Resume

• 2005

  Yale University

  Northern Arizona University

  Flagstaff

  Arizona

  Lecturer

  Northern Arizona University

  Train to do independent scientific research in biological and biomedical science and publish primary research articles.

  Yale University

  W. L. Gore & Associates

  Flagstaff

  Arizona Area

  Research Scientist

  Institute for Engineering in Medicine Dynamic Cell Biomarkers Group 2013-\nBiophysical Society Early Careers Committee Member\t2014-\nMinnesota Academy of Science

  Winchell Undergraduate Research Judge\t2013\nInstitute of Physics Physical Biology Referee\t2012-

  Postdoctoral Associate

  Greater Minneapolis-St. Paul Area

  University of Minnesota

  Ph.D.

  Molecular Biophysics and Biochemistry

  McDougal Center Graduate Student Fellow

  Yale University

• 2000

  BS

  Physics

  Chemistry

  UW College of Arts and Sciences Student Advisory Board (2004)\nUW Putnam Team Member (2004)\nAmerican Chemical Society UW Student Chapter: VP

  President (2003-2004)\n\tSociety of Physics Students UW Chapter: VP

  President (2002-2003)

  University of Wyoming

• Digital Image Processing

  Research

  Fluorescence Spectroscopy

  Mechanics

  Protein Chemistry

  Science

  Biophysics

  Protein Expression

  Quantitative Research

  Microscopy

  Biochemistry

  PCR

  Scientific

  Molecular Biology

  Biological Physics

  Fluorescence Microscopy

  Protein Engineering

  Quantitative Models

  Protein Purification

  Molecular Cloning

  Multi-Platform Compatible Software for Analysis of Polymer Bending Mechanics.

  Austin ElamCau

  Hyeran KangAustin

  Cytoskeletal polymers play a fundamental role in the responses of cells to both external and internal stresses. Quantitative knowledge of the mechanical properties of those polymers is essential for developing predictive models of cell mechanics and mechano-sensing. Linear cytoskeletal polymers

  such as actin filaments and microtubules

  can grow to cellular length scales at which they behave as semiflexible polymers that undergo thermally-driven shape deformations. Bending deformations are often modeled using the wormlike chain model. A quantitative metric of a polymer's resistance to bending is the persistence length

  the fundamental parameter of that model. A polymer's bending persistence length is extracted from its shape as visualized using various imaging techniques. However

  the analysis methodologies required for determining the persistence length are often not readily within reach of most biological researchers or educators. Motivated by that limitation

  we developed user-friendly

  multi-platform compatible software to determine the bending persistence length from images of surface-adsorbed or freely fluctuating polymers. Three different types of analysis are available (cosine correlation

  end-to-end and bending-mode analyses)

  allowing for rigorous cross-checking of analysis results. The software is freely available and we provide sample data of adsorbed and fluctuating filaments and expected analysis results for educational and tutorial purposes.

  Multi-Platform Compatible Software for Analysis of Polymer Bending Mechanics.

  Jean-Louis Martiel

  Laurent Blanchoin

  We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven

  two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 microm

  corresponding to a flexural rigidity of 0.040 pN microm(2). Cofilin binding lowers the persistence length approximately 5-fold to a value of 2.2 microm and the filament flexural rigidity to 0.0091 pN microm(2). That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics

  together with our previous analysis of torsional stiffness

  provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding

  and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity.

  Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics.

  Laurent Blanchoin

  Enrique M. De La Cruz

  Martiel JL

  Guérin C

  Reymann AC

  Boujemaa-Paterski R

  Roland J

  Cristian Suarez

  Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs)

  many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin

  one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro

  binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments

  as well as cofilin severing

  limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide

  direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits

  which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments

  thereby controlling the overall filament length

  depending on cofilin binding density.

  Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries.

  Laurent Blanchoin

  Emil Reisler

  Jean-Louis Martiel

  Cristian Suarez

  Christine K Chen

  The actin regulatory protein

  cofilin

  increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries

  thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here

  we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility

  and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments

  analogous to failure of some nonprotein materials.

  Cofilin-linked changes in actin filament flexibility promote severing.

  Enrique M. De La Cruz

  Emil Reisler

  Elena E. Grintsevich

  Anaëlle Pierre

  The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology

  including intracellular transport

  cell motility

  cell division

  determining cellular shape

  and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However

  the molecular origins of these salt-dependent effects

  particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions

  are unknown. Here

  we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics

  respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.

  Identification of cation binding sites on actin that drive polymerization and modulate bending stiffness

  David Thomas

  Anaelle Pierre

  Ewa Prochniewicz

  The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin (CaM) light chains. We have used transient phosphorescence anisotropy to monitor the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin with strongly bound myosin VI (MVI) and to evaluate the effect of MVI-bound CaM light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca(2+)- and CaM-dependent manner

  as indicated from an increase in the final anisotropy and a decrease in the correlation time of transient phosphorescence anisotropy decays. MVI with bound apo-CaM or Ca(2+)-CaM weakly affects actin filament microsecond dynamics

  relative to other myosins (e.g.

  muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e.

  increases the torsional rigidity)

  such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca(2+)-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin binding site(s)

  which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca(2)(+)- and CaM-dependent regulation of myosin VI motility and ATP utilization.

  Actin filament dynamics in the actomyosin-VI complex is regulated allosterically by calcium-calmodulin light chain

  Jean-Louis Martiel

  Laurent Blanchoin

  Jeremy Roland

  Actin filaments are semiflexible polymers that display large-scale conformational twisting and bending motions. Modulation of filament bending and twisting dynamics has been linked to regulatory actin-binding protein function

  filament assembly and fragmentation

  and overall cell motility. The relationship between actin filament bending and twisting dynamics has not been evaluated. The numerical and analytical experiments presented here reveal that actin filaments have a strong intrinsic twist-bend coupling that obligates the reciprocal interconversion of bending energy and twisting stress. We developed a mesoscopic model of actin filaments that captures key documented features

  including the subunit dimensions

  interaction energies

  helicity

  and geometrical constraints coming from the double-stranded structure. The filament bending and torsional rigidities predicted by the model are comparable to experimental values

  demonstrating the capacity of the model to assess the mechanical properties of actin filaments

  including the coupling between twisting and bending motions. The predicted actin filament twist-bend coupling is strong

  with a persistence length of 0.15-0.4 μm depending on the actin-bound nucleotide. Twist-bend coupling is an emergent property that introduces local asymmetry to actin filaments and contributes to their overall elasticity. Up to 60% of the filament subunit elastic free energy originates from twist-bend coupling

  with the largest contributions resulting under relatively small deformations. A comparison of filaments with different architectures indicates that twist-bend coupling in actin filaments originates from their double protofilament and helical structure.

  Origin of twist-bend coupling in actin filaments.

  Central to cell motility is the actin filament severing activity of cofilin

  which is essential to the viability of cell. However

  it is unknown how cofilin severs an actin filament. I determined that cofilin decreases the elastic modulus of actin filaments

  which makes them more flexible. Based on these results

  I proposed that cofilin severs actin filaments when partially decorated by accumulating stress from thermally-driven shape fluctuations at bare (rigid) and cofilin-decorated (compliant) boundaries. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. I tested this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale correlate with an increase in the flexibility of actin filaments by cofilin. Indeed

  severing is attenuated when cofilin binds but does not increase actin filament flexibility. I also revealed that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully-decorated filaments. These results support a mechanism for actin filament severing by cofilin in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments

  analogous to failure of some non-protein materials.

  Brannon

  McCullough

  Ph.D.

  W. L. Gore & Associates

  University of Minnesota