Name: pip-on-a-chip: a label-free study of protein-phosphoinositide interactions
Uploaded: 2017-07-27T13:00:00.000+00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

DS 및 CEC는 부분적으로는 보조금 AI053531 (NIAID, NIH)에 의해 지원되었다. SS 및 PSC는 보조금 N00014-14-1-0792 (ONR)에 의해 지원되었습니다.

저자는 공개 할 것이 없습니다.

각각의 PIP 변이체는 저농도 임에도 불구하고 특정 세포 소기관의 세포질 표면에 존재하며, 세포막 고유의 물리적 구성과 기능적 특이성의 형성에 기여합니다 1 . PIP를의 가장 중요한 용도 중 하나는 특정 세포 내 현지화 및 / 또는 활성화 6, 7을 필요로하는 단백질의 다수에 대한 특정 도킹 플랫폼이다. 세포 생리학 및 질병에서의 역할?...

무수한 상호 작용과 생화학 적 과정은 2 차원 유체 막 표면에서 일어난다. 진핵 세포의 멤브레인 - 둘러싸인 세포 기관은 생화학 적 과정과 관련 프로테옴뿐만 아니라 지질 조성에서도 독특합니다. 예외적으로 인지질의 한 종류는 phosphoinositides (PIPs)입니다. 이들이 세포질 리피도미스의 1 %만을 차지한다고 할지라도, 이들은 신호 변환,자가 식욕 (autophagy), 막 인신 매매 (membrane trafficking)에서 중요한 역할을한다. 그 중에서도 1 , 2 , 3 , 4 가 중요하다. 셀룰러 PIP 키나제하여 이노시톨 헤드 그룹의 동적 인산화 일곱 PIP 모노있다 headgroups, 비스 -, 또는 트리스 - (5)의 인산화를 일으킨다. 또한, PIP는 세포막의 세포 내 신원을 정의하고 하나 이상의 포스 포이 노를 함유하는 단백질 / 효소에 대한 특화된 막 도킹 사이트로서 작용한다예를 들어, 도메인을 itide 결합, 플렉스 트린 상 동성 (PH) Phox 동성 (PX)과 epsin N 말단 동성 (ENTH) 6,7. 가장 많이 연구 된 PIP 결합 도메인 중 하나는 특히 높은 나노 몰 낮은 마이크로 몰 범위의 친 화성 8에서 포스파티딜 이노시톨 4,5- 비스 포스페이트 (PI (4,5) P 2)과 상호 작용하는 포스 포 리파제 C (PLC) -δ1의 PH 도메인이고 , 9 , 10 , 11 . 다양한 정성 및 정량 시험관 방법이 개발되어 이러한 상호 작용의 메커니즘, 열역학 및 특이성을 연구합니다. 가장 일반적으로 사용되는 PIP 결합 분석에는 표면 플라스 몬 공명 (SPR), 등온 열량계 (ITC), 핵 자기 공명 (NMR) 분광기, 리포좀 부유 / 침강 분석법 및 지질 블롯 (Fat-blots / PIP-strips)12 , 13 . 이것들이 광범위하게 이용 되더라도 그들은 모두 많은 단점을 가지고 있습니다. 예를 들어, SPR, ITC 및 NMR은 많은 양의 시료, 값 비싼 계측 및 / 또는 숙련 된 인원 12 , 13을 필요로 합니다. 항체 기반 lipid-blots와 같은 일부 분석 형식은 PIP의 수용성 형태를 사용하고 비 생리 학적 방식으로 12 , 14 , 15 , 16로 표시 합니다. 또한, 지질 블롯은 신뢰할 수있게 정량 될 수 없으며 종종 가양 성 / 음성 관찰 12 , 17 , 18을 초래합니다. 이러한 어려움을 극복하고 현재 도구 세트를 개선하기 위해 지원되는 지질 이중층 (lipid bilayer, SLB)을 기반으로 새로운 라벨없는 방법이 am<html> ( 그림 1 ) 19 단백질 - PIP 상호 작용의 연구에 성공적으로 적용된 마이크로 유체 플랫폼. 단백질 -PIP 상호 작용을 검출하기 위해 사용 된 전략은 pH 변조 감지에 기초한다. 이것은 phosphatidylethanolamine lipid head group 20에 직접 conjugated ortho -sulforhodamine B ( o SRB)가있는 pH 민감성 염료를 포함합니다. 오의 SRB-POPE 프로브 (도 2a)는 낮은 pH에서 높은 형광 및 7.5 몰 %의 PI (4,5) P (2) 함유 SLB 수 (도 5b)에서 약 6.7의 pKa 높은 pH에서 켄칭된다. PLC-δ1 PH 도메인은 PI (4,5) P 2 ( 도 5A )에 대한 높은 특이성으로 인해 단백질 -IP 결합 방법의 유효성 확인에 광범위하게 사용되어왔다 ( 도 5A ) 21 , 22 ,&quot;&gt; 23, 24, 25 .Hence, 우리는 PLC-δ1의 PH 도메인은 그것의 PI 결합 테스트하는데 사용될 수 있다는 추론 (4,5)가 PIP 온 칩 분석을 통해 P 2. 산도 도메인 구조 본 연구에서 사용은 순 양전하 PI (8.4)을 가지며, 따라서 OH 유치 -.받는 이온 - PI (4,5) P (2) 함유 SLB 수 결합시 이온 (도 5c)를 상기 PH 도메인은 OH를 가져온다 결과적으로 계면 전위를 변조하여 O SRB-POPE (도 5C) 26. 산도 도메인 농도의 함수로서, 형광 켄칭된다 (도 6A)의 양성자 화 된 상태를 시프트 막 표면. 최종적으로, 정규화 된 데이터는 산도 도메인 PI (4,5) P (2)의 상호 작용 (도 6b,도 6c).의 친 화성을 결정하기 위해 결합 등온선에 맞게 &lt;/ p&gt; 이 연구에서는 미세 유체 플랫폼 내에서 PIP를 함유 한 SLB에 단백질 결합을 수행하기위한 상세한 프로토콜이 제공됩니다. 이 프로토콜은 독자가 microfluidic 장치와 소낭 준비를 SLB 형성과 단백질 바인딩으로 조립하는 것을 필요로합니다. 또한, 데이터 분석을위한 지시는 PLC-δ1의 PH 도메인-PI (4,5) P (2)의 상호 작용이 제공되는 선호도에 대한 정보를 추출한다.

