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PubMed1998

Morphological transformation of liposomes caused by assembly of encapsulated tubulin and determination of shape by microtubule-associated proteins (MAPs).

To examine the role of cytoskeletons in cellular morphogenesis, we generated liposomes encapsulating tubulin, with or without microtubule-associated proteins (MAPs), and observed their transformation using dark-field microscopy. When tubulin was polymerized with MAPs in liposomes, liposomes were transformed into a "bipolar" shape with a central sphere and two tubular membrane protrusions that aligned in a straight line. On the other hand, when pure tubulin was polymerized in liposomes without MAPs, they initially transformed into a bipolar shape but subsequently re-transformed into a "monopolar" shape, i.e. a sphere with only one straight tubular portion. This re-transformation occurred in two ways: first, by shortening of one of the tubular portions due to microtubule disassembly; or second, by fluctuation of the central sphere toward one of the ends without shortening of the tube portion. MAPs prevented this re-transformation, and their role in stabilizing the shape of transformed liposomes was studied by the co-sedimentation method. The results show that MAPs, particularly MAP1 and MAP2, mediate binding between microtubules and the liposomal membrane. However, MAP2 by itself did not bind to liposomes, but was able to stabilize bipolar liposomes. This stabilization is caused not only by direct links between microtubules and liposomes, but also by prevention of Brownian motion of microtubules through an increase in friction.

Liposomes↗

PubMed1997

Photonic force microscope based on optical tweezers and two-photon excitation for biological applications.

A new scanning probe microscope, the photonic force microscope (PFM), based on optical tweezers and two-photon absorption processes for biological applications is described. Optical tweezers are used to trap a fluorescent latex bead with a diameter of 200 nm in an aqueous solution in all three dimensions. The fluorescent dye is chosen to fulfill the two-photon absorption criterion for the 1064-nm line of a Nd:YVO4 laser. The intensity of the fluorescence emission is utilized as a very sensitive position sensor along the optical axis. Two-dimensional images are formed by laterally scanning the trapped latex bead across biological samples while recording the two-photon-induced fluorescences intensity. A scanning probe image of the outer surface of a small neurite from a cultured rat hippocampal neuron is shown, which is hardly visible under differential interference contrast microscopy. The lateral resolution is given by the bead diameter; the axial resolution is 40 nm. Under the experimental conditions the maximal imaging force applied by the probe is below 5 pN.

Animals↗

PubMed1994

Haemophilus ducreyi adheres to human keratinocytes.

Haemophilus ducreyi, Moraxella catarrhalis and a non-piliated Escherichia coli K-12 strain were studied for their ability to bind to human keratinocytes in vitro. Epidermal cells isolated from neonatal foreskins were grown to confluence in serum-free keratinocyte media. Probing of the monolayers with anti-cytokeratin antibody showed that 97% of cells were keratinocytes. Bacteria were grown to mid-log phase and seeded onto the monolayers. At various time-points monolayers were washed with PBS to remove non-adherent bacteria, and the monolayers were quantitatively cultured. After 120 min, 15 to 23% of the H. ducreyi inocula bound to the monolayer, while less than 1% of the M. catarrhalis or E. coli controls bound. Wet mounts of fixed monolayers observed with differential interference contrast microscopy confirmed the quantitative data. We conclude that H. ducreyi binds to keratinocytes and that this process may play a role in the initiation of chancroid.

Bacterial Adhesion↗

PubMed2006

Novel biophysical techniques for investigating long-term cell adhesion dynamics on biomaterial surfaces.

Cell adhesion on biomaterial surface is crucial for the regeneration and function of clinically viable cell and tissues. In turn, the cellular phenotypes, following the mechanochemical transduction of adherent cells on biomaterials, are directly correlated to the biophysical responses of cells. However, the lack of an integrated bio-analytical system for probing the cell-substrate interface poses significant obstacles to understanding the behavior of cells on biomaterial surface. We have developed a novel method, based on the principle of confocal reflectance interference contrast microscopy (C-RICM) that has enabled us to study the biomechanical deformation of cells on biomaterial surfaces. In this article, we would like to describe our recent development of the C-RICM system that integrates a confocal fluorescence microscope, phase contrast microscope and GFP expression system. We shall demonstrate the system by determining the adhesion contact kinetics, initial deformation rate, cytoskeleton structures of adherent cells on extracellular matrices (e.g., collagen and fibronectin) and biodegradable polymer (e.g., poly(lactic acid)) during long-term culture. We shall demonstrate that this unique approach could provide valuable biophysical information necessary for designing optimized biomaterial surfaces for cell/tissue regeneration applications.

Animals↗

PubMed1990

Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization.

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus Kc of the membrane and of the cytoplasmic viscosity eta were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I0 passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of Kc/R3(0) and mu.R0 (R0 = equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of Kc = 2-3 x 10(-19) Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, Kc = 3.4 x 10(-20) Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (Kc = 5 x 10(-19) Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent Kc and eta values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocyte-stomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCl solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) Kc and eta are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent Kc and eta after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

In vitro changes in human spermatozoa exposed to gastric juice: laboratory findings as a support for forensic practice.

Samples of complete human semen were incubated in gastric juice for different time periods at 37 degrees C, and by simulating the post-mortem temperature decrease of the human body. The changes in the spermatozoa were similar in both experiments. Short incubation specimens were examined directly with interference contrast microscopy and showed an almost immediate immobilization of spermatozoa when they were brought in contact with gastric juice. Specimens with longer incubation periods were stained with alcalic fuchsine and examined by immersion microscopy. There was a morphologically stable plateau for the heads of the spermatozoa for up to 6 h of incubation. The tails disappeared progressively in the first 45 min. After more than 6 h of incubation a progressive swelling and lysis of the heads was observed. Spermatozoa could be recognized for up to 7 days of incubation.

Cell Survival↗

PubMed1974