gradient-strain-chip-for-stimulating-cellular-behaviors-cell-laden

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

National Tsing Hua University

Vacuolar H(+)-translocating inorganic pyrophosphatase [vacuolar H(+)-pyrophosphatase (V-PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen, at the expense of PP(i) hydrolysis, for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function, although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton-translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 +/- 0.3 nm and 26.3 +/- 4.7 nm, whereas those of the luminal protrusions were 1.2 +/- 0.1 nm and 21.7 +/- 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6xHis tags on C-terminal ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 2.2 +/- 1.4 nm. Thus, our study is the first to provide structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.

The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique.

Homodimeric H(+)-pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H(+)-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP(i)), for H(+) translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP(i) binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H(+)-PPase are 49.3 + or - 4.0 and 67.2 + or - 5.7 A, respectively. Furthermore, putative PP(i) binding motifs on individual subunits are found to be relatively far away from each other (70.8 + or - 4.8 A), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H(+)-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H(+)-PPase upon substrate binding.

Distance variations between active sites of H(+)-pyrophosphatase determined by fluorescence resonance energy transfer.

This paper describes a Self-Aligned Wet (SAW) cell suitable for direct-cell or bacteria incubation and observation in a wet environment inside a transmission electron microscope. This SAW cell is fabricated by a bulk-micromachining process and composed of two structurally complementary counterparts (an out-frame and an in-frame), where each contain a silicon nitride film based observation window. The in- and out-frames can be self-aligned via a mechanism of surface tension from a bio-sample droplet without the aid of positioning stages. The liquid chamber is enclosed between two silicon nitride membranes that are thin enough to allow high energy electrons to penetrate while also sustaining the pressure difference between the TEM vacuum and the vapor pressure within the liquid chamber. A large field of view (150 μm × 150 μm) in a SAW cell is favored and formed from a larger sized observation window in the out-frame, which is fabricated using a unique circular membrane formation process. In this paper, we introduce a novel design to circumvent the challenges of charging/heating problems in silicon nitride that arise from interactions with an electron beam. This paper also demonstrates TEM observations of D. Radiodurans growth in a liquid environment within a thicker chamber (20 μm) within a SAW cell.

Self-aligned wet-cell for hydrated microbiology observation in TEM.

Cell alignment is a critical factor to govern cellular behavior and function for various tissue engineering applications ranging from cardiac to neural regeneration. In addition to physical geometry, strain is a crucial parameter to manipulate cellular alignment for functional tissue formation. In this paper, we introduce a simple approach to generate a range of gradient static strains without external mechanical control for the stimulation of cellular behavior within 3D biomimetic hydrogel microenvironments. A glass-supported microfluidic chip with a convex flexible polydimethylsiloxane (PDMS) membrane on the top was employed for loading the cells suspended in a prepolymer solution. Following UV crosslinking through a photomask with a concentric circular pattern, the cell-laden hydrogels were formed in a height gradient from the center (maximum) to the boundary (minimum). When the convex PDMS membrane retracted back to a flat surface, it applied compressive gradient forces on the cell-laden hydrogels. The concentric circular hydrogel patterns confined the direction of hydrogel elongation, and the compressive strain on the hydrogel therefore resulted in elongation stretch in the radial direction to guide cell alignment. NIH3T3 cells were cultured in the chip for 3 days with compressive strains that varied from ~65% (center) to ~15% (boundary) on hydrogels. We found that the hydrogel geometry dominated the cell alignment near the outside boundary, where cells aligned along the circular direction, and the compressive strain dominated the cell alignment near the center, where cells aligned radially. This study developed a new and simple approach to facilitate cellular alignment based on hydrogel geometry and strain stimulation for tissue engineering applications. This platform offers unique advantages and is significantly different from the existing approaches owing to the fact that gradient generation was accomplished in a miniature device without using an external mechanical source.

Article	Year
The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique. The FEBS journal\| PubMed ID: 19614743	2009
Distance variations between active sites of H(+)-pyrophosphatase determined by fluorescence resonance energy transfer. The Journal of biological chemistry\| PubMed ID: 20511234	2010
Self-aligned wet-cell for hydrated microbiology observation in TEM. Lab on a chip\| PubMed ID: 22130521	2012
Gradient static-strain stimulation in a microfluidic chip for 3D cellular alignment. Lab on a chip\| PubMed ID: 24253194	2013

Tsu-Wei Huang

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