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Biofabricated mass-producible arteries containing layered and aligned cells and matrix exhibit enhanced cellular function., Changes in structure and function of small muscular arteries play a major role in the pathophysiology of pulmonary hypertension, a burgeoning public health challenge. Improved anatomically mimetic in vitro models of these microvessels are urgently needed because nonhuman vessels and previous models do not accurately recapitulate the microenvironment and architecture of the human microvascular wall. Here, we describe parallel biofabrication of photopatterned self-rolled biomimetic pulmonary arterial microvessels of tunable size and infrastructure. These microvessels feature anatomically accurate layering and patterning of aligned human smooth muscle cells, extracellular matrix, and endothelial cells and exhibit notable increases in endothelial longevity and nitric oxide production. Computational image processing yielded high-resolution 3D perspectives of cells and proteins. Our studies provide a new paradigm for engineering multicellular tissues with precise 3D spatial positioning of multiple constituents in planar moieties, providing a biomimetic platform for investigation of microvascular pathobiology in human disease.

Effects of 3D confinement on cellular growth and matrix assembly are important in tissue engineering, developmental biology, and regenerative medicine. Polydimethylsiloxane wells with varying anisotropy are microfabicated using soft-lithography. Microcontact printing of bovine serum albumin is used to block cell adhesion to surfaces between wells. The orientations of fibroblast stress fibers, microtubules, and fibronectin fibrils are examined 1 day after cell seeding using laser scanning confocal microscopy, and anisotropy is quantified using a custom autocorrelation analysis. Actin, microtubules, and fibronectin exhibit higher anisotropy coefficients for cells grown in rectangular wells with aspect ratios of 1:4 and 1:8, as compared to those in wells with lower aspect ratios or in square wells. The effects of disabling individual cytoskeletal components on fibroblast responses to anisotropy are then tested by applying actin or microtubule polymerization inhibitors, Rho kinase inhibitor, or by siRNA-mediated knockdown of AXL or cofilin-1. Latrunculin A decreases cytoskeletal and matrix anisotropy, nocodazole ablates both, and Y27632 mutes cellular polarity while decreasing matrix anisotropy. AXL siRNA knockdown has little effect, as does siRNA knockdown of cofilin-1. These data identify several specific cytoskeletal strategies as targets for the manipulation of anisotropy in 3D tissue constructs.

CD8 + T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8 + T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8 + T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8 + T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8 + T cells and signaling by G protein-coupled receptors, although CD8 + T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8 + T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8 + T cells.

Summary Introduction Contractile networks are fundamental to many cellular functions, particularly cytokinesis and cell motility. Contractile networks depend on myosin-II mechanochemistry to generate sliding force on the actin polymers. However, to be contractile, the networks must also be crosslinked by crosslinking proteins, and to change the shape of the cell, the network must be linked to the plasma membrane. Discerning how this integrated network operates is essential for understanding cytokinesis contractility and shape control. Here, we analyzed the cytoskeletal network that drives furrow ingression in Dictyostelium . Results We establish that the actin polymers are assembled into a meshwork and that myosin-II does not assemble into a discrete ring in the Dictyostelium cleavage furrow of adherent cells. We show that myosin-II generates regional mechanics by increasing cleavage furrow stiffness and slows furrow ingression during late cytokinesis as compared to myoII nulls. Actin crosslinkers dynacortin and fimbrin similarly slow furrow ingression and contribute to cell mechanics in a myosin-II-dependent manner. By using FRAP, we show that the actin crosslinkers have slower kinetics in the cleavage furrow cortex than in the pole, that their kinetics differ between wild-type and myoII null cells, and that the protein dynamics of each crosslinker correlate with its impact on cortical mechanics. Conclusions These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change.

The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews [1–4]). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.

The cytoplasm of living cells is filled with cytoskeletal filament networks that dictate the shape and motility of cells. The dynamic and heterogeneous nature of the cytoskeletal network has been visualized, but the limited accessibility of living cytoplasm prevents easy characterization of its physical properties. From the simple observation that the Brownian motion of a particle reveals the mechanics of its microenvironment, a new approach, microrheology, emerged and successfully applied to living cells. Here, we review recent progress in microrheology and the implication for the understanding of subcellular processes in biology.

