Cellular mechanics

Coupling and Elastic Loading Affect the Active Response by the Inner Ear Hair Cell Bundles
Clark Elliott Strimbu, Lea Fredrickson-Hemsing, Dolores Bozovic
Active hair bundle motility has been proposed to underlie the amplification mechanism in the auditory endorgans of non-mammals and in the vestibular systems of all vertebrates, and to constitute a crucial component of cochlear amplification in mammals. We used semi-intact in vitro preparations of the bullfrog sacculus to study the effects of elastic mechanical loading on both natively coupled and freely oscillating hair bundles. For the latter, we attached glass fibers of different stiffness to the stereocilia and observed the induced changes in the spontaneous bundle movement. When driven with sinusoidal deflections, hair bundles displayed phase-locked response indicative of an Arnold Tongue, with the frequency selectivity highest at low amplitudes and decreasing under stronger stimulation. A striking broadening of the mode-locked response was seen with increasing stiffness of the load, until approximate impedance matching, where the phase-locked response remained flat over the physiological range of frequencies. When the otolithic membrane was left intact atop the preparation, the natural loading of the bundles likewise decreased their frequency selectivity with respect to that observed in freely oscillating bundles. To probe for signatures of the active process under natural loading and coupling conditions, we applied transient mechanical stimuli to the otolithic membrane. Following the pulses, the underlying bundles displayed active movement in the opposite direction, analogous to the twitches observed in individual cells. Tracking features in the otolithic membrane indicated that it moved in phase with the bundles. Hence, synchronous active motility evoked in the system of coupled hair bundles by external input is sufficient to displace large overlying structures.
JPEGs: Coupling and Elastic Loading Affect the Active Response.jpg (1.37 MB)
PDFs: journal.pone.0033862.pdf (11.78 MB)
Research categories: Cellular mechanics

Distribution of Frequencies of Spontaneous Oscillations in Hair Cells of the Bullfrog Sacculus
Under in vitro conditions, hair bundles of the amphibian inner ear show spontaneous oscillation. We used a high-speed camera to track these active movements, following multiple hair cells in a single field of view. Our techniques enabled us to acquire records on over 100 actively oscillating bundles per epithelium and show the oscillations to be mutually uncorrelated.
JPEGs: Distribution of Frequencies of Spontaneous Oscillations in Hair Cells.jpg (1.17 MB)
PDFs: Ramunno09.pdf (10.57 MB)
Research categories: Cellular mechanics, Nonequilibrium physics

Elasticity theory and shape transitions of viral shells
T. T. Nguyen, R. F. Bruinsma, and W. M. Gelbart, Elasticity theory and shape transitions of viral shells, Phys. Rev. E 72, 051923 (2005).
From decades of high-resolution crystallography and electron microsopy studies, viral capsids have been shown to exhibit a cross-over – upon increase in radius – from spherical to faceted icosahedral structures. Lidmar, Mirny and Nelson showed in 2003 that this “buckling” transition can be accounted for by continuum elasticity theory in terms of a competition between stretching and bending energies of the curved, closed, protein shells. Nguyen et al. generalize this approach by allowing for nonzero spontaneous curvature of the shell, and for nonicosahedral shapes. They find a continuous or weakly first-order transition from icosahedral to spherocylindrical symmetry near the onset of the buckling transition, driven by increase in the ratio of stretching to bending modulus, consistent with experimentally observed variations in the shapes of a variety of viral capsids.
PDFs: shape transitions.pdf (7.44 MB)
PNGs: Elasticity theory and shape transitions of viral shells.png (201.05 KB)
Research categories: Cellular mechanics

