Biomedical subjects

Daniel A Fletcher

Publications and source records attributed to Daniel A Fletcher.

13 recordsLinked to original sources

PubMed2007

Reversible stress softening of actin networks.

The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.

Actins↗

PubMed2006

Chemotherapy exposure increases leukemia cell stiffness.

Deformability of blood cells is known to influence vascular flow and contribute to vascular complications. Medications for hematologic diseases have the potential to modulate these complications if they alter blood cell deformability. Here we report the effect of chemotherapy on leukemia cell mechanical properties. Acute lymphoblastic and acute myeloid leukemia cells were incubated with standard induction chemotherapy, and individual cell stiffness was tracked with atomic force microscopy. When exposed to dexamethasone or daunorubicin, leukemia cell stiffness increased by nearly 2 orders of magnitude, which decreased their passage through microfluidic channels. This stiffness increase occurred before caspase activation and peaked after completion of cell death, and the rate of stiffness increase depended on chemotherapy type. Stiffening with cell death occurred for all cell types investigated and may be due to dynamic changes in the actin cytoskeleton. These observations suggest that chemotherapy itself may increase the risk of vascular complications in acute leukemia.

Actins↗

PubMed2006

Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures.

The treelike structures of many organs, including the mammary gland, are generated by branching morphogenesis, a reiterative process of branch initiation and invasion from a preexisting epithelium. Using a micropatterning approach to control the initial three-dimensional structure of mouse mammary epithelial tubules in culture, combined with an algorithm to quantify the extent of branching, we found that the geometry of tubules dictates the position of branches. We predicted numerically and confirm experimentally that branches initiate at sites with a local minimum in the concentration of autocrine inhibitory morphogens, such as transforming growth factor-beta. These results reveal that tissue geometry can control organ morphogenesis by defining the local cellular microenvironment, a finding that has relevance to control of invasion and metastasis.

Algorithms↗

PubMed2006

Actin polymerization serves as a membrane domain switch in model lipid bilayers.

The ability of cells to mount localized responses to external or internal stimuli is critically dependent on organization of lipids and proteins in the plasma membrane. Involvement of the actin cytoskeleton in membrane organization has been documented, but an active role for actin networks that directly links internal organization of the cytoskeleton with membrane organization has not yet been identified. Here we show that branched actin networks formed on model lipid membranes enriched with the lipid second messenger PIP(2) trigger both temporal and spatial rearrangement of membrane components. Using giant unilamellar vesicles able to separate into two coexisting liquid phases, we demonstrate that polymerization of dendritic actin networks on the membrane induces phase separation of initially homogenous vesicles. This switch-like behavior depends only on the PIP(2)-N-WASP link between the membrane and actin network, and we find that the presence of a preexisting actin network spatially biases the location of phase separation. These results show that dynamic, membrane-bound actin networks alone can control when and where membrane domains form and may actively contribute to membrane organization during cell signaling.

Actins↗

PubMed2006

Force microscopy of nonadherent cells: a comparison of leukemia cell deformability.

Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E(infinity) = 855 +/- 670 Pa for HL60 cells, E(infinity) = 48 +/- 35 Pa for Jurkat cells, E(infinity) = 156 +/- 87 for neutrophils, mean +/- SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia.

Acute Disease↗

PubMed2006

Fast, cheap and somewhat in control.

Efforts to manipulate living organisms have raised the question of whether engineering principles of hierarchy, abstraction and design can be applied to biological systems. Here, we consider the practical challenges to controlling living organisms that must be surmounted, or at least managed, if synthetic biology and cellular bioengineering are to be productive.

Animals↗

PubMed2005

Loading history determines the velocity of actin-network growth.

Directional polymerization of actin filaments in branched networks is one of the most powerful force-generating systems in eukaryotic cells. Growth of densely cross-linked actin networks drives cell crawling, intracellular transport of vesicles and organelles, and movement of intracellular pathogens such as Listeria monocytogenes. Using a modified atomic force microscope (AFM), we obtained force-velocity (Fv) measurements of growing actin networks in vitro until network elongation ceased at the stall force. We found that the growth velocity of a branched actin network against increasing forces is load-independent over a wide range of forces before a convex decline to stall. Surprisingly, when force was decreased on a growing network, the velocity increased to a value greater than the previous velocity, such that two or more stable growth velocities can exist at a single load. These results demonstrate that a single Fv relationship does not capture the complete behaviour of this system, unlike other molecular motors in cells, because the growth velocity depends on loading history rather than solely on the instantaneous load.

Actin Cytoskeleton↗

PubMed2005

Spiroplasma swim by a processive change in body helicity.

Microscopic organisms must rely on very different strategies than their macroscopic counterparts to swim through liquid. To date, the best understood method for prokaryotic swimming employs the rotation of flagella. Here, we show that Spiroplasma, tiny helical bacteria that infect plants and insects, use a very different approach. By measuring cell kinematics during free swimming, we find that propulsion is generated by the propagation of kink pairs down the length of the cell body. A processive change in the helicity of the body creates these waves and enables directional movement.

