이 작품은 국립 보건원에서 루이스 빌과 CA135123 대학 (CH까지)에서 시작 자금에 의해 지원됩니다.

1. 세포 배양 및 Transfection <ol><li> COS-7 세포는 4.5 g / L 포도당와 10 % 태아 소 혈청 (FBS)와 보충 Dulbecco 수정일 독수리의 중간 (DMEM)에서 배양되어 있습니다. </li><li> 플라스미드 transfection는 제조업체의 지시 사항 (Invitrogen)에 따라 Lipofectamine하여 이루어집니다. </li></ol> 2. 세포 표면 스네어의 표현의 형광 미세 분석 <ol><li> transfection 전날, 3 × 10 4 COS-7 세포는 24 잘 플레이트에 포함 된 멸균 12 mm 유리 coverslips (피셔 과학)에 놓는 있습니다. </li><li> TTA을 인코딩 그 플라스미드의 각 잘, 0.25 μg의 V-세포의 경우 (pTet 오프, CLONTECH) Express는 VAMPs을 뒤집은하는 plasmids의 0.25 μg과 cotransfected 있습니다. </li><li> 각 잘에 t-세포를 들어, 플라스미드 인코딩 트레 - LacZ (pBI-G, CLONTECH)의 0.25 μg는 0.25 μg 익스프레스 SNAP-25와 syntaxins 1 4 뒤집힌있는 plasmids의 각을 cotransfected 있습니다. </li><li> t 후 24 시간ransfection, 세포는 PBS에 4 % paraformaldehyde + + 10 분에 (PBS는 0.1 g / L CaCl 2, 0.1 g / L MgCl 2 보충) 한 다음 PBS에서 10 % FBS에서 차단 + + 30 분에 고정되어 있습니다. </li><li> 세포는 안티 - Myc 단클론 항체 9E10 (깔끔한 하이 브리 도마 문화 표면에 뜨는)에 1.5 시간에 incubated 수 있습니다. </li><li> 4 개의 세차 후 PBS + +, 셀 1:500의 희석에 FITC-복합 차 항체 (잭슨 Immunoresearch 연구소)와 1 시간에 incubated 수 있습니다. </li><li> 4 개의 세차 후 PBS + +, 레이블 셀 골드 antifade 시약 (Invitrogen)를 연장으로 장착되어 있습니다. </li><li> 공 촛점 이미지는 올림푸스 레이저 스캐닝 공 촛점 현미경에서 수집됩니다. 이미지는 어도비 포토샵 소프트웨어로 처리됩니다. </li></ol> 3. 세포 표면 올무 식의 FACS 분석 <ol><li> transfection 전날, 2 × 10 5 COS-7 세포는 6 잘 플레이트의 각 우물에 놓는 있습니다. </li><li> tra 후 24 시간과 nsfection은 스네어, pTet 오프 및 pBI-G의 plasmids, 방석을 뒤집은 세포는 PBS에 1 % paraformaldehyde와 + + 15 분에 고정하고 PBS에서 10 % FBS에서 + + 15 분에 차단됩니다. </li><li> 세포는 60 분에 반 Myc 단클론 항체 9E10와 incubated 수 있습니다. </li><li> 든 세 세차 후 PBS + +, 셀이 45 분에 FITC-복합 차 항체 (1:200 희석)으로 분류됩니다. </li><li> PBS 든 세 세차는 + +, 세포는 세포 스크레이퍼로 접시에서 긁어 후. </li><li> 15000 세포는 FACSCalibur 흐름 cytometer (BD Biosciences)를 사용하여 분석하고 있습니다. 각 샘플의 평균 형광 강도는 CellQuest Pro 소프트웨어를 사용하여 얻어진다. </li></ol> 4. 효소 세포 융합 분석 <ol><li> transfection 전날, 1.2 × 10 6 COS-7 세포는 각각 100 mm 조직 배양 접시에 놓는, 그리고 2아르 × 10 5 COS-7 세포는 6 잘 플레이트의 각 우물에 놓는 있습니다. </li><li> 5 μg flipp의 각 - V-세포, 0.5에 대한플라스미드 에드 즉석 반주는 각 100 mm 배양 접시에있는 세포에 pTet 오프 5 μg과 cotransfected 있습니다. 제어 세포는 빈 벡터 pcDNA3.1 (+)와 pTet 오프로 cotransfected 있습니다. VAMPs 1, 4, 5, 7, 8, tunicamycin의 N-글리코 실화를 방지하기 위해 (10 μg / ml) 쇼핑은 transfection시 세포 배양 매체에 포함되어 있습니다. </li><li> 6 잘 플레이트의 각 잘에 t-세포의 경우, 1 μg은 SNAP-25 플라스미드를 뒤집힌 및 pBI-G는 0.5 μg과 cotransfected 아르는 플라스미드 syntaxin4을 뒤집은의 플라스미드 syntaxin1 또는 0.05 μg을 뒤집어 졌죠. </li><li> 24 시간 transfection 후, V-세포는 효소가없는 세포 분리 버퍼 (Invitrogen)와 문화 요리에서 분리됩니다. 단독 세포가 hemacytometer로 간주하고에 resuspended 아르 HEPES 버퍼 DMEM 10 % FBS, 6.7 μg / ML의 tunicamycin 및 0.67 밀리미터 DTT와 함께 보충. </li><li> 4.8 × resuspended 10 5 V-셀은 각도 이미 t-세포를 포함에 추가됩니다. </li><li> 6, 12 시간 또는 24 시간 쥐 37 이후 ° C 5 % CO 2 </sub&gt;에서 β-갈 락토의 표현은 제조업체의 지침 (Promega)에 따라 리포터 용해 버퍼와 β-갈 락토 효소 분석 시스템을 사용하여 측정된다. 간단히, 세포는 PBS로 두 번 세척 된 다음 리포터 용해 버퍼에 lysed. 세포 lysates는 분석이 × 버퍼의 동등한 볼륨과 혼합되어 있습니다. 빈 컨트롤으로, 리포터 용해 버퍼는 분석이 × 버퍼과 혼합되어 있습니다. </li><li> 90 분 후, colorimetric 반응은 1 M 나트륨 탄산을 추가하여 중지됩니다. </li><li> 420 나노 미터의 흡광도는 히타치 100-40 분광 광도계를 사용하여 측정된다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