Bacteria Use Type IV Pili to Walk Upright and Detach from Surfaces
M. L. Gibiansky, J. C. Conrad, F. Jin, V. D. Gordon, D. A. Motto, M. A. Mathewson, W. G. Stopka, D. C. Zelasko, J. Shrout, G. C. L. Wong, “Bacteria use type IV pili to stand, walk upright, and detach from surfaces”, Science, 330, 197 (2010).
Maxsim L. Gibiansky, Jacinta C. Conrad, Fan Jin, Vernita D. Gordon, Dominick A. Motto, Margie A. Mathewson, Wiktor G. Stopka, Daria C. Zelasko, Joshua D. Shrout, Gerard C. L. Wong
Bacterial biofilms are structured multicellular communities involved in a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near surfaces is crucial for understanding the
transition between planktonic and biofilm phenotypes. By translating microscopy movies into searchable databases of bacterial behavior, we identified fundamental type IV pili–driven mechanisms for Pseudomonas aeruginosa surface motility involved in distinct foraging strategies. Bacteria stood upright and “walked” with trajectories optimized for two-dimensional surface exploration. Vertical orientation facilitated surface detachment and could influence biofilm morphology.
JPEGs: Gerard Wong group discovers walking bacteria.jpg (397.02 KB)
PDFs: Science Gibiansky Conrad Wong 2010.pdf (21.73 MB)
Research categories: Cellular mechanics, Tissues and Organisms, Nonequilibrium physics

Depiction of a localized retinal delamination
Chou and Siegel, The mechanics of retinal detachment, Submitted to: Physical Biology, (2012).
Chou, Siegel
We present a model of the mechanical and fluid forces associated with retinal detachments where the retinal photoreceptor cells separate from the underlying retinal pigment epithelium (RPE). We determine the conditions under which the subretinal fluid pressure exceeds the maximum yield stress holding the retina and RPE together, giving rise to an irreversible, extended retinal delamination. For detachments induced by traction forces, we find a critical radius beyond which the blister is unstable to growth. Growth of a detached blister can also be driven by inflamed tissue within which, for example, the hydraulic conductivities of the retina or choroid increase, the RPE pumps fail, or the adhesion properties change. We determine the parameter regimes in which the blister either becomes unstable to growth, remains stable and finite-sized, or shrinks, allowing possible healing. The corresponding stable blister radius and shape are calculated. Our analysis provides a quantitative description of the physical mechanisms involved in exudative retinal detachments and can help guide the development of retinal reattachment protocols or preventative procedures.
PNGs: Depiction of a localized retinal delamination.png (153.42 KB)
Cellular mechanics, Tissues and Organisms, Nonequilibrium physics

Bacteria use type IV pili to slingshot on surfaces
F. Jin, J. C. Conrad, M. L. Gibiansky, G. C. L. Wong, “Bacteria use type IV pili to slingshot on surfaces”, Proc. Nat. Acad. Sci. USA, 108 12617-12622 (2011).
Fan Jina, Jacinta C. Conrad, Maxsim L. Gibianskya, and Gerard C. L. Wong
Bacteria optimize the use of their motility appendages to move efficiently on a wide range of surfaces prior to forming multicellu- lar bacterial biofilms. The “twitching” motility mode employed by many bacterial species for surface exploration uses type-IV pili (TFP) as linear actuators to enable directional crawling. In addition to linear motion, however, motility requires turns and changes of direction. Moreover, the motility mechanism must be adaptable to the continually changing surface conditions encountered during biofilm formation. Here, we develop a novel two-point tracking algorithm to dissect twitching motility in this context. We show that TFP-mediated crawling in Pseudomonas aeruginosa consistently alternates between two distinct actions: a translation of constant velocity and a combined translation-rotation that is approximately 20× faster in instantaneous velocity. Orientational distributions of these actions suggest that the former is due to pulling by multiple TFP, whereas the latter is due to release by single TFP. The release action leads to a fast “slingshot” motion that can turn the cell body efficiently by oversteering. Furthermore, the large velocity of the slingshot motion enables bacteria to move efficiently through environments that contain shear-thinning vis- coelastic fluids, such as the extracellular polymeric substances (EPS) that bacteria secrete on surfaces during biofilm formation.
JPEGs: Wong group discovers ‘slingshoting’ bacteria.jpg (363.20 KB)
PDFs: PNAS Jin Wong 2011.pdf (7.95 MB)
Research categories: Cellular mechanics, Tissues and Organisms, Nonequilibrium physics