Spanning all size levels, regulating biological forces and transport are fundamental life processes. Used by various investigators over the last dozen years, optical techniques offer unique advantages for studying biological forces. The most mature of these techniques, optical tweezers, or the single-beam optical trap, is commercially available and is used by numerous investigators. Although technical innovations have improved the versatility of optical tweezers, simple optical tweezers continue to provide insights into cell biology. Two new, promising optical technologies, laser-tracking microrheology and the optical stretcher, allow mechanical measurements that are not possible with optical tweezers. Here, I review these various optical technologies and their roles in understanding mechanical forces in cell biology.

To understand the microscopic mechanical properties of actin networks, we monitor the motion of embedded particles with controlled surface properties. The highly resolved Brownian motions of these particles reveal the viscoelastic character of the microenvironments around them. In both non-cross-linked and highly cross-linked actin networks, particles that bind F-actin report viscoelastic moduli comparable to those determined by macroscopic rheology experiments. By contrast, particles modified to prevent actin binding have weak microenvironments that are surprisingly insensitive to the introduction of filament cross-links. Even when adjacent in the same cross-linked gel, actin-binding and nonbinding particles report viscoelastic moduli that differ by two orders of magnitude at low frequencies (0.5-1.5 rad/s) but converge at high frequencies (> 10(4) rad/s). For all particle chemistries, electron and light microscopies show no F-actin recruitment or depletion, so F-actin microheterogeneities cannot explain the deep penetration (approximately 100 nm) of nonbinding particles. Instead, we hypothesize that a local depletion of cross-linking around nonbinding particles explains the phenomena. With implications for organelle mobility in cells, our results show that actin binding is required for microenvironments to reflect macroscopic properties, and conversely, releasing actin enhances particle mobility beyond the effects of mere biochemical untethering.

The actin-based motility of the bacterium, Listeria monocytogenes, is a model system for understanding motile cell functions involving actin polymerization. Although the biochemical and genetic aspects of Listeria motility have been intensely studied, biophysical data are sparse. Here we have used high-resolution laser tracking to follow the trailing ends of Listeria moving in the lamellae of COS7 cells. We found that pauses during motility occur frequently and that episodes of step-like motion often show pauses spaced at about 5.4 nm, which corresponds to the spatial periodicity of F-actin. We occasionally observed smaller steps (

To establish laser-tracking microrheology (LTM) as a new technique for quantifying cytoskeletal mechanics, we measure viscoelastic moduli with wide bandwidth (5 decades) within living cells. With the first subcellular measurements of viscoelastic phase angles, LTM provides estimates of solid versus liquid behavior at different frequencies. In LTM, the viscoelastic shear moduli are inferred from the Brownian motion of particles embedded in the cytoskeletal network. Custom laser optoelectronics provide sub-nanometer and near-microsecond resolution of particle trajectories. The kidney epithelial cell line, COS7, has numerous spherical lipid-storage granules that are ideal probes for noninvasive LTM. Although most granules are percolating through perinuclear spaces, a subset of perinuclear granules is embedded in dense viscoelastic cytoplasm. Over all time scales embedded particles exhibit subdiffusive behavior and are not merely tethered by molecular motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2)) are more rigid than viscoelastic perinuclear regions (330 +/- 250 dyne/cm(2), p < 0.0001), but spectra converge at high frequencies. Although the actin-disrupting agent, latrunculin A, softens and liquefies lamellae, physiological levels of F-actin, alone (11 +/- 1.2 dyne/cm(2)) are approximately 70-fold softer than lamellae. Therefore, F-actin is necessary for lamellae mechanics, but not sufficient. Furthermore, in time-lapse of apparently quiescent cells, individual lamellar granules can show approximately 4-fold changes in moduli that last >10 s. Over a broad range of frequencies (0.1-30, 000 rad/s), LTM provides a unique ability to noninvasively quantify dynamic, local changes in cell viscoelasticity.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks’ rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.

We present a new method for measuring the linear viscoelastic shear moduli of complex fluids. Using photodiode detection of laser light scattered from a thermally excited colloidal probe sphere, we track its trajectory and extract the moduli using a frequency-dependent Stokes-Einstein equation. Spectra obtained for polyethylene oxide in water are in excellent agreement with those found mechanically and using diffusing wave spectroscopy. Since only minute sample volumes are required, this method is well suited for biomaterials of high purity, as we demonstrate with a concentrated DNA solution.