원래 세포 융합 분석 6 형광 현미경으로 올무로 인한 세포 융합 이벤트를 결정합니다. 여기 β-갈 락토 및 spectrometric 측정의 활성화 표현으로 올무로 인한 세포 융합 이벤트를 단정 지을 혁신적인 분석을 설명합니다. 이 분석을 사용하여, 우리는 정기적으로 15 분석 - 20 V-와 t-올무 한 실험에서 조합. 세포 표면에 올무 식을 측정하는 유동 세포 계측법을 사용하여, 우리는 VAMPs의 표현 수준을 ...

<table border="1"><tbody><tr><td> 시약 및 장비 명 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12800-017 </td></tr><tr><td> COS-7 세포 </td><td> ATCC </td><td> CRL-1651 </td></tr><tr><td> tunicamycin </td><td> 시그마 - 알드리치 </td><td> T7765-5MG </td></tr><tr><td> pTet 오프 </td><td> Clontech </td><td> K1620-A </td></tr><tr><td> pBI-G </td><td> Clontech </td><td> 6150-1 </td></tr><tr><td> lipofectamine </td><td> Invitrogen </td><td> 18324-012 </td></tr><tr><td> 효소가없는 세포 분리 솔루션 </td><td> Invitrogen </td><td> 13151-014 </td></tr><tr><td> 토끼 반 TET 억제 polyclonal 항체 </td><td> Millipore </td><td> AB3541 </td></tr><tr><td> FITC - 복합 당나귀 안티 - 마우스 IgG (H + L) </td><td> Jackson의 ImmunoResearch </td><td> 715-095-150 </td></tr><tr><td> 레이저 스캐닝 공 촛점 현미경 </td><td> 하늘 </td><td> FV1000 </td></tr><tr><td> FACSCalibur 흐름 cytometer </td><td> BD Biosciences </td><td></td></tr><tr><td> 리포터의 용해 버퍼와 β-갈 락토 효소 분석 시스템 </td><td> Promega </td><td> E2000 </td></tr><tr><td> 모델 100-40 UV-VIS 분광 광도계 </td><td> 히타치 </td><td> C740843 </td></tr></tbody></table>