<table><tbody><tr><td>&lt;strong&gt;Coverslip&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glass Coverslips: Rectangles</td><td>Fisher Scientific</td><td>12-544B</td><td>22 x 40 x 0.16 - 0.19 mm, No. 1 1/2; Borosilicate Glass</td></tr><tr><td>7X Cleaning Solution</td><td>MP Biomedicals</td><td>976670</td><td>Detergent</td></tr><tr><td>PYREX Crystallizing Dish</td><td>Corning</td><td>3140-190</td><td>Borosilicate glass dish with a flat bottom; Diameter x Height (190 x 100 mm); Distributor: VWR (89090-700)</td></tr><tr><td>Sentry Xpress 2.0</td><td>Paragon Industries</td><td>SC-2</td><td>Kiln</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;PDMS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>4019862</td><td>Polydimethylsiloxane (PDMS); Distributor: Ellsworth Adhesives</td></tr><tr><td>PYREX Desiccator</td><td>VWR</td><td>89134-402</td><td>Vacuum Rated</td></tr><tr><td>Biopsy punch</td><td>Harris</td><td>15110-10</td><td>Harris Uni-Core; 1.0 mm diameter; Miltex Biopsy Punch with Plunger (Cat. No. 15110-10) can be used as an alternative</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Device&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasma Cleaning System</td><td>PlasmaEtch</td><td>PE25-JW</td><td>2-stage Direct Drive Oil Vacuum Pump, O2 service (Krytox Charged)</td></tr><tr><td>Digital Hot Plate</td><td>Benchmark</td><td>H3760-H</td><td>Purchased through Denville Scientific (Cat. No. 1005640)</td></tr><tr><td>Frosted Micro Slides</td><td>VWR</td><td>48312-003</td><td>Frosted, Selected, and Precleaned; Made of Swiss Glass; Thickness: 1 mm; Dimensions: 75 x 25 mm; GR 144</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mold&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AutoCAD</td><td>Autodesk</td><td>v.2016</td><td>Drafting software for the photomask design</td></tr><tr><td>Photomask</td><td>CAD/Art Services</td><td>N/A</td><td>Design with black background and clear features was printed at 20k dpi resolution on a transparent mask (5 x 7 in) by CAD/Art Services</td></tr><tr><td>Silicone Wafers</td><td>University Wafer</td><td>1575</td><td>Prime Grade, Single Side Polished; 100 mm (4 inch) Diameter; 525 um Thickness</td></tr><tr><td>SU-8 50</td><td>MicroChem Corp.</td><td>N/A</td><td>Negative Tone Photoresist; Penn State Nanofabrication Facility Property</td></tr><tr><td>SU-8 Developer</td><td>MicroChem Corp.</td><td>N/A</td><td>Penn State Nanofabrication Facility Property</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;SUV&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850457C</td><td>POPC</td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4-phosphate</td><td>Avanti Polar Lipids</td><td>840045X</td><td>PI4P</td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate</td><td>Avanti Polar Lipids</td><td>840046X</td><td>PI(4,5)P2</td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine</td><td>Avanti Polar Lipids</td><td>850757C</td><td>POPE; Required for the synthesis of oSRB-POPE</td></tr><tr><td>Lissamine Rhodamine B Sulfonyl Chloride (mixed isomers)</td><td>ThermoFisher Scientific</td><td>L-20</td><td>Required for the synthesis of oSRB-POPE</td></tr><tr><td>pH Sensitive Fluorescent Lipid Probe (oSRB-POPE)</td><td>In-house</td><td>N/A</td><td>In-house Synthesis (Huang D. et al. 2013)</td></tr><tr><td>Glass Scintillation Vial</td><td>VWR</td><td>66022-065</td><td>20 mL volume capacity</td></tr><tr><td>Aquasonic 250D</td><td>VWR</td><td>N/A</td><td>Ultrasonic Water Bath</td></tr><tr><td>Nuclepore Track-Etched Membranes</td><td>Whatman</td><td>110605</td><td>Polycarbonate Membrane; Diameter: 25 mm; Pore Size: 0.1 um; Distributor: Sigma-Aldrich</td></tr><tr><td>Chloroform</td><td>VWR</td><td>CX1054-6</td><td>HPLC grade</td></tr><tr><td>LIPEX Extruder</td><td>Transferra Nanosciences</td><td>T.001</td><td>LIPEX 10 mL Thermobarrel Extruder</td></tr><tr><td>Viscotek 802 DLS</td><td>Malvern Instruments</td><td>N/A</td><td>Dynamic Light Scattering; Penn State X-Ray Crystallography Facility Property</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Data Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>v.6</td><td>Curve-fitting software for data analysis</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Axiovert 200M Epifluorescence Microscope</td><td>Carl Zeiss Microscopy</td><td>N/A</td><td>Microscope</td></tr><tr><td>AxioCam MRm Camera</td><td>Carl Zeiss Microscopy</td><td>N/A</td><td>Camera</td></tr><tr><td>X-Cite 120</td><td>Excelitas Technologies</td><td>N/A</td><td>Light Source</td></tr><tr><td>Alexa 568 Filter Set</td><td>Carl Zeiss Microscopy</td><td>N/A</td><td>Ex/Em 576/603 nm</td></tr><tr><td>AxioVision LE64 v.4.9.1.0 Software</td><td>Carl Zeiss Microscopy</td><td>N/A</td><td>Image Processing Software</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tips</td><td>VWR</td><td>10034-132</td><td>200 uL pipette tips; Thin and smooth tip for applying the protein solution into the microfluidic channel</td></tr><tr><td>Tips</td><td>VWR</td><td>53509-070</td><td>10 uL pipette tips; Thin and smooth tip for applying the vesicle solution into the microfluidic channel</td></tr><tr><td>Orion Star A321 pH meter</td><td>Thermo Scientific</td><td>STARA3210</td><td>pH meter</td></tr><tr><td>Orion micro pH probe</td><td>Thermo Scientific</td><td>8220BNWP</td><td>micro pH probe</td></tr><tr><td>N-(2-Hydroxyethyl)-Piperazine-N&#039;-(2-Ethanesulfonic Acid)</td><td>VWR</td><td>VWRB30487</td><td>HEPES, Free Acid</td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>BDH8014-2.5KGR</td><td>NaCl</td></tr><tr><td>Tubing</td><td>Allied Wire &amp;amp; Cable</td><td>TFT-200-24 N</td><td>Internal Diameter: 0.020-0.026 inches (0.051-0.066 cm); Wall Thickness: 0.010 inches (0.025 cm); Flexible Polytetrafluoroethylene Thin-Wall Tubing; Natural Color</td></tr><tr><td>Nitrogen Gas - Industrial</td><td>Praxair</td><td>N/A</td><td>Local Provider</td></tr><tr><td>Oxygen Gas - Industrial</td><td>Praxair</td><td>N/A</td><td>Local Provider</td></tr><tr><td>Liquid Nitrogen</td><td>Praxair</td><td>N/A</td><td>Local Provider</td></tr></tbody></table>