Optical tweezers, or the single-beam optical gradient force trap, is becoming a major tool in biology for noninvasive micromanipulation on an optical microscope. The principles and practical aspects that influence construction are presented in an introductory primer. Quantitative theories are also reviewed but have yet to supplant user calibration. Various biological applications are summarized, including recent quantitative force and displacement measurements. Finally, tantalizing developments for new, nonimaging microscopy techniques based on optical tweezers are included.

MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G₂) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G₂/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G₂/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G₁) to synthesis (S), as described previously, as well as G₂ to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events(1, 2). Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time(3). In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac(4), Cdc42(4), RhoA(4) and Ras(5), in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells(6). Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level(6).

The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half-time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport.

Proximity to membranes is required of actin networks for many key cell functions, including mechanics and motility. However, F-actin rigidity should hinder a filament's approach to surfaces. Using confocal microscopy, we monitor the distribution of fluorescent actin near nonadherent glass surfaces. Initially uniform, monomers polymerize to create a depletion zone where F-actin is absent at the surface but increases monotonically with distance from the surface. At its largest, depletion effects can extend35 microm, comparable with the average, mass-weighted filament length. Increasing the rigidity of actin filaments with phalloidin increases the extent of depletion, whereas shortening filaments by using capping protein reduces it proportionally. In addition, depletion kinetics are faster with higher actin concentrations, consistent with faster polymerization and faster Brownian-ratchet-driven motion. Conversely, the extent of depletion decreases with actin concentration, suggesting that entropy is the thermodynamic driving force. Quantitatively, depletion kinetics and extent match existing actin kinetics, rigidity, and lengths. However, explaining depletion profiles and concentration dependence (power-law of -1) requires modifying the rigid rod model. Within cells, surface depletion should slow membrane-associated F-actin reactions another approximately 10-fold beyond hydrodynamically slowed diffusion of filaments (approximately 10-fold). In addition, surface depletion should cause membranes to bend spontaneously toward filaments. Such depletion principles underlie the thermodynamics of all surface-associated reactions with mechanical structures, ranging from DNA to filaments to networks. For various functions, cells must actively resist the thermodynamics of depletion.

We demonstrate a quantitative optical scatter imaging (OSI) technique, based on Fourier filtering, for detecting alterations in the size of particles with wavelength-scale dimensions. We generate our scatter image by taking the ratio of images collected at high and low numerical aperture in central dark-field microscopy. Such an image spatially encodes the ratio of wide to narrow angle scatter and hence provides a measure of local particle size. We validated OSI on sphere suspensions and live cells. In live cells, OSI revealed biochemically induced morphological changes that were not apparent in unprocessed differential interference contrast images. Unlike high-resolution imaging methods, OSI can provide size information for particles smaller than the camera's spatial resolution.

Cell cortices rearrange dynamically to complete cytokinesis, crawlin response to chemoattractant, build tissues, and make neuronal connections. Highly enriched in the cell cortex, actin, myosin II, and actin crosslinkers facilitate cortical movements. Because cortical behavior is the consequence of nanoscale biochemical events, it is essential to probe the cortex at this level. Here, we use high-resolution laser-based particle tracking to examine how myosin II mechanochemistry and dynacortin-mediated actin crosslinking control cortex dynamics in Dictyostelium . Consistent with its low duty ratio, myosin II does not directly drive active bead motility. Instead, myosin II and dynacortin antagonistically regulate other active processes in the living cortex.

During cytokinesis, global and equatorial pathways deform the cell cortex in a stereotypical manner, which leads to daughter cell separation. Equatorial forces are largely generated by myosin-II and the actin crosslinker, cortexillin-I. In contrast, global mechanics are determined by the cortical cytoskeleton, including the actin crosslinker, dynacortin. We used direct morphometric characterization and laser-tracking microrheology to quantify cortical mechanical properties of wild-type and cortexillin-I and dynacortin mutant Dictyostelium cells. Both cortexillin-I and dynacortin influence cytokinesis and interphase cortical viscoelasticity as predicted from genetics and biochemical data using purified dynacortin proteins. Our studies suggest that the regulation of cytokinesis ultimately requires modulation of proteins that control the cortical mechanical properties that establish the force-balance that specifies the shapes of cytokinesis. The combination of genetic, biochemical, and biophysical observations suggests that the cell's cortical mechanical properties control how the cortex is remodeled during cytokinesis.