Methylcellulose↗

PubMed2005

Photopatterning of actin filament structures.

We report a general strategy for spatiotemporal control of actin polymerization in vitro using photoactivatable actin. Caged actin was synthesized by chemically modified lysine residues on monomeric actin and released with focused ultraviolet (UV) illumination. Epifluorescence microscopy revealed nucleation and elongation of individual actin filaments (8 nm in diameter) after localized release of caged actin. We also used this strategy to generate branched filament structures by releasing caged actin in the presence of actin binding proteins. Controlled self-assembly of actin filaments represents a versatile "bottom-up" technique for constructing structural building blocks and functional templates for nanoscience applications.

Actin Cytoskeleton↗

PubMed2004

Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis.

Uropathogenic Escherichia coli (UPEC) are capable of forming complex intracellular bacterial communities (IBC) within the superficial umbrella cells of the bladders of C3H and BALB/c mice. By using time-lapse fluorescence videomicroscopy to observe infected mouse bladder explants, we discovered that IBCs formed by uropathogenic E. coli progressed through four distinct developmental stages that differed with respect to growth rate, bacterial length, colony organization, motility, and its eventual dispersal. In the first phase, bacteria in the IBC were nonmotile, rod shaped, and grew rapidly in loosely organized colonies free in the cytoplasm of the bladder superficial umbrella cells. In the second phase, the loose collection of bacteria in the IBC matured into a slower growing, highly organized biofilm-like community consisting of coccoid bacteria that ultimately filled most of the cytoplasm. In the third phase, bacteria in the biofilm-like state in the IBC switched to a motile rod-shaped phenotype allowing detachment from the community and eventual fluxing out of the host cell. During the fourth phase, the bacteria filamented. Filamentation appeared to be in response to a Toll-like receptor 4-mediated innate defense mechanism. Bacteria that fluxed out of the superficial umbrella cells were able to reenter the IBC developmental cascade but with slower kinetics and ultimately a quiescent reservoir was established. Intracellular growth and filamentation provided an advantage to the bacteria in evading infiltrating polymorphonuclear leukocytes. This work has developed a technique to observe live infected organs and revealed a complex differentiation pathway that facilitates bacterial persistence in the urinary tract.

Animals↗

PubMed2004

An introduction to cell motility for the physical scientist.

Directed, purposeful movement is one of the qualities that we most closely associate with living organisms, and essentially all known forms of life on this planet exhibit some type of self-generated movement or motility. Even organisms that remain sessile most of the time, like flowering plants and trees, are quite busy at the cellular level, with large organelles, including chloroplasts, constantly racing around within cellular boundaries. Directed biological movement requires that the cell be able to convert its abundant stores of chemical energy into mechanical energy. Understanding how this mechanochemical energy transduction takes place and understanding how small biological forces generated at the molecular level are marshaled and organized for large-scale cellular or organismal movements are the focus of the field of cell motility. This tutorial, aimed at readers with a background in physical sciences, surveys the state of current knowledge and recent advances in modeling cell motility.

Animals↗

PubMed2003

Compression forces generated by actin comet tails on lipid vesicles.

Polymerizing networks of actin filaments generate force for a variety of movements in living cells, including protrusion of filopodia and lamellipodia, intra- and intercellular motility of certain bacterial and viral pathogens, and motility of endocytic vesicles and other membrane-bound organelles. During actin-based motility, coexisting populations of actin filaments exert both pushing and retarding forces on the moving cargo. To examine the distribution and magnitude of forces generated by actin, we have developed a model system where large artificial lipid vesicles coated with the protein ActA from the bacterial pathogen Listeria monocytogenes are propelled by actin polymerization in cytoplasmic extract. We find that motile vesicles associated with actin comet tails are significantly deformed due to an inward compression force exerted by actin polymerization orthogonal to the direction of motion, which is >10-fold greater in magnitude than the component of the force exerted in the direction of motion. Furthermore, there is a spatial segregation of the pushing and retarding forces, such that pushing predominates along the sides of the vesicle, although retarding forces predominate at the rear. We estimate that the total net (pushing minus retarding) force generated by the actin comet tail is approximately 0.4-4 nN. In addition, actin comet tail formation is associated with polarization of the ActA protein on the fluid vesicle surface, which may reinforce the persistence of unidirectional motion by helping to maintain a persistent asymmetry of actin filament density.

Actins↗

PubMed2002

Intravascular drug delivery with a pulsed liquid microjet.

Occlusions of the retinal veins and arteries, associated with diseases such as hypertension and arteriosclerosis, are a major cause of severe and irreversible loss of vision. Treatments for retinal vascular diseases have been unsatisfactory owing in part to the difficulty of delivering drugs to the site of disease within the eye. In this article, we demonstrate that a new device, the vapor bubble-driven pulsed liquid microjet, can deliver drugs into the lumen of small vessels such as those found in the retina. A 15- micro m-diameter liquid jet traveling at more than 60 m/s was shown to penetrate and deliver fluid through the wall of a blood vessel that was 60 micro m in diameter. Perforation of the wall of the blood vessel did not extend beyond the jet diameter.

Allantois↗