analysis of snare-mediated membrane fusion using an enzymatic cell fusion assay

스네어 (들 oluble N-ethylmaleimide에 민감한 요소 ttachment 단백질 재를 ceptor) 소포 (V-그물)에 및 대상 멤브레인에 단백질의 상호 작용 (T-올무) 세포 소포의 융합 1-4 catalyze. Reconstitution의 assays는 올무로 인한 막 융합 (5)의 체계 및 규정을 해부에 필수적입니다. 세포 융합 분석 6,7에서 스네어 단백질은 세포 표면에 ectopically 표현됩니다. 이러한 올무가 세포 세포막을 융합하기에 충분한 것으로 입증, 스네어 단백질 드라이브 세포 세포 융합 &quot;을 뒤집힌.&quot; 세포 융합 분석은 현미경 분석을 기반으로하기 때문에 여러 V-와 t-올무 상호 작용 양적 분석하는 데 사용하면 덜 효율적이다. 여기 β-갈 락토의 활성화 표현으로 올무로 인한 세포 융합 이벤트를 단정 지을만한 새로운 검정 8 설명합니다. 두테트라 사이클린 제어 transactivator (TTA) 및 테트라 사이클린 - 응답 요소의 제어 (트레 - LacZ)에서 LacZ 유전자를 인코딩 플라스미드 기자 : Tet 오프 유전자 발현 시스템 9의 구성 요소는 판독 시스템으로 사용됩니다. 익스프레스 익스프레스는 세포 표면에 t-올무 단백질을 뒤집은 것이 COS-7 세포 (T-세포)로 세포 표면의 V-스네어 단백질 (V-세포)와 transfect 트레 - LacZ를 뒤집힌 것을 COS-7 세포에 우리 transfect TTA . 트레 전사 LacZ의 활성화 및 β-갈 락토 표현에 TTA의 바인딩의 V-및 T-세포 결과의 올무에 의존 융합. β-갈 락토의 활동은 420 nm의에서 흡수하여 colorimetric 방법을 사용하여 정량하고 있습니다. 소포 - 관련 막 단백질 (VAMPs)는 다양한 사후 Golgi 기공을 갖는 구획에 10-15을 거주 V-올무 있습니다. 동일한 수준의 VAMPs 1, 3, 4, 5, 7, 8을 표현함으로써, 우리는 막 융합을 비교효소 세포 융합 분석을 사용하여 활동. spectrometric 측정을 바탕으로,이 분석은 스네어로 인한 막 융합을 분석 및 높은 처리량을 연구 양적 접근 방식을 제공합니다.

양적 세포 융합 분석을 개발하기 위해, 우리는 트레에 TTA의 바인딩에 의해 강한 전사 활성을 활용할 수 있습니다. TTA의 부재에서, 트레-LacZ에 LacZ 유전자의 전사는 침묵입니다. TTA가 존재하면, 트레에 바인딩와 LacZ의 전사를 활성화합니다. 그림 1은 효소 세포 융합 분석의 순서도를 보여줍니다. TTA는 세포 표면의 특급 V-스네어 단백질, 그리고 트레 - LacZ가 T-세포로 t...

Watch this Scientific Journal Video about Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay at JoVE.com

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

우리는 β-갈 락토의 활성화 표현으로 올무로 인한 막 퓨전 이벤트를 단정 지을 세포 융합 분석을 개발했습니다.

효소 세포 융합 분석을 사용하여 올가미로 인한 막 퓨전 분석

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

analysis-snare-mediated-membrane-fusion-using-an-enzymatic-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.9K 조회수.  University of Louisville School of Medicine. 우리는 β-갈 락토의 활성화 표현으로 올무로 인한 막 퓨전 이벤트를 단정 지을 세포 융합 분석을 개발했습니다.

비디오: Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

셀 연락처에서 단백질 단백질 상호 작용의 형광 변동 분광학 분석 결과

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

연구

생화학

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

지질 혼합 애신을 위한 리포솜으로 재조합 초파리 아틀라스틴의 세제 지원 재구성

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

미토콘드리아 멤브레인 역학 을 재구성하기위한 모델 멤브레인 플랫폼

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

단일 입자 수준에서 바이러스성 퓨전 속도론의 측정 방법

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

생물학

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

세포는 형광 현미경 Electrofusion와 시각

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

결합 된 방법론을 활용하여 축삭 분기 및 신경 세포 형태 형성 동안 플라즈마 멤브레인 배달의 역할을 정의합니다

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

신경과학

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

편광 TIRF 현미경에 의해 감시 미세 유동 흐름 전지에 닿는 지원 이중층 단일 테오의 SNARE 매개 퓨전

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

면역학 및 감염병학

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

병렬 공촛점 현미경을 사용하여 여러 세포의 운동 데이터를 취득 빠르고 높은 해상도, mtPA-GFP 기반 Mitochondrial 퓨전 분석

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

부신 크로마핀 세포에서 융합 기공 역학의 세 가지 모드를 측정하는 공초점 현미경 검사

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...