pip-on-a-chip: a label-free study of protein-phosphoinositide interactions

수많은 세포 단백질이 막 표면과 상호 작용하여 필수 세포 과정에 영향을줍니다. 이러한 상호 작용은 phosphoinositides (PIPs)의 경우처럼 특정 세포 내 지방화 및 / 또는 활성화를 보장하기 위해 멤브레인 내의 특정 지질 성분을 향하게 할 수 있습니다. PIP와 세포질 PIP 결합 도메인은 세포 생리학에서 그들의 역할을 더 잘 이해하기 위해 광범위하게 연구되어 왔습니다. 우리는 protein-PIP 상호 작용을 연구하기위한 도구로서지지 된 지질 이중층 (SLBs)에 pH 조절 분석을 적용했습니다. 이 연구에서, pH 민감성 ortho- sulforhodamine B conjugated phosphatidylethanolamine은 protein-PIP 상호 작용을 검출하는 데 사용됩니다. 단백질이 PIP 함유 멤브레인 표면에 결합하면, 계면 전위가 조절되어 ( 즉 , 국소 pH 변화), 프로브의 양성자 화 상태가 변하게됩니다. pH 조절 분석의 성공적인 사용에 대한 사례 연구는 phospholipase C delta1 Pleckstr상동 (PLC-δ1의 PH) 도메인 및 예로서, 포스파티딜 이노시톨 -4,5- 비스 포스페이트 (PI (4,5) P 2)의 상호 작용이다. 이 상호 작용에 대한 겉보기 해리 상수 ( K d, app )는 0.39 ± 0.05 μM이었고 , 다른 것들에 의해 얻어진 K d 값과 유사했다. 이전에 관찰 된 바와 같이, PLC-δ1 PH 도메인은 PI (4,5) P 2 특이 적이며, 포스파티딜 이노시톨 4- 인산염에 대한 약한 결합을 나타내며, 순수한 포스파티딜콜린 SLB에 대한 결합은 보이지 않는다. PIP-on-a-chip 분석은 낮은 샘플 부피 및 리간드 / 수용체 표지 요구 사항이 없지만 작은 및 낮은 친화도 막 상호 작용을 테스트 할 수있는 능력을 포함하되 이에 국한되지 않는 전통적인 PIP 결합 분석보다 유리합니다. 큰 분자 및 개선 된 신호 대 잡음비. 따라서, PIP-on-a-chip 접근법의 사용은 광범위한 막 상호 작용 메커니즘의 해명을 용이하게 할 것이다. 또한이 방법은 잠재적으로세포막과 상호 작용할 수있는 단백질의 능력을 조절하는 치료제를 확인하는데있어

우리는 PIP-on-a-chip 마이크로 디바이스 ( 그림 1 ) 내에서 PLC-δ1 PH 도메인 -PI (4,5) P 2 상호 작용을 연구하기 위해 pH 변조 분석을 사용했습니다. 상세한 프로토콜을 통해 마이크로 유체 소자 구성 요소를 준비 및 조립하고 소형 단층 소포 (SUV)를 만들고 ( 그림 2 ) 장치 내 SLB를 형성하고 ( 그림 3 ) PIP를 ?...

Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

여기서 우리는 pH 조절에 기반한 라벨없는 방법을 사용하여 단백질 - 포스 포이 노시 티드 상호 작용을 연구하기위한 마이크로 유체 플랫폼의 맥락에서지지 된 지질 이중층을 제시합니다.

PIP-on-a-chip : 단백질 - 포스 포이 노시 티드 상호 작용에 대한 라벨없는 연구

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

pip-on-chip-label-free-study-protein-phosphoinositide

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Bioengineering

9.4K 조회수.  The Pennsylvania State University.  여기서 우리는 pH 조절에 기반한 라벨없는 방법을 사용하여 단백질 - 포스 포이 노시 티드 상호 작용을 연구하기위한 마이크로 유체 플랫폼의 맥락에서지지 된 지질 이중층을 제시합니다. 

비디오: PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned 표면은 암세포와 Hyaluronic 산성 상호 작용을 연구하기 위해

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

연구

생체공학

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

소설 형광 프로브와 플라즈마 막에서 포스파티딜 이노시톨 -4,5- 비스 포스페이트의 단일 분자 슈퍼 해상도 이미징

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

생화학

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Hematologic 질환의 Microvascular 상호 작용 유학을위한 Endothelialized Microfluidics

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

라만 분광학을 사용하여 세포와 세포외 기질의 비 접촉, 레이블이없는 모니터링

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

고성능 액체 크로마토 그래피 결합 흐름 섬광에 의한 방사성 표지 및 Phosphoinositides의 세포 수준의 정량화

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

화학

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

바이오-레이어 간섭계 분석 결과 의해 작은 분자와 이온 채널 단백질의 상호 작용 활동을 캡처

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

이노시톨 인산염 또는 인산성교단백질의 동정은 서부 블롯 또는 질량분광법에 결합된 친화성 크로마토그래피에 의한 단백질

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

형광 기반 측정 인스파디딜세린/포스파디딜리노시톨 4-인산염 교환 막 간

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

pH Modulation Assay on Supported Lipid Bilayers to Detect Protein-Phosphoinositide Interactions

Source: Shengjuler, D., et al., <a href="https://app.jove.com/v/55869">PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions.</a> J. Vis. Exp. (2017)
In this video, we demonstrate protein-phosphoinositide interactions in a microfluidic pattern using the pH modulation assay, using pH-sensitive fluorescent dye tagged to phosphatidylethanolamine lipid of a supported lipid bilayer, called a microfluidic platform. The modulations in pH after protein binding to phospholipid cause fluorescence quenching.

단백질-포스포이노시티드 상호작용을 검출하기 위해 지원되는 지질 이중층에 대한 pH 조절 분석

Source: Shengjuler, D., et al., PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions. J. Vis. Exp. (2017)
In this video, we ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-lipid interactions

Phosphoinositides and PIPs

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane compartments imparts &#8220;surface identity&#8221; to the cell membrane. The phosphorylated heads of PIPs are recognized and bound by particular proteins, including proteins that help in vesicle coat assembly.
For example, the cytosolic side of the plasma membrane has high concentrations of the PI(4,5)P2, which helps recruit proteins such as dynamin and AP2 for clathrin-mediated endocytosis. PI(4,5)P2 is rapidly formed and destroyed by localized enzymes at particular locations and at specific times. This helps control the precise timing of clathrin-coated vesicle formation. Once the coated vesicle pinches off the plasma membrane during endocytosis, PI(4,5)P2 is destroyed from the site.
Many such PIs are involved in the secretory or endocytic pathways, such as&#160; PI(3)P localized at early endosomes and intraluminal vesicles of late endosomes,&#160; PI(4)P localized at the TGN, secretory granules, and synaptic vesicles; and PI(3,5)P2 localized at the late endosome boundary membrane.

포스포이노시티드 및 PIP

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ...