An In Vitro Model System to Test Mechano-Microbiological Interactions Between Bacteria and Host Cells.

The aim of this chapter is to present an innovative technique to visualize changes of the F-actin cytoskeleton in response to locally applied force. We developed an in vitro system that combines micromanipulation of force by magnetic tweezers with simultaneous live cell fluorescence microscopy. We applied pulling forces to magnetic beads coated with the Neisseria gonorrhoeae Type IV pili in the same order of magnitude than the forces generated by live bacteria. We saw quick and robust F-actin accumulation in individual cells at the sites where pulling forces were applied. Using the magnetic tweezers, we were able to mimic the local response of the F-actin cytoskeleton to bacteria-generated forces. In this chapter, we describe our magnetic tweezers system and show how to control it in order to study cellular responses to force.

Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy.

PURPOSE: To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA photochemical corneal cross-linking (CXL) for therapeutic tissue cross-linking of the cornea. METHODS: Eleven fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. After epithelial debridement, the tissue was exposed to 1/4 max (9.8 mM) or 1/3 max (13 mM) SMG at pH 8.5 for 30 minutes or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Postexposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (The Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts using a standardized algorithm. RESULTS: The ΔTm after CXL, 1/3 SMG, and 1/4 SMG was 2.2 ± 0.9°C, 1.3 ± 0.5°C, and 1.1 ± 0.5°C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high-power field. The values were 3 ± 1.7% (control) and 8.9 ± 11.1% (CXL) (P = 0.390); 1 ± 0.2% (control) and 19.5 ± 32.2% (1/3 max SMG) (P = 0.426); and 2.7 ± 2.4% (control) and 2.8 ± 2.2% (1/4 max SMG) (P = 0.938). The values for endothelial toxicity were then indexed over the shift in Tm to yield a toxicity/fixation index. The values were as follows: 2.7 for CXL, 14 for 1/3 max, and 0.1 for 1/4 max. CONCLUSIONS: Quarter max (1/4 max = 9.8 mM) SMG effectively cross-linked tissue and was nontoxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.

An In Vitro Model System to Test Mechano-microbiological Interactions Between Bacteria and Host Cells.

The aim of this chapter is to present an innovative technique to visualize changes of the f-actin cytoskeleton in response to locally applied force. We developed an in vitro system that combines micromanipulation of force by magnetic tweezers with simultaneous live cell fluorescence microscopy. We applied pulling forces to magnetic beads coated with the Neisseria gonorrhoeae Type IV pili in the same order of magnitude than the forces generated by live bacteria. We saw quick and robust f-actin accumulation at the sites where pulling forces were applied. Using the magnetic tweezers we were able to mimic the local response of the f-actin cytoskeleton to bacteria-generated forces. In this chapter we describe our magnetic tweezers system and show how to control it in order to study cellular responses to force.

The fission yeast cytokinetic contractile ring regulates septum shape and closure.

During cytokinesis, fission yeast and other fungi and bacteria grow a septum that divides the cell in two. In fission yeast closure of the circular septum hole by the ß-glucan synthases (Bgs) and other glucan synthases in the plasma membrane is tightly coupled to constriction of an actomyosin contractile ring attached to the membrane. It is unknown how septum growth is coordinated over scales of several microns to maintain septum circularity. Here, we documented the shapes of ingrowing septum edges by measuring the roughness of the edges, a measure of the deviation from circularity. The roughness was small, with spatial correlations indicative of spatially coordinated growth. We hypothesized that Bgs-mediated septum growth is mechanosensitive and coupled to contractile ring tension. A mathematical model showed that ring tension then generates almost circular septum edges by adjusting growth rates in a curvature-dependent fashion. The model reproduced experimental roughness statistics and showed that septum synthesis sets the mean closure rate. Our results suggest that the fission yeast cytokinetic ring tension does not set the constriction rate but regulates septum closure by suppressing roughness produced by inherently stochastic molecular growth processes.

The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis.

The functions of the actin-myosin-based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells.

Role of turgor pressure in endocytosis in fission yeast.

Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane.

Anomalous diffusion in living yeast cells.

The viscoelastic properties of the cytoplasm of living yeast cells were investigated by studying the motion of lipid granules naturally occurring in the cytoplasm. A large frequency range of observation was obtained by a combination of video-based and laser-based tracking methods. At time scales from 10(-4) to 10(2) s, the granules typically perform subdiffusive motion with characteristics different from previous measurements in living cells. This subdiffusive behavior is thought to be due to the presence of polymer networks and membranous structures in the cytoplasm. Consistent with this hypothesis, we observe that the motion becomes less subdiffusive upon actin disruption.