Morphological Phase Diagram for Lipid Membrane Domains with Entropic Tension
Rim, J.E., Ursell, T.S., Phillips, R., and Klug, W.S., “Morphological Phase Diagram for Lipid Mem- brane Domains with Entropic Tension”, Phys. Rev. Lett., 106(5):057801 (2011)
Circular domains in phase-separated lipid vesicles with symmetric leaflet composition commonly exhibit three stable morphologies: flat, dimpled, and budded. However, stable dimples (i.e., partially budded domains) present a puzzle since simple elastic theories of domain shape predict that only flat and spherical budded domains are mechanically stable in the absence of spontaneous curvature. We argue that this inconsistency arises from the failure of the constant surface tension ensemble to properly account for the effect of entropic bending fluctuations. Formulating membrane elasticity within an entropic tension ensemble, wherein tension represents the free energy cost of extracting membrane area from thermal bending of the membrane, we calculate a morphological phase diagram that contains regions of mechanical stability for each of the flat, dimpled, and budded domain morphologies.
PDFs: Rim-PRL-2011.pdf (4.59 MB)
PNGs: Morphological Phase Diagram for Lipid Mem- brane Domains with Entropic Tension.png (984.66 KB)
Research categories: Cellular mechanics, Soft and fragile matter

The Mechanics and Affine-Nonaffine Transition in Polydisperse Semi-flexible Networks
Bai, M., Missel, A.R., Klug, W.S., and Levine, A.J., “The Mechanics and Affine-Nonaffine Transition in Polydisperse Semi-flexible Networks”, Soft Matter, 7(3):907–914 (2011)
Semiflexible gels are composed of a crosslinked network of filaments that can support both bending and extensional forces. We study numerically the mechanical effect of adding a low density of highly incompliant semiflexible filaments to a random network of softer semiflexible filaments. Such heterogeneous networks form simple models of the mechanics of cytoskeletal networks composed primarily of F-actin but containing a low density of significantly stiffer microtubules. Networks composed solely of these two filament types were recently studied in the in vitro experiments of Lin et al. Here we determine the effect of the stiffer impurity filaments generally on the collective mechanics of the heterogeneous filament network and, more specifically, on the affine to non-affine (A/NA) crossover in the softer filament matrix, which occurs in semiflexible networks as a function of their network density. We show that the addition of a small fraction of longer and stiffer filaments to a nonaffine network leads to a significant increase in its collective elastic moduli, even though the stiff filaments do not themselves form a stress bearing network. We also determine the relationship between the density of the stiff filaments and the geometric measure of nonaffinity for the network. Here the effect of the stiffer impurity filaments is complex: their addition makes affine networks slightly more affine, but highly nonaffine networks even more nonaffine. Moreover, there is a strong negative spatial correlation between density of the stiff filaments and local geometric measure of nonaffinity. Taken together, these two observations show that the stiffer filaments serve to locally suppress nonaffine deformation but redistribute it to regions of the network where the stiffer filaments are sparse.
PDFs: Bai-SoftMatter-2011.pdf (4.67 MB)
PNGs: The Mechanics and Affine-Nonaffine Transition in Polydisperse Semi-flexible Networks.png (7.64 MB)
Research categories: Cellular mechanics, Soft and fragile matter

Affine to Nonaffine Transition in Networks of Nematically Ordered Semiflexible Polymers
Missel A.R., Bai M., Klug W.S., and Levine A.J., “Affine to Nonaffine Transition in Networks of Nematically Ordered Semiflexible Polymers”, Phys. Rev. E, 82:041907 (2010)
We study the mechanics of nematically ordered semiflexible networks showing that they, like isotropic networks, undergo an affine to nonaffine crossover controlled by the ratio of the filament length to the nonaffinity length. Deep in the nonaffine regime, however, these anisotropic networks exhibit a much more complex mechanical response characterized by a vanishing linear-response regime for highly ordered networks and a dependence of the shear modulus on shear direction at both small ͑linear͒ and finite ͑nonlinear͒ strains that is different from the affine prediction of orthotropic continuum linear elasticity. We show that these features can be understood in terms of a generalized floppy modes analysis of the nonaffine mechanics and a type of cooperative Euler buckling.
PDFs: Missel-PRE-2010.pdf (4.17 MB)
PNGs: Cooperative Euler buckling.png (2.86 MB)
Research categories: Cellular mechanics, Soft and fragile matter

