The authors acknowledge Dr. Mark Shaaya for his contribution to the development of LightR enzymes and associated protocols. pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573), pcDNA3.1 Floxed-STOP mCherry was a gift from Mositoshi Sato (Addgene plasmid #122963), bRaf-Venus construct bearing V600E mutation was a gift from Dr. John O&#39;Bryan (MUSC); ERK2 gene from pCEFL-ERK2 (a gift from Dr. Channing Der&#39;s lab, UNC) was cloned into mCherry-C1 backbone to obtain mCherry-ERK2 plasmid; and pmiRFP670-N1 was a gift from Vladislav Verkhusha (Addgene plasmid # 79987). The work was supported by NIH grants R33CA258012, R35GM145318, and P01HL151327 to AK. This work was further supported by the T32 VBST training fellowship T32HL144459 to NL.

Optogenetic Control of Enzyme Activity Using Light-Regulated Allosteric Switch

The authors have nothing to disclose.

Our study presents an optogenetic approach for the investigation of diverse signaling pathways and demonstrates its wide applicability in addressing different biological questions. The LightR tool system provides several essential advantages: (1) Allosteric regulation of protein activity, (2) Tight temporal control of activity that can be tuned to achieve different kinetics of activation and inactivation, (3) Spatial resolution of activity at the subcellular level, (4) Specificity of signaling modulation and biological a...