Isometric forces generated by single molecules of the mechanochemical enzyme kinesin were measured with a laser-induced, single-beam optical gradient trap, also known as optical tweezers. For the microspheres used in this study, the optical tweezers was spring-like for a radius of 100 nanometers and had a maximum force region at a radius of approximately 150 nanometers. With the use of biotinylated microtubules and special streptavidin-coated latex microspheres as handles, microtubule translocation by single squid kinesin molecules was reversibly stalled. The stalled microtubules escaped optical trapping forces of 1.9 +/- 0.4 piconewtons. The ability to measure force parameters of single macromolecules now allows direct testing of molecular models for contractility.

We have developed microtubule binding and motility assays for Cin8p, a kinesin-related mitotic spindle motor protein from Saccharomyces cerevisiae. The methods examine Cin8p rapidly purified from crude yeast cell extracts. We created a recombinant form of CIN8 that fused the biotin carrying polypeptide from yeast pyruvate carboxylase to the carboxyl terminus of Cin8p. This form was biotinated in yeast cells and provided Cin8p activity in vivo. Avidin-coated glass surfaces were used to specifically bind biotinated Cin8p from crude extracts. Microtubules bound to the Cin8p-coated surfaces and moved at 3.4 +/- 0.5 micrometer/min in the presence of ATP. Force production by Cin8p was directed toward the plus ends of microtubules. A mutation affecting the microtubule-binding site within the motor domain (cin8-F467A) decreased Cin8p's ability to bind microtubules to the glass surface by10-fold, but reduced gliding velocity by only 35%. The cin8-3 mutant form, affecting the alpha2 helix of the motor domain, caused a moderate defect in microtubule binding, but motility was severely affected. cin8-F467A cells, but not cin8-3 cells, were greatly impaired in bipolar spindle forming ability. We conclude that microtubule binding by Cin8p is more important than motility for proper spindle formation.

Publisher Summary This chapter discusses local heating by optical tweezers. Because most optical tweezers have been custom-built, if not customized for particular applications, estimates of aqueous heating are likely to vary between apparatuses. The chapter presents a simple, direct method to estimate the local heating by optical tweezers that will be useful to all practitioners of the technology. A number of methods exist for monitoring the local temperature near the field of observation. Microthermocouples can be inserted near the field of view, but accidental irradiation of the thermocouple by the optical tweezers laser can cause temperature jumps more than 500°C, which vaporize the local media.

Background: Cholangiocarcinomas (CCA) are cancers with dismal prognosis, thought to arise from epithelial cells lining the biliary tree. An improved understanding of the pathogenesis of CCA, as well as novel diagnostic and therapeutic approaches are direly needed. MicroRNAs (miRs) are short, single-stranded sequences of RNA, that were recently demonstrated to play a major role in the regulation of virtually all cellular processes. Several previous studies identified miRs that are dysregulated in CCA as well as distinct roles played by miRs in CCA genesis or progression. Methods: We performed microRNA-arrays on 5 normal biliary duct epithelias and 5 CCAs that had been obtained at surgery. After data analysis, we selected mir-494 for all subsequent analysis among top 5 miRs. To confirm the initial miR array data, quantitative real time RT-PCR analysis was performed using 12 human CCA as well as 5 normal cholangiocyte specimens. And then, we transfected 2 CCA cell lines with miR-494 and a non-specific mimic, respectively, and performed mRNA arrays to identify in an unbiased fashion the genes whose expression is downregulated by miR-494. Furthermore, we performed fluorescence activated cell sorting, as well as differential interference contrast microscopy. Results: miR-494 is significantly downregulated in human CCA specimens. Cells transfected with miR-494 showed a significant decrease in growth in the cell proliferation assay. As for the results of mRNA array, Ingenuity Pathway Analysis also indicated that miR-494 appears to coordinately affect several genes involved in the Mitotic Roles of Polo-like Kinase and Cell cycle: G2/M Checkpoint Regulation canonical pathways. We verified that miR-494 modulates the protein level of six genes involved in the G2/M transition. Next, we identified direct binding of miR-494 to the open reading frame (ORF), and downregulation of PTTG1 and TOP2A. Conclusions: our findings suggest that miR-494 has a global regulatory role in cell cycle progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. Citation Format: Sumitaka Yamanaka, Nathaniel R. Campbell, Scot C. Kuo, Esteban Mezey, Anirban Maitra, Florin Selaru. A microRNA downregulated in human cholangiocarcinoma induces G2/M arrest through multiple targets. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5284. doi:10.1158/1538-7445.AM2013-5284