On the Role of the Filament Length Distribution in the Mechanics of Semiflexible Networks
Bai, M., Missel, A.R., Levine, A.J., and Klug, W.S.,“On the Role of the Filament Length Distribution in the Mechanics of Semiflexible Networks”, Acta Biomaterialia, 7(5):2109–2118 (2011)
This paper explores the effects of filament length polydispersity on the mechanical properties of semiflexible crosslinked polymer networks. Extending previous studies on monodisperse networks, we compute numerically the response of crosslinked networks of elastic filaments of bimodal and exponential length distributions. These polydisperse networks are subject to the same affine to nonaffine (A/NA) transition observed previously for monodisperse networks, wherein the decreases in either crosslink density or bending stiffness lead to a shift from affine, stretching-dominated deformations to nonaffine, bendingdominated deformations. We find that the onset of this transition is generally more sensitive to changes in the density of longer filaments than shorter filaments, meaning that longer filaments have greater mechanical efficiency. Moreover, in polydisperse networks, mixtures of long and short filaments interact cooperatively to generally produce a nonaffine mechanical response closer to the affine prediction than comparable monodisperse networks of either long or short filaments. Accordingly, the mechanical affinity of polydisperse networks is dependent on the filament length composition. Overall, length polydispersity has the effect of sharpening and shifting the A/NA transition to lower network densities. We discuss the implications of these results on experimental observation of the A/NA transition, and on the design of advanced materials.
PDFs: On the role.pdf
PNGs: Filament Length Distribution in the Mechanics of Semiflexible Networks.png (13.04 MB)
Research categories: Cellular mechanics, Soft and fragile matter

The physics of retinal detachments
Tom Chou, Michael Siegel
We develop mathematical model describing the mechanical and fluid forces associated with ex- udative retinal detachments. We assume that the retina adheres to the underlying retinal pigment epitelium (RPE) cells layer via an attractive interaction potential that can be irreversibly destroyed. By computing the total water flow arising from transretinal, vascular, and retinal pigment epithe- lium (RPE) pump currents, we determine the conditions under which the subretinal fluid pressure exceeds the maximum yield stress holding the retina and RPE together, giving rise to an extended retinal delamination. We also investigate localized, blister-like retinal detachments by balancing mechanical tension in the retina with both the chorioretinal adhesion energy and the pressure jump across the retina. For detachments formed by traction, we find a critical radius beyond which the blister is unstable to unbounded growth. On the other hand, if growth of the detached blister is further driven by inflamed choroidal tissue (in which e.g., the RPE pumps do not function), we find in certain cases the blister size depends simply on two parameters, the normal-tissue, dimensionless RPE pump flux, and a dimensionless combination comprising the retinal stretching elasticity, the retina-RPE adhesion energy, and the area of the inflamed lesion. We find parameter regimes which lead to either a finite or infinite blister radii, and to the corresponding blister shape. Our model provides a mathematical description of the physical mechanisms involved in exudative retinal de- tachments and macular edema and can guide further development of retinal reattachment protocols or preventative procedures.
JPEGs: csr-oct.jpg (3.01 MB)

Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
Timothee Lionnet, Michelle M. Spiering, Stephen J. Benkovic, David Bensimon, Vincent Croquette
Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (400 bp/s), its unwinding rate extrapolated to zero force is much slower (30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.
JPEGs: Real-time observation of bacteriophage T4 gp41.jpg (390.16 KB)
PDFs: Real-time observation of bacteriophage T4 gp41.pdf (8.38 MB)
Research categories: Biological Macromolecules, Cellular mechanics, Nonequilibrium physics, Cellular mechanics, Tissues and Organisms, Nonequilibrium physics, Soft and fragile matter

Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers
Francesco Mosconi, Jean Francois Allemand, David Bensimon, and Vincent Croquette
We deduced the torque applied on a single stretched and twisted DNA by integrating the change in the molecule’s extension with respect to force as it is coiled. While consistent with previous direct measurements of the torque at high forces (F > 1 pN), this method, which is simple and does not require a sophisticated setup, allows for lower force estimates. We used this approach to deduce the effective torsional modulus of DNA, which decreases with force, and to estimate the buckling torque of DNA as a function of force in various salt conditions.
JPEGs: Torque on a Single Stretched and Twisted DNA.jpg (527.58 KB)
PDFs: Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers.pdf (8.96 MB)
Research categories: Biological Macromolecules, Cellular mechanics, Experimental probes