The ability of the cell to interpret external signals and convert them into specific responses in physiological or pathological contexts is directed by dedicated groups of proteins. The contribution of any protein to such complex responses is often defined by its subcellular location, level of expression, and the timing of transient, sustained, or oscillatory activation. Dissecting the role of these individual parameters in the regulation of signaling demands methods capable of replicating intricate spatiotemporal control of protein activity at the subcellular level. Traditional techniques such as genetic manipulation and small molecule inhibitors fall short in this regard. In contrast, optogenetic techniques promise the potential for the dissection of biological processes by manipulating or mimicking physiological and pathological processes. However, current tools often lack broad applicability or subcellular control. Several existing strategies achieve tight regulation of protein localization and interactions; but lack direct control over enzymatic activity1,2,3,4. Others enable regulation of enzymatic activity but may lack subcellular control or have limited applicability across different enzyme classes5,6,7,8,9. In this protocol paper, we describe a novel protein engineering method that combines the advantages of optogenetics into one tool: tight temporal regulation, tunable kinetics, subcellular control, and broad enzyme applicability10. We engineered a Light-regulated (LightR) domain that functions as an allosteric switch when inserted into the target protein of interest. This strategy enables tight spatiotemporal control of the activation and deactivation of a protein of interest in living cells.
Here, the design and application strategy for the LightR optogenetic tool across several enzyme classes is discussed. This study offers a step-by-step protocol for the development, characterization, and application of light-regulated tyrosine kinase Src (LightR-Src). The study further demonstrates the tunability of LightR switch inactivation kinetics. The slow-inactivating LightR switch can maintain enzyme activity with reduced frequency of illumination, whereas fast cycling version, FastLightR, requires more frequent illumination for activation but shows fast inactivation when illumination is turned off. Activation/inactivation of FastLightR-Src in living cells induces cycles of cell spreading and retraction. FastLightR-Src-induced cell spreading agrees with the physiological role of Src kinase11,12. Due to fast inactivation kinetics, FastLightR-Src can also be regulated at a subcellular level, resulting in stimulation of local protrusions and cell polarization. To demonstrate the applicability of the LightR tool to other types of enzymes, we briefly walk the readers through similar success with engineered light-regulated kinase bRaf (LightR-bRaf, FastLightR-bRaf) and a site-specific DNA recombinase Cre (LightR-Cre). Overall, this approach has the potential to advance our understanding of complex signaling pathways shaping the pathophysiology of diverse diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>#1.5 Glass Coverslips 25 mm Round</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0715&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Tubes&#160;</td>
			<td>USA Scientific&#160;</td>
			<td>cc7682-3394</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Buffer</td>
			<td></td>
			<td></td>
			<td>For 500 mL: 5.18 g of&#160; Tris-HCL, 131.5 mL of glycerol, 52.5 mL of 20% SDS, 0.5 g of bromophenol blue, final pH 6.8</td>
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		<tr>
			<td>4-20% Mini-PROTEAN TGX Precast Gel</td>
			<td>Biorad</td>
			<td>4561096</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Phusion Plus Buffer&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>F538L</td>
			<td></td>
		</tr>
		<tr>
			<td>60 LED Microscope Ring Light</td>
			<td>Boli Optics</td>
			<td>ML46241324</td>
			<td>Blue LED, 60 mm diameter, 5 W</td>
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		<tr>
			<td>Agarose&#160;</td>
			<td>GoldBiotech</td>
			<td>A-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Erk 1/2 Antibody</td>
			<td>Cell Signaling</td>
			<td>9102</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAPDH Antibody</td>
			<td>invitrogen</td>
			<td>AM4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP</td>
			<td>Clontech</td>
			<td>632380</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mCherry Antibody</td>
			<td>invitrogen</td>
			<td>M11217</td>
			<td></td>
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		<tr>
			<td>Anti-MEK</td>
			<td>Cell Signaling</td>
			<td>9122</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p130Cas</td>
			<td>BD Biosciences</td>
			<td>610271</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-paxillin</td>
			<td>Cell Signaling</td>
			<td>2542</td>
			<td></td>
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			<td>Anti-phospho-Erk 1/2 T202/Y204 Antibody</td>
			<td>Cell Signaling</td>
			<td>9101</td>
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			<td>Anti-phospho-pY249 p130Cas</td>
			<td>Cell Signaling</td>
			<td>4014</td>
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			<td>Anti-phospho-Y118 Paxillin</td>
			<td>Cell Signaling</td>
			<td>2541</td>
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			<td>Anto-phospho-S217/221 MEK</td>
			<td>Cell Signaling</td>
			<td>9121</td>
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			<td>Arduino Compatable Power Supply</td>
			<td>Corporate Computer</td>
			<td>LJH -186</td>
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			<td>Arduino Uno Rev3</td>
			<td>Arduino</td>
			<td>&#8206;A000066</td>
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			<td>Attofluor Cell Chamber</td>
			<td>invitrogen</td>
			<td>A7816</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchmark Fetal Bovine Serum (FBS)&#160;</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td>Heat Inactivated Triple 0.1 &#181;m sterile-filtered&#160;</td>
		</tr>
		<tr>
			<td>bRaf-V600E-Venus</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. John O&#39;Bryan, MUSC</td>
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		<tr>
			<td>BSA&#160;</td>
			<td>GoldBiotech</td>
			<td>A-420&#160;</td>
			<td></td>
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		<tr>
			<td>Carbenicillin (Disodium)</td>
			<td>Gold Biotechnology</td>
			<td>C-103-25</td>
			<td></td>
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		<tr>