Publisher Summary This chapter discusses methods used for tracking nanometer movements of single motor molecules with high precision. General principles are described. Approaches based on video are most convenient; however, improved temporal resolution and spatial sensitivity are achieved by custom photodiode detection systems with custom illumination. In the case of microtubules gliding on motor-coated glass, translocation while pivoting around a nodal point is a strong indicator of motility by a single motor molecule. On the other hand, in the case of bead movements statistical arguments have been used to show that the movements observed are driven by single motors. The use of the optical tweezers in those assays is important to make an unbiased sample of the population of beads. To record the position of particles—moving by single motors—requires a major commitment of resources, either to a specialized system or to a high-quality video-enhanced differential interference contrast (DIC) microscope. The lack of image formation in the photodiode systems is compensated by their greater temporal and spatial resolution. However, in contrast, the video-enhanced DIC systems have lower resolution and give additional information about the microtubule position and allow the use of fiducial marker beads in the system.

Alterations in plasma membrane structure and function seem to be of primary importance in the pathogenesis of cell injury, calling for more understanding of the changes in plasma membrane lipid structure (e.g., lipid order, lateral diffusion, dependence of phase states, and viscoelasticity) during the evolution of hypoxic injury in hepatocytes using multiple fluorescent spectroscopic techniques. Following hypoxic injury, fluorescence recovery after photobleaching was used to monitor plasma membrane lipid diffusion, resonance energy transfer microscopy was used to detect the lipid topography (domain formation), and the laser trapping technique was used to measure the plasma membrane viscoelasticity. The use of these different kinds of fluorescent spectroscopic techniques coupled with the authors' previous studies using digitized fluorescence polarization microscopy which was used to measure lipid order (fluidity) allowed the delineation of alterations in membrane structure during hypoxic injury and a model of membrane architecture during hypoxic injury, which could not be obtained from the use of any of these techniques alone. A model is proposed in which gel- and fluid-phase lipid islands form during hypoxic cell injury. Formation of these lipid domains promotes cell surface bleb formation, with eventual weakening of plasma membrane integrity, bleb rupture, and cell death. 11

Proximity to membranes is required of actin networks for many key cell functions, including mechanics and motility. However, F-actin rigidity should hinder a filament's approach to surfaces. Using confocal microscopy, we monitor the distribution of fluorescent actin near non-adherent glass surfaces. Initially uniform, monomers polymerize to create a depletion zone where F-actin is absent at the surface but increases monotonically with distance from the surface. At its largest, depletion effects can extend >35 μm, comparable to mass-weighted filament lengths. Increasing the rigidity of actin filaments with phalloidin increases the extent of depletion, whereas shortening filaments using capping protein reduces it proportionally. In addition, depletion kinetics are faster with higher actin concentrations, consistent with faster polymerization and faster Brownian-ratchet-driven motion. Conversely, the extent of depletion decreases with actin concentration, suggesting that entropy is the thermodynamic driving force. Quantitatively, depletion kinetics and extent match existing actin kinetics, rigidity and lengths. However, explaining depletion profiles and concentration-dependence (power-law of -1) requires modifying the rigid rod model. Dynamically crosslinked and dendritic (ARP2/3) networks either slow or enhance the extent of depletion, respectively. In cells, surface depletion should slow membrane-associated F-actin reactions another ∼10-fold beyond hydrodynamic considerations, and to favor membrane invaginations by decreased surface tension. Similar depletion principles underlie the thermodynamics of all surface-associated reactions with mechanical structures, ranging from DNA to filaments to networks. For various functions, cells must actively resist the thermodynamics of depletion.

