This study was supported by NIH research funding (DK090868 and GM092930 to T.I.). There is a pending patent on a caged rapamycin analog.

1. Transfection of Plasmid DNA
<ol>
	<li>Add plasmid DNAs, 0.5 &mu;g membrane-tethered FRB (hereafter referred to as LDR) and 0.5 &mu;g FKBP-Tiam1 (T-cell lymphoma invasion and metastasis-inducing protein 1: Rac activator) tagged with fluorescent protein to 37.5 &mu;l dH2O. Add 1 &mu;l FuGENE HD.</li>
	<li>Vortex and incubate at room temperature for 20 minutes. Meanwhile, proceed to steps 1.3-1.8.</li>
	<li>Wash each well of an 8-well chamber with 50 &mu;l poly-D-Lysine.</li>
	<li>Wash the center of each well with dH2O.</li>
	<li>Trypsinize 80-85% confluent NIH-3T3 cells and dilute to 10 ml culture medium (DMEM with 10% FBS).</li>
	<li>Remove 375 &mu;l of suspension and centrifuge at 1750 rpm for 3 minutes.</li>
	<li>Aspirate the media, being careful not to disturb the pellet.</li>
	<li>Add 750 &mu;l of DMEM w/ 10% FBS and resuspend.</li>
	<li>Add cell suspension to dH2O/DNA/FuGENE HD solution and mix gently.</li>
	<li>Add 250 &mu;l of cell/DNA suspension to each well (will fill up to 3 wells).</li>
	<li>Incubate the transfected cells at 37 &deg;C and 5% CO2 for 15-18 hr.</li>
</ol>
2. Preparation of cRb-A Solution
<ol>
	<li>Synthesize caged rapamycin-biotin adduct (cRb). See "Synthetic scheme of cRb" for details. Briefly, caging and biotin moieties are prepared separately. The caging moiety is then conjugated to rapamycin (LC lab), the product subsequently coupled to the biotin moiety by a means of click reaction7 to afford cRb.</li>
	<li>Prepare a 1 &mu;M stock solution of cRb in DMSO.</li>
	<li>Dissolve 1mg avidin in 250 &mu;l of PBS in a 0.5 ml Eppendorf tube.</li>
	<li>Add 1 &mu;l of cRb stock solution to dissolved avidin. Mix by inverting several times.</li>
	<li>Incubate at room temperature for 15 min to yield cRb-avidin conjugate (cRb-A) solution.</li>
	<li>Exclude unbound small molecules and purify cRb-A using a size-exclusion column.</li>
</ol>
3. Microscopy and Light Illumination at Subcellular Level
<ol>
	<li>Serum starve transfected cells in DMEM without FBS for 12 hours.</li>
	<li>Gently aspirate the media off the cells and add 250 &mu;l of the prepared 400 nM cRb-A solution (from step 2.6).</li>
	<li>To visualize protein dynamics in living cells at subcellular resolution before and after light treatment, spinning disk microscopy was used (Figure 2a). UV illumination was achieved by coupling optics for UV LED light (365 nm) with epi-fluorescence light (Figure 2b).</li>
	<li>MetaMorph software was used to capture fluorescence images of transfected cells every 15 seconds.</li>
	<li>Define the coordinates and size of an illumination spot by using a glass plate coated with fluorescent dyes excited by UV light. The size can be varied by applying objective lens with different magnifications and by selecting optical fibers with different core size.</li>
	<li>After determining background levels of membrane ruffling, irradiate UV light at the periphery of cells. The following two steps are to contrast preceding localized effects.</li>
	<li>Globally irradiate cells with 365 nm light provided by a mercury lamp.</li>
	<li>Dissolve uncaged, thus original rapamycin in DMSO and then in PBS (400 nM final), and add the solution to the imaging chamber to induce global effects.</li>
</ol>
4. Representative Results
An example of representative results is shown in Figure 3. UV light irradiation at a small region near a cellular edge rapidly induced formation of localized ruffles only in and near the irradiated area. To confirm that this was indeed local activity, we then globally irradiated cells with UV, or added rapamycin to the bulk media, both of which induced global ruffle formation throughout the membrane. For a quantitative analysis, we divided target cells into four quadrants, and counted the frequency of ruffle formation in each quadrant upon local as well as following global stimulation under various conditions including controls (Figure 4). It is only this combination of UV irradiation, cRb-A, and appropriate FKBP-FRB constructs that induces local ruffling.
<img alt="Figure 1" src="/files/ftp_upload/3794/3794fig1.jpg" /> 
Figure 1. Schematic of spatially confined protein dimerization using caged rapamycin-avidin conjugate (cRb-A) and UV light. UV irradiation cleaves the linker between rapamycin and biotin, resulting in the release of chemical dimerizer (HE-Rapa, C40-O-(2-hydroxyethyl) rapamycin) and its by-product. The released dimerizer then diffuses into cells and induces dimerization between FKBP-POI (protein of interest) and plasma membrane anchored FRB only in the proximity of the irradiated region (blue circle). Without the UV irradiation, FKBP and FRB do not associate (red cross), hence producing no effect. (Copyright@ACS2011) 6
<img alt="Figure 2" src="/files/ftp_upload/3794/3794fig2.jpg" /> 
Figure 2. Picture of the customized confocal fluorescence microscope. (a) Frontal view of the confocal microscope (inverted Axiovert 200, Zeiss). (b) Close-up, top view of a coupling unit for EPI fluorescence and UV illumination.
<img alt="Figure 3" src="/files/ftp_upload/3794/3794fig3.jpg" /> 
Figure 3. Spatially confined ruffle formation in a transfected NIH3T3 cell induced by localized UV irradiation. Confocal fluorescence images of a NIH3T3 fibroblast cell that is transfected with YFP-tagged FKBP-Tiam1 (Rac activator) and LDR (membrane-anchored FRB) underwent irradiation in a confined area near its edge with LED light at 365 nm for 15 sec starting at 210 sec in the presence of cRb-A conjugate in the bulk media. This treatment was followed by global illumination with mercury lamp for 1 sec at 1063 sec and global addition of rapamycin final 400 nM at 1560 sec. The cell showed almost no ruffling activity at the beginning (a) but showed spatially confined ruffling after localized illumination (yellow circle) (b). The cell showed global ruffle formation after global illumination and addition of rapamycin(c-d), indicating that the local ruffle formation was caused by spatially confined activation of Rac. Scale bar: 10 &mu;m. (Copyright@ACS2011) 6
<img alt="Figure 4" src="/files/ftp_upload/3794/3794fig4.jpg" /> 
Figure 4. Frequency of ruffle formation in quadrants of transfected NIH3T3 cells. Local UV irradiation with cRb-A and following global UV irradiation were performed on 13 cells. cRb-A addition (no UV irradiation), local UV irradiation with or without avidin were implemented on 13, 5 and 6 cells, respectively. None of the observed cells showed ruffle formation with cRb-A without UV irradiation, or local UV irradiation with or without avidin in bulk media. We collected data from cells with low background ruffle formation but which had the capacity of rapamycin-induced ruffle formation, which we checked after each experiment by confirming global ruffle formation as a result of addition of rapamycin (final 400 nM). These results showed that cRb-A and local UV irradiation are necessary for local ruffle formation in the cells. (Copyright@ACS2011) 6
Abbreviations:
FKBP: FK506-binding protein 
FRB: FKBP-rapamycin-binding protein 
Tiam1: T-cell lymphoma invasion and metastasis-inducing protein 1 (Rac GEF) 
LDR: Lyn-DSAG linker-FRB (Lyn: N-terminal 11 amino acids of Lyn kinase) 
HE-Rapa: C40-O-(2-hydroxyethyl) rapamycin 
cRb: caged rapamycin with biotin moiety 
cRb-A: cRb conjugated to avidin