Collaborative coupling between polymerase and helicase for leading-strand synthesis
Maria Manosas, Michelle M. Spiering, Fangyuan Ding, Vincent Croquette and Stephen J. Benkovic
Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions.
JPEGs: Collaborative coupling.jpg (1.06 MB)
PDFs: Collaborative coupling.pdf (7.69 MB)
Research categories: Biological Macromolecules, Cellular mechanics, Experimental probes

Criterion for Amino Acid Composition of Defensins and Antimicrobial Peptides Based on Geometry of Membrane Destabilization
N. W. Schmidt, A. Mishra, G. H. Lai, M. Davis, L. K. Sanders, D. Tran, A. Garcia, K. P. Tai, P. B. McCray Jr., A. J. Ouellette, M. E. Selsted, G. C. L. Wong, “Criterion for amino acid composition of defensins and antimicrobial peptides based on geometry
Nathan W. Schmidt, Abhijit Mishra, Ghee Hwee Lai, Matthew Davis, Lori K. Sanders, Dat Tran, Angie Garcia, Kenneth P. Tai, Paul B. McCray, Jr., Andre J. Ouellette, Michael E. Selsted, Gerard C. L. Wong
Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small-angle X-ray scattering to investigate
interactions between heterogeneous membranes and members of the defensin subfamilies, R-defensins (Crp-4), β-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay (“negative Gaussian”) membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, because it is a necessary condition for processes such as pore formation, blebbing, budding, and vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This “design rule” is consistent with the amino acid compositions of 1080 known cationic AMPs.
JPEGs: Wong group discovers selection rule for antimicrobial peptide sequences.jpg (126.45 KB)
PDFs: JACS Schmidt Wong 2011.pdf (23.51 MB)
Research categories: Biological Macromolecules, Cellular mechanics

Translocation of TAT peptide and analogs induced by multiplexed membrane and cytoskeletal interactions
A. Mishra, G. H. Lai, N. W. Schmidt, V. Z. Sun, A. Rodriguez, R. Tong, L. Tang, J. J. Cheng, T. J. Deming, D. T. Kamei, G. C. L. Wong, “Translocation of TAT peptide and analogs induced by multiplexed membrane and cytoskeletal interactions”, Proc. Nat.
Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using “nunchuck” CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay (“negative Gaussian”) membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.
JPEGs: Wong group finds molecular mechanisms for cell penetrating peptides.jpg (700.20 KB)
PDFs: PNAS Zasloff Mishra Wong 2011.pdf (6.61 MB)
Research categories: Biological Macromolecules, Cellular mechanics

Viral Capsid Equilibrium Dynamics Reveals Nonuniform Elastic Properties
May, E.R., Aggarwal, A., Klug, W.S., and Brooks III, C.L., “Viral Capsid Equilibrium Dynamics Reveals Nonuniform Elastic Properties”, Biophysical Journal, 100(11):L59–L61 (2011)
The long wavelength, low-frequency modes of motion are the relevant motions for understanding the continuummechanical properties of biomolecules. By examining these low-frequency modes, in the context of a spherical harmonic basisset, we identify four elastic moduli that are required to describe the two-dimensional elastic behavior of capsids. This is incontrast to previous modeling and theoretical studies on elastic shells, which use only the two-dimensional Young’s modulus(Y) and the bending modulus (k) to describe the system. Presumably, the heterogeneity of the structure and the anisotropyof the biomolecular interactions lead to a deviation from the homogeneous, isotropic, linear elastic shell theory. We assign func-tional relevance of the various moduli governing different deformation modes, including a mode primarily sensed in atomic forcemicroscopy nanoindentation experiments. We have performed our analysis on the T 1⁄4 3 cowpea chlorotic mottle virus and ourestimate for the nanoindentation modulus is in accord with experimental measurements.
PDFs: May-BPJ-2011.pdf (10.53 MB)
PNGs: harmonic spectra of the protein shells of viruses.png (3.18 MB)
Research categories: Viruses, Cellular mechanics