			<td>CellMask Deep Red plasma membrane dye</td>
			<td>invitrogen</td>
			<td>c10046</td>
			<td></td>
		</tr>
		<tr>
			<td>Colony Screen MasterMix&#160;</td>
			<td>Genesee</td>
			<td>42-138</td>
			<td></td>
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		<tr>
			<td>DH5a competent cells&#160;</td>
			<td>NEB&#160;</td>
			<td>C2987H</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Corning&#160;</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Ladder&#160;</td>
			<td>GoldBio&#160;</td>
			<td>D010-500</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs&#160;</td>
			<td>NEB</td>
			<td>N04475</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpn1 Enzyme&#160;</td>
			<td>NEB</td>
			<td>R01765</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>GoldBio</td>
			<td>DTT10</td>
			<td>DL-Dithiothreitol, Cleland&#39;s Reagents&#160;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros&#160;</td>
			<td>409910250</td>
			<td></td>
		</tr>
		<tr>
			<td>FastLightR-bRaf-mVenus</td>
			<td>Addgene</td>
			<td>#162155</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma&#160;</td>
			<td>Sigma&#160;</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE(R) 6 Transfection Reagent&#160;</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Gel Green Nucleic Acid Stain&#160;</td>
			<td>GoldBio</td>
			<td>G-740-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye Purple 6x&#160;</td>
			<td>NEB</td>
			<td>B7024A</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneJET Gel extraction Kit&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>K0692</td>
			<td>Gel Extraction Kit&#160;</td>
		</tr>
		<tr>
			<td>GeneJET Plasmid Miniprep Kit</td>
			<td>Thermo Scientific&#160;</td>
			<td>K0502</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>GlutaMAX-l (100x) 100 mL&#160;</td>
		</tr>
		<tr>
			<td>HEK 293T Cells&#160;</td>
			<td>ATCC&#160;</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa Cells&#160;</td>
			<td>ATCC&#160;</td>
			<td>CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fischer&#160;</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Iot Relay</td>
			<td>Digital Loggers</td>
			<td>DLI 705020645490</td>
			<td>AC/DC control relay for illumination</td>
		</tr>
		<tr>
			<td>Kanamycin Monosulfate</td>
			<td>Gold Biotechnology</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma&#160;</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 1x</td>
			<td>Corning&#160;</td>
			<td>10-045-CV&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar&#160;</td>
			<td>Fisher</td>
			<td>BP1425-2</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Grow Light System</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td>LED panel lamp system&#160;</td>
		</tr>
		<tr>
			<td>LightR-bRaf-mVenus</td>
			<td>Addgene</td>
			<td>#162154</td>
			<td></td>
		</tr>
		<tr>
			<td>LightR-iCre-miRFP670</td>
			<td>Addgene</td>
			<td>#162158</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2024a</td>
			<td>Software for running CellGEO Scripts</td>
		</tr>
		<tr>
			<td>Metamorph</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Imaging Analysis Software</td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma&#160;</td>
			<td>M5310</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniPrep Kit&#160;</td>
			<td>Gene Choice&#160;</td>
			<td>96-308</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Precast Gels 12 well</td>
			<td>Bio-Rad</td>
			<td>4561085</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water&#160;</td>
			<td>Corning&#160;</td>
			<td>46-000-CV&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiband Polychroic Mirror</td>
			<td>89903BS</td>
			<td>Chroma</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Fisher Chemical</td>
			<td>S271-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o Ca and Mg&#160;</td>
			<td>Corning&#160;</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-iCre</td>
			<td>Addgene</td>
			<td>#89573</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1_Floxed-STOP mCherry</td>
			<td>Addgene</td>
			<td>#122963</td>
			<td></td>
		</tr>
		<tr>
			<td>pCEFL-ERK2</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Channing Der&#39;s Lab, UNC</td>
		</tr>
		<tr>
			<td>PCR Tubes&#160;</td>
			<td>labForce</td>
			<td>1149Z65</td>
			<td>0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps&#160;</td>
		</tr>
		<tr>
			<td>Phusion Plus DNA Polymerase</td>
			<td>Thermo Scientific&#160;</td>
			<td>F630S</td>
			<td></td>
		</tr>
		<tr>
			<td>pmiRFP670-N1</td>
			<td>Addgene</td>
			<td>#79987</td>
			<td></td>
		</tr>
		<tr>
			<td>Polygon 400 Patterned Illuminator</td>
			<td>Mightex</td>
			<td>DSI-G-00C</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Membranes</td>
			<td>BioRad</td>
			<td>1620219</td>
			<td>Immun-Blot PVDF/Filter Paper Sandwiches&#160;</td>
		</tr>
		<tr>
			<td>T0.25% Trypsin, 2.21 mM, eDTA, 1x [-] sodium</td>
			<td>Corning&#160;</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Acetate-EDTA (TAE) 50x&#160;</td>
			<td>Fischer&#160;</td>
			<td>BP1332-1&#160;</td>
			<td>For electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>UPlanSApo 40x Microscope Objective</td>
			<td>Olympus</td>
			<td>1-U2B828</td>
			<td></td>
		</tr>
		<tr>
			<td>USB TTL Box</td>
			<td>National Instruments</td>
			<td>6501</td>
			<td>For TTL interface</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Fisher Chemical&#160;</td>
			<td>O3446I-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

spatiotemporal control of protein activity through optogenetic allosteric regulation

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme&#39;s catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain&#39;s structure and enzyme activity. In this manuscript, we discuss design strategies&#160;to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

The LightR-Src is designed and generated following the strategy described in Figure 1A,B. Biochemical analysis of LightR-Src accesses the phosphorylation of known endogenous Src substrates, p130Cas (Y249)22 and paxillin (Y118)23 in response to blue light at 60 min of continuous illumination (Figure 2B). Notably, no background activation of Src kinase is observed when LightR-Src is expressed only in...

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most ...

spatiotemporal-control-protein-activity-through-optogenetic

Research

JoVE Journal

Biology

422 Views.  University of Illinois at Chicago. This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Video: Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...