubule assembly by oligocations: cooperativity between charged groups. , 1998, Biochemistry.

[72]  E. Mandelkow,et al.  Microtubules and microtubule-associated proteins. , 1995, Current opinion in cell biology.

[73]  D. Sackett,et al.  Cation selective promotion of tubulin polymerization by alkali metal chlorides , 1996, Protein science : a publication of the Protein Society.

[74]  E. Salmon,et al.  How calcium causes microtubule depolymerization. , 1997, Cell motility and the cytoskeleton.

[75]  C. Dekker,et al.  Electrophoresis of individual microtubules in microchannels , 2007, Proceedings of the National Academy of Sciences.

[76]  E. Nogales,et al.  Cryo-electron microscopy of GDP-tubulin rings , 2007, Cell Biochemistry and Biophysics.

[77]  B. S. Manjunath,et al.  Tau isoform‐specific modulation of kinesin‐driven microtubule gliding rates and trajectories as determined with tau‐stabilized microtubules , 2010, Cytoskeleton.

[78]  Viola Vogel,et al.  Molecular self-assembly of "nanowires"and "nanospools" using active transport. , 2005, Nano letters.

[79]  Jay X. Tang,et al.  Ion multivalence and like-charge polyelectrolyte attraction. , 2003, Physical review letters.

[80]  V. Bloomfield,et al.  Condensation of DNA by multivalent cations: Considerations on mechanism , 1991, Biopolymers.

[81]  D. Needleman,et al.  Higher-order assembly of microtubules by counterions: from hexagonal bundles to living necklaces. , 2004, Proceedings of the National Academy of Sciences of the United States of America.