Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged “canoe” shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features—cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence—that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes—which had VASP highly enriched at their leading edges and migrated fast with straight trajectories—to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior phenotypes as large-scale consequences of kinetic contributions of VASP to actin filament growth and protection from capping at the leading edge. This work shows that the local effects of actin-remodeling proteins on cytoskeletal dynamics and organization can manifest as global modifications of the shape and behavior of migrating cells and that mathematical modeling can elucidate these large-scale cell behaviors from knowledge of detailed multiscale protein interactions.

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.The organization and molecular architecture of plant cell walls represent some of the most challenging problems in plant biology. Although much is known about general aspects of assembly and biosynthesis of the plant cell wall, the detailed three-dimensional molecular cell wall structure remains poorly understood. The highly complex and dynamic nature of the plant cell wall has perhaps limited the generation of such detailed structural models. This information is pivotal for the successful implementation of novel approaches for conversion of biomass to liquid biofuels, given that one of the critical processing steps in biomass conversion involves systematic deconstruction of cell walls. Therefore, a comprehensive understanding of the architecture and chemical composition of the plant cell wall will not only help develop molecularscale models, but will also help improve the efficiency of biomass deconstruction.The composition and molecular organization of the cell wall is species and cell type dependent (Vorwerk et al., 2004). Thus, the development of a model plant system, which utilizes a single cell type, has enhanced our capacity to understand cell wall architecture. The ability to genera...

Intracellular Listeria monocytogenes actin-based motility is characterized by significant individual variability, which can be influenced by cytoarchitecture. L. monocytogenes was used as a probe to transmit information about structural variation among subcellular domains defined by mitochondrial density. By analyzing the movement of a large population of L. monocytogenes in PtK2 cells, we found that mean speed and trajectory curvature were significantly larger for bacteria moving in mitochondria-containing domains (generally perinuclear) than for bacteria moving in mitochondria-free domains (generally peripheral). Analysis of bacteria that traversed both mitochondria-containing and mitochondria-free domains revealed that these motile differences were not intrinsic to bacteria themselves. Disruption of mitochondrial respiration did not affect bacterial mean speed, speed persistence, or trajectory curvature. In contrast, microtubule depolymerization lead to decreased mean speed per bacterium and increased mean speed persistence of L. monocytogenes moving in mitochondria-free domains compared with untreated cells. L. monocytogenes were also observed to physically collide with mitochondria and push them away from the bacterial path of motion, causing bacteria to slow down before rapidly resuming their speed. Our results show that subcellular domains along with microtubule depolymerization may influence the actin cytoskeleton to affect L. monocytogenes speed, speed persistence, and trajectory curvature.

ETOC: We reconstitute actin-based motility using ellipsoidal particles mimicking the rod shape of Listeria monocytogenes and systematically analyze bead motile behaviors. By combining features of elastic propulsion and tethered-ratchet actin-polymerization models, we can explain our observations with a comprehensive new biophysical model.

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.

Scanning Laser Image Correlation (SLIC) is a technique to measure the flow of small particles and to observe local flow patterns over an area. SLIC can be applied to situations including blood cells flowing through blood vessels or tracer particles flowing through microfluidic channels. The main advantage of SLIC over other flow measurement techniques is that SLIC can be scaled to measure flow in areas ranging from microns to centimeters wide. To accomplish this, an image is first acquired through laser scanning and analyzed with number and brightness analysis (N&B) to identify regions of flow. This is used as a guide to manually (or potentially automatically) select a pattern within the image, such as a line along the center of a channel, that is then scanned repeatedly with the laser beam. Since the entire image is not scanned in each measurement SLIC measurements can be obtained quickly and efficiently. The results of these scanned patterns are analyzed with the recently developed pair correlation technique to extract the rate of flow and to identify characteristic flow patterns such as turbulence, particles that adhere to the channel walls, and variable velocity along the length of the channel. In the work presented here we demonstrate the effectiveness of SLIC by measuring blood flow in a zebra fish model. With SLIC, we are able to obtain blood flow measurements equivalent to those obtained with other techniques. We are also able to map the rate of flow and to observe variability in flow rate over time. This indicates that SLIC has potential to measure blood flow in other animals as well and may hold potential as the basis of a medical device.