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	<li>Etienne-Manneville, S., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+cell+biology.">Rho GTPases in cell biology.</a> Nature. 420, 629-635 (2002).</li><li>Takai, Y., Sasaki, T., Matozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTP-binding+proteins.">Small GTP-binding proteins.</a> Physiol. 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No conflicts of interest declared.

We described a technique that employs a novel caged compound together with the FKBP-FRB heterodimerization system in order to manipulate Rho GTPase activity at a precise subcellular location on a time scale of seconds. 
There are three limitations of the present approach. Firstly, because the method is based on preventing or allowing diffusion of dimerizer across the plasma membrane, the target cellular location needs to be plasma membrane or its vicinity. Further optimization of the chemical ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Rapamycin</td>
 <td>Tecoland</td>
 <td>RAPA99</td>
 </tr>
 <tr>
 <td>FuGENE HD</td>
 <td>Roche</td>
 <td>04709691001</td>
 </tr>
 <tr>
 <td>Avidin</td>
 <td>Sigma</td>
 <td>A9275-10MG</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650-5X5ML</td>
 </tr>
 <tr>
 <td>Size-exclusion column</td>
 <td>GE Healthcare</td>
 <td>Spin Trap G-25</td>
 </tr>
 <tr>
 <td>Glass bottom 8-well chamber</td>
 <td>Thermo Scientific</td>
 <td>12-565-47</td>
 </tr>
 <tr>
 <td>Inverted Fluorescence microscope</td>
 <td>Zeiss</td>
 <td>Axiovert200M</td>
 </tr>
 <tr>
 <td>UV LED light source</td>
 <td>Rapp Opto Electronics</td>
 <td>UVILED</td>
 </tr>
 <tr>
 <td>Confocal spinning disk</td>
 <td>Yokogawa Electric Corp.</td>
 <td>CSU10</td>
 </tr>
 <tr>
 <td>CCD camera</td>
 <td>Hamamatsu Photonics</td>
 <td>ORCA-ER</td>
 </tr>
 <tr>
 <td>100x Objective lens</td>
 <td>Zeiss</td>
 <td>Plan Apochromat</td>
 </tr>
 </tbody>
</table>
 Synthetic scheme of cRb (Copyright ACS2011) 6
 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="Scheme 1" /></a> 
Synthetic conditions: (a) BnBr, K2CO3, DMF (b) f.HNO3, AcOH (c) TFA (d) Propargyl-Br, K2CO3, DMF, (e) glycerol, cat. pTsA, Toluene (f) NaBH3CN, TiCl4, MeCN (g) Tf2O, Pyridin (h) methanol HCl, (i) LiAlH4, THF (j) TsCl, Pyridine, CH2Cl2 (k) NaN3, DMF (l) 8, 2,6-di-t-butylpyridine, CH2Cl2 (m) 13, CuSO4, Ascorbate, 2-propanol, H2O, CH2Cl2.

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. 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 Rac4, Cdc424, RhoA4 and Ras5, 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 cells6. 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 level6.

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

spatio-temporal-manipulation-small-gtpase-activity-at-subcellular

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Bioengineering

10.8K Views.  Johns Hopkins University. A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Video: Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...