Cell membranes often are patchy, composed of lateral domains. These domains may be formed by barriers within or on either side of the membrane bilayer. Major histocompatibility complex (MHC) class 1 molecules that were either transmembrane- (H-2Db) or glycosylphosphatidylinositol (GPI)-anchored (Qa2) were labeled with antibody-coated gold particles and moved across the cell surface with a laser optical tweezers until they encountered a barrier, the barrier-free path length (BFP). At room temperature, the BFPs of Qa2 and H-2Db were 1.7 +/- 0.2 and 0.6 +/- 0.1 (micrometers +/- SEM), respectively. Barriers persisted at 34 degrees C, although the BFP for both MHC molecules was fivefold greater at 34 degrees C than at 23 degrees C. This indicates that barriers to lateral movement are primarily on the cytoplasmic half of the membrane and are dynamic.

Please provide author affiliations Figure 1. Optical Scatter Imaging. Representative cell undergoing programmed cell death (apoptosis). Differential Interference Contrast (DIC) images (left panels) and OSI images (right panels) are displayed at times t = -7 and 37 min. before and after apoptosis induction at t=0. "C" and "N" respectively point to the cytoplasm and nucleus. The decreased scatter ratio within the cytoplasm at t = 37 is consistent with an increase in the average particle size within the cytoplasm, and might be attributable to a loss in the volume homeostasis of mitochondria. OSI complements current microscopic techniques such as DIC by revealing subcellular alterations that might otherwise remain undetected in conventional microscopic images. OSIR = Optical Scatter Image Ratio.

Alterations in plasma membrane structure and function are considered of primary importance in the pathogenesis of cell injury. Multiple microscopic techniques are employed to detail alterations in plasma membrane lipid structure during hypoxic injury in individual rat hepatocytes. Multiparameter digitized video microscopy, fluorescence quenching imaging, and fluorescence resonance energy transfer imaging are used to measure and monitor lipid domain formation and topography; laser tweezers are used to monitor the plasma membrane viscoelasticity. These microscopic techniques indicate that hypoxic injury in hepatocytes leads to alterations in plasma membrane lipid topography with the eventual formation of lipid domains. In concert with previous data generated with digitized fluorescence polarization microscopy and fluorescence recovery after photobleaching (FRAP), a model is proposed where formation of the distinct lipid domains promotes loss of the plasma membrane permeability barrier and cell death.

The flaA (cheC) locus from Escherichia coli is important in controlling the rotational direction of flagella during chemotaxis. The locus was sequenced, and a site of transcriptional initiation was determined. Two reading frames, flaAI and flaAII, span the locus. flaAII corresponds to certain flaA and cheC mutations, and has some unusual features in the predicted secondary structure. flaAI, however, has not been identified previously, but a flaAI deletion, which produced a truncated FlaAI peptide in minicells, clearly identified the FlaAI protein.

The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.

To understand output control in bacterial chemotaxis, we varied the levels of expression of cellular cheY and cheZ genes and found that the overproduction of the corresponding proteins affected Escherichia coli swimming behavior. In the absence of other signal-transducing gene products, CheY overproduction made free-swimming cells tumble more frequently. A plot of the fraction of the population that are tumbling versus the CheY concentration was hyperbolic, with half of the population tumbling at 30 microM (25,000 copies per cell) CheY monomers in the cytosol. Overproduction of aspartate receptor (Tar) by 30-fold had a negligible effect on CheY-induced tumbling, so Tar does not sequester CheY. CheZ overproduction decreased tumbling in all tumbling mutants except certain flaAII(cheC) mutants. In the absence of other chemotaxis gene products, CheZ overproduction inhibited CheY-induced tumbling. Models for CheY as a tumbling signal and CheZ as a smooth-swimming signal to control flagellar rotation are discussed.

By means of a computerized video processing system, the flagellar motors of Escherichia coli were shown to have multiple kinetic states for each rotational direction. High-resolution analysis of flagellar motors revealed new kinetic states both in wild-type cells and in a strain deleted of other signal-transducing genes to which CheY had been introduced. This strain, RP1091, retained residual kinase activity that could phosphorylate CheY, complicating the biochemical identification of certain kinetic states. The behavioral effect of CheY on single flagellar motors was ultrasensitive, with an apparent Hill coefficient of 5.5 +/- 1.9 (SD) and a half-maximal effect at 10.1 +/- 0.5 (SD) microM CheY. Based on the CheY concentration dependence, a two-state model is clearly excluded, even for the simpler system of CheY-induced rotational reversals in the deletion strain. The data are best described by a four-state model, with two clockwise and two counterclockwise states.