Session P9 - Subcellular Dynamics.
ORAL session, Wednesday morning, March 05
Room 4ABC, Austin Convention Center

[P9.001] E. coli's division decision: modeling Min-protein oscillations I

E. coli is a rod-shaped bacterium that grows and divides into two equivalent daughter cells. One mechanism that regulates the central placement of the division site is the Min-protein system, which prevents division near the cell ends. A surprising discovery in recent years is that the Min system is an oscillator involving wholesale shifts of proteins from one end of the cell to the other. We present a complete model of the Min system, using only known properties of the proteins, which accurately reproduces the observed oscillations. The oscillations are driven by hydrolysis of ATP: (1) MinD binds ATP and the complex binds to the membrane; (2) MinE binds to the MinD:ATP, induces ATP hydrolysis, and all constituents are released from the membrane; (3) MinD:ADP releases ADP, completing the cycle.

[P9.002] E. coli's division decision: modeling Min-protein oscillations II

We present results of our model of the Min-protein oscillations in E. coli. The model correctly reproduces the central experimental observations: (1) In each oscillation, the protein MinD accumulates in the cell membrane in a ``polar zone'' at the end of the cell. This polar zone then shrinks toward the end of the cell, as a new accumulation forms at the opposite pole; (2) the protein MinE forms a ring at the boundary of the MinD endcap; and (3) the oscillation period is proportional to the ratio of the total amounts of MinD and MinE in the cell. Our model explains why MinD accumulates at the poles of the cell without special targets at the cell ends and predicts the formation of a MinE ring without any MinE-MinE interaction. Other successes of the model are the doubling of the spatial oscillation pattern in long cells (>10 microns), and the dramatic increase of the oscillation period and elimination of the MinE ring in a MinE mutant strain. Finally, we propose an answer to the fundamental question of why E. coli needs an oscillator.

[P9.003] Diffusion-limited reactions on the cell surface

Fibroblast growth factors (FGF) stimulates proliferation of many cell types, and are crucial in such processes as eg. wound healing. Cells have specific receptor (R) protein molecules on their surface which bind FGF for this purpose. FGF is also bound by Heparan Sulfate Proteoglycan (HSPG) molecules which are present on the cell surface. In isolation, both these complexes are unstable, with half-life of the order of 10-20 minutes, wheras in intact cells, the half-life of FGF-R complex is nearly 5 hours! To account for this increased stability, it has been proposed that R-FGF complex combines with HSPG via surface diffusion and forms the triad R-FGF-HSPG. We examine the feasibility of this reaction using the well-known Smoluchowski theory and Monte Carlo simulations. Our results support the triad formation theory, and are in qualitative agreement with experimental results. We also discuss the effects of slowing down of surface diffusion of these molecules by such factors as eg. the cytosekeletal network and anchored proteins.

[P9.004] Minimal Assumptions Comprehensive Electrostatic Model for Mitotic Motions

Primitive biological cells had to divide using very few biological mechanisms. This work proposes physicochemical mechanisms based on nanoscale electrostatics which explain and unify the basic motions during mitosis: (1) assembly of the asters, (2) motion of asters to poles, (3) chromosome attachment, (4) separation of sister chromatids, (5) prometaphase monovalent attachment motions, (6) chromosome congression to the cell equator, (7) metaphase oscillations, and (8) anaphase A poleward chromosome motion. In the cytosol of cells, electrostatic fields are subject to strong attenuation by ionic screening. However, the presence of microtubules within cells changes the situation completely. Microtubule dimer subunits are electric dipolar structures, and can act as intermediaries which extend the reach of the electrostatic interaction over cellular distances. Experimental studies have shown that intracellular pH rises to a peak at mitosis, and decreases through cytokinesis. This result, in conjunction with the electric dipole nature of microtubule subunits is sufficient to explain the dynamics of the above events and motions, including their timing and sequencing. The physicochemical methods utilized by primitive eukaryotic cells could provide important clues regarding our understanding of cell division in modern eukaryotic cells.

[P9.005] Microtubule self-organized spindle formation induced by motor-proteins

Recent experiments in vitro (Heald et al. Nature, Vol 382, 420 (1996)) have shown that the formation of the mitotic spindle during cell division does not depend on the presence of chromosomes and centrosomes, but that the bipolar spindle can form around DNA-coated beads. It was suggested there that motor proteins (MP) induce a self-organized spindle formation. We have shown that a simple non-equilirium model that uses only (+) and (-) ended MP can produce spindle structures. We discuss the different types of structures that can self-organize as a function of the ratios of the two types of MP to MT. We suggest further experiments that can test the validity of the model, and thereby, the hypothesis of motor induced spindle formation.

[P9.006] Chromatin motion and nucleosome dynamics in live cells and isolated nuclei observed using two-photon standing wave photobleaching

DNA in eukaryotic cells is wrapped around histone proteins to form chromatin fibers. Fluctuations in the local structure of these chromatin fibers are believed to control the transcriptional availability of DNA. Such fluctuations also result in the constrained diffusive motion of the chromatin in live cells observed using high resolution fluorescence microscopy. Using a two-photon standing wave fluorescence photobleaching method, we have measured the short-range (100 nm) diffusive motion of labeled DNA both in vivo and in vitro. The motion observed in living cells is absent from isolated nuclei. By varying the ionic strength of nuclear environment, we can modify the binding of the core histones to the DNA. We can also use chemical or photochemical reactions to irreversibly crosslink the histones to the DNA. Both experiments provide evidence that chromatin diffusion is controlled by the chemistry of the histone interactions, as opposed to thermal fluctuations or polymer entanglement.

[P9.007] STRUCTURE AND MECHANICS OF ACTIN CORTEX CONTAINED IN VESICLES

We designed giant phospholipid vesicles containing actin filaments as an elementary mechanical cell model. G-actin is polymerized inside the vesicles through ionophore-mediated Mg++ entry and the filaments are bound electrostatically to the membrane through lipids with amino-polyethyleneglycol (PEG) headgroups forming a shell beneath the membrane. The density of this cortex is varied by changing the initial actin concentration. A magnetic micrometric bead attached on the top of a sedimented vesicle is pulled vertically while horizontal and vertical displacements of the bead are simulatenously tracked by microscopy. Linear response allows to determine the bending and shear moduli of the actin-membrane complexe.

[P9.008] The Role of Actin Networks in Eukaryotic Cells

The actin cytoskeleton plays an important role in cell motility, where its ability to change its structural strength by transitioning from the gel-like (solid) state to the sol-like (liquid) state is crucial. One way the in vivo network can achieve the essential process of fluidization is by depolymerizing the actin network, via severing, fragmenting and capping proteins such as severin, gelsolin and CapZ. We modify existing actin polymer models and apply them to the in vivo actin cortex, to estimate its strength. We focus on transient crosslinking – a different mechanism that achieves this transition without depolymerization. Our work shows that a wide range of in vivo shear moduli from 1 Pa to 1 KPa that spans the viscoelastic behavior witnessed in eukaryotic cells can be achieved through transient crosslinking and trans-cellular protein migration alone. The transiently crosslinked actin network’s structural contribution is maximized, when both actin and the crosslinking protein are colocalized into a nearly nematic network, while its strength is low when both proteins are homogeneously distributed in vivo. Such an alternative mechanism to depolymerization is supported by experiments on modified fibroblast and dictyostellium cells which lack severing and fragmenting proteins, but are still observed to display cell motility and other normal cell functions.

[P9.009] Self Assembly and Spatial Structure in Actin Networks

Actin is an abundant cellular protein that self-assembles into filaments with lengths of several microns. Actin filaments in solution form complex networks in which interactions between polymer chains become important. Interesting concentration-dependent phenomena occur in these systems, which are currently being investigated via a simple experimental in vitro model. The concentration regime in which a partial phase transition to a nematic liquid crystalline state occurs is focused upon. As predicted by Onsager, isotropic and anisotropic domains coexist in this region. Larger scale spatial structure, which is not predicted by theory, has also been observed. This structure is suggestive of pattern formation. The dissipative biochemical reactions responsible for the phenomenon know as treadmilling guarantee that the actin networks under consideration are not in thermodynamic equilibrium. Preliminary results suggest that energy dissipative processes are correlated with the observed large scale structural variation. Therefore, investigation of the role of nonequilibrium processes in the creation of large scale ordering in this in vitro system are being pursued via advanced microscopy techniques. Using such methods, including polarization, confocal and multi-photon fluorescence, the structural variation can be quantitatively characterized.

[P9.010] Mimicking Temperature Through Molecular Machines

All eukaryotic cells depend on mechanisms of self-assembly of protein filaments to form a cytoskeleton within the cell. The need for motility and reaction by cells to stimuli additionally requires the existence of pathways which serve to restructure and disassemble cytoskeletal structures. Temperature-driven increases in disorder are the most physically fundamental method for breaking down complex structures, yet would play a destructive role in cellular dynamics. A similar situation is seen on the genetic level with the unfolding of DNA strands for replication and cell division – while temperature-driven unfolding of the strands stands as the most simple pathway, molecular machinery are present to perform the same function without heat-induced damage to the cell (Lodish et al, 2000). We report experimental evidence of a similar mechanism functioning on actin cytoskeletal dynamics, involving collections of the actin-specific molecular motor Myosin II. While crosslink-driven bundling self-assembles complex actomyosin structures (including bundles, asters, and large aggregates) in the near-chemical-equilibrum state, an activation of the motors causes a rapid disassembly of all structures. Such a mechanism is not only harmless to cell function, but occurs on a very rapid timescale which is favorable for quick cytoskeletal dynamics.

[P9.011] Fluctuations in the Internal Viscoelasticity of Living Bovine Endothelial Cells

Understanding the internal mechanical properties of living cells is essential to gain insight into basic cellular functions such as cell motility. We use an oscillating optical tweezers technique to determine the frequency dependent mechanical moduli of bovine endothelial cells. By optically trapping structures intrinsic to the interior of the cells, we determine the viscoelastic moduli with minimal intrusion. At fixed oscillation frequencies, large fluctuations in the viscoelastic moduli surrounding the optically trapped cellular structure for healthy, living cells were observed. Conversely, nutrient depleted cells show a reduction in both the magnitude and fluctuation of the elastic moduli. For frequencies ranging from 1 to 100 rad/s, the viscoelastic moduli for the nutrient depleted cells show a weak dependence on frequency. In addition, the magnitude of the elastic modulus is greater than the viscous modulus for frequencies ranging from 1 to 1000 rad/s. These final results are consistent with those found in non-localized measurements.

[P9.012] Correlation of Cell and Substrate Mechanical Properties

The mechanical properties of neonatal rat ventricular fibroblasts plated onto elastomer surfaces were studied in vitro and correlated to the mechanical response of the substrate. In order to differentiate the response of the cells to mechanical as opposed to mechanical modifications in their environment, only the rheological properties of the substrates were modified. In the case of entangled polymers this can be accomplished either by varying the molecular weight or the thickness of polymer films spun cast onto rigid supports. Scanning lateral force microscopy, which has been shown to be an effective technique for measuring relative modulii of surfaces(1) was used to track the mechanical response of the substrates as a function of processing procedures, molecular weight, both in liquid, air, and following fibronectin incubation. The response of the living cells was then compared to that of the underlying substrate. The samples were then stained and the distribution of actin correlated to the mechanical response. 1. S. Ge et al. Phys. Rev. Lett. 11, (2000)2340

[P9.013] Correlation between local viscoleastic behavior and lamellipodia extension

The lamellipodium is an actin-rich, protrusive region of a cell reaching in the direction of locomotion. The actin cytoskeleton supports the lamellipodia protrusion through actin polymerization with other accessory proteins. The protrusive force of the lamellipodia can be quantitatively understood by determining the cell’s local viscoelastic properties. We investigated the correlation between local viscoelastic properties and lamellipodia extension. As for the viscoelastic study, we used our AFM microrheology technique, which allows local viscoelastic measurements for very thin regions such as the lamellipodium. We quantified the lamellipodia extension by discussing parameters such as the extension rate, the directionality and the retracting time. We obtained time-lapse phase contrast images and analyzed them using image processing techniques. Our previous measurements showed differences in Young’s modulus as well as lamellipodia extension between the Balb 3T3 fibroblasts and the SVT2 transformed fibroblasts. We generalize this relation by investigating other cell lines, which were known to show different cell motility.

[P9.014] Universal strain stiffening in biological gels and tissues

Unlike most synthetic materials, many biological materials get stiffer as they are deformed. This nonlinear elastic response, critical for physiologic function of tissues such as the blood vessel wall, has been documented since at least the 19th century but the molecular structure and the design principles responsible for it are unknown. In various systems, different hypotheses ranging from complex multiphase structures to tensegrity models have been proposed to explain strain-stiffening in biological gels and tissues, and in these cases the specific viscoelastic properties depend critically on the detailed assembly and geometry of the highly ordered material. In this presentation we show that a much simpler molecular theory accounts for the most dramatic forms of strain stiffening found in a wide range of molecularly distinct biopolymer gels ranging from purified cytoskeletal and extracellular matrix gels to intact tissues such as the mesentery. The theory shows that the physics of semi flexible chains arranged in an open crosslinked meshwork invariably stiffen at low strains independent of the need for a specific architecture or multiple elements with different intrinsic stiffness. These findings explain why stiff polymers are chosen over more flexibler ones in tissues where only a limited range of deformation is appropriate.

[P9.015] A Microfluidic Optical Stretcher As A Diagnostic Tool

By combining the Optical Stretcher, a two beam laser trap that measures the viscoelastic properties of cells, with current microfabrication techniques, we have developed a device able to differentiate cell types within a heterogeneous population. A micro-peristaltic pump controls the flow of cells through channels constructed by PDMS soft lithography. Cells are individually trapped and deformed by two divergent, counter-propagating laser beams aligned perpendicular to the flow, and are measured using videomicroscopy. Viscoelastic properties of a cell depend strongly on its cytoskeleton, which along with a cell’s optical properties determine the amount of stretching, or optical deformability, for a given laser power. This serves as a new, inherent cell marker capable, for example, of detecting less elastic cancer cells among a population of healthy cells. As higher levels of integration become possible the experiment will progress towards the ultimate goal of a lab-on-a-chip. Increasingly more advanced microfluidic systems will incorporate cell sorting, medium swapping, and DNA microarrays, and will help span the gap between genomics, proteomics, and cellomics.

Part P of program listing