Name: investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays
Uploaded: 2016-06-18T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

We thank the Dictyostelium Stock Center for strains and reagents. This study was financed by grants from the Swiss National Science Foundation and the Canton of Basel.

<ol><li> 시간 경과 현미경에 의해 Dictyostelium의 discoideum의 초기 기아 응답을 관찰한다. <ol><li> HPO 4 5 g의 proteose 펩톤 5 g thiotone의 E 펩톤 10 g 글루코스, 5 g의 효모 추출물, 0.35 g이 나트륨 : 1 L에 대해 HL-5 매체 (함유의 Erlenmayer 플라스크 DH1.10 세포 또는 CORA 결핍 세포 성장 * 7H 2 O, 0.35 g의 KH 160 rpm의 회전 진탕 배양기에서 22 ° C에서 2 PO 4, 0.05 g의 dihydrostreptomycin - 황산, 산도 6.6). 0.01 × 106 세포 / ㎖, 2 × 106 세포 / ml 사이의 밀도로 세포를 유지한다. </li><li> 수확 로그 단계 성장 DH1.10 셀 (21) 또는 22 ° C에서 HL-5 매체와 문화를 흔들어 성장 코라 결핍 셀 DH1.10 배경에서 발생 (10)에 의해, 세포 응집을 검사합니다. 이를 위해 400 XG에서 3 분 동안 세포 (통상 10 내지 50 ㎖), 원심 분리기의 적당량을 취하고 BSS (10 mM의 염화나트륨, 10 mM의 KCl을 두 번 세포를 씻어2.5 mM의 CaCl2를, pH가 6.5). </li><li> 혈구를 사용하여 세포를 계산합니다. 계속해서, (5, 10, 20, 40)의 밀도로 접시의 세포를 24- 웰 플레이트에 104 세포 / cm × 2. 그들이 BSS에서 22 ° C에서 1 시간 동안 부착 할 수 있습니다. </li><li> 이미지마다 135 초를 복용, 10 전에 설명 된 바와 같이 시간 경과 현미경에 의해 집계를 시각화합니다. 설정 5X 목적과 적절한 소프트웨어에 의해 자동으로 전자 승산 전하 결합 소자 카메라를 구비 한 생균 이미징을 사용하여 (재료 표 참조). </li></ol></li><li> 기아 동안 Dictyostelium의 discoideum 세포의 캠프 펄스. <ol><li> 세포를 수확하여 DH1.10 셀 21 DH1.10 CORA 결핍 셀 (10)의 개발에 대한 외부로부터인가 된 cAMP 펄스의 효과를 조사한다. 이렇게하려면 400 XG에 3 분 동안 적절한 세포의 양 (보통 10 ~ 50 ㎖), 원심 분리기를 가지고 BSS에 두 번 씻는다. </li><li> COUBSS에서 1 × 107 세포 / ml의 농도로 세포를 혈구를 사용하여 재현 탁 NT 세포. 펄스를 적용하기 전에 2 시간 동안 22 ° C에서 배양 (160 rpm으로)를 흔들어. 타이머 제어 연동 펌프를 사용하여 캠프 펄스를 추가합니다. 5 시간에 걸쳐 5 초 펄스 50 nM의 캠프 (최종 농도) 15 μL 매 6.5 분을 제공하는 펌프를 프로그래밍. </li><li> 혈구를 사용하여 세포를 계산합니다. 계속해서, (5, 10, 20, 40)의 밀도로 세포를 플레이트를 24- 웰 플레이트에 104 세포 / cm × 2. BSS에서 1 시간 동안 부착 할 수 있습니다. </li><li> 5 배 목표를 사용하여 밝은 필드 현미경으로 22 ° C에서 16 시간 후 집계를 시각화합니다. </li></ol></li><li> 에어컨 매체에 노출을 통해 Dictyostelium discoideum 개발 유도. <ol><li> (22) 한 바와 같이 신선한 컨디셔닝 매체를 준비합니다. 숭배를 흔들어에서 로그 상 DH1.10 세포 21 DH1.10 코라 결핍 셀 (10)를 수집400 XG에서 3 분 동안 피펫, 원심 분리기를 이용하여 22 ° C에서 HL-5 매체와 URES 및 PBM에서 세포를 세 번 씻어 (0.02 M 인산 칼륨, 10 μM 염화칼슘 2, 1 ㎜의 MgCl 2, pH를 6.1). </li><li> 혈구를 사용하여 세포를 카운트 ml의 1 × 10 7 세포 /의 밀도로 PBM에서 다음을 재현 탁하고 110 회전 / 22 ° C에서 20 시간 동안 흔들어. </li><li> 3 분 동안 400 × g에서 원심 분리 후 조정 배지를 수집하고 4 ℃에서 15 분 동안 8,000 × g에서 원심 분리하여 명확히. </li><li> 0.45 μm의 필터를 통해 필터 에어컨 매체 (재료 표 참조) PBM에서 배를 희석. </li><li> (5, 10, 20, 40)의 밀도 혈구 접시를 사용하여 세포를 카운트 24- 웰 플레이트에 104 세포 / cm × 2. 그들이 22 ° C에서 1 시간 동안 부착 할 수 있습니다. </li><li> 이전에 제조 된 배지로 교환 세포 상등액. </li><li> 집계 a를 시각화5 배 목표를 사용하여 밝은 필드 현미경에 의한 따고 16 시간. </li></ol></li></ol>

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No conflicts of interest declared.

coronin 단백질은 진핵 계통 군의 대부분 분류군에서 발견된다. Dictyostelium의 discoideum의 coronin A는 포유류 coronin (1)의 동체는 세포 초기 발달 동안 응집 중심을 형성 할 수없는 A-결핍을 coronin 때문에, 초기 기아 반응에 관여 사이클 10. 정량적으로 정확하게 균주 개발 사이의 지연을 평가할 수 있도록, 자동 스테이지 컨트롤러 현미경 생균 촬상 설정 필수적인 도구이다. <p class="jove_cont...

단백질의 coronin 제품군은 높은 진핵 생물에 걸쳐 보존됩니다. 이 단백질은 아미노 말단 트립토판 아스파 테이트 (WD) 반복 함유 카르복시 말단 꼬인 코일 도메인 (13, 14)에 접속 된 고유 영역 다음 영역 (도 1)의 존재를 특징으로한다. Coronins는 골격 조절 및 신호 전달 (12)을 포함한 세포 기능의 다양성에 연루되어왔다. 포유 동물에서, 최대 6 개의 짧은 coronin 분자 (coronin 1-6)뿐만 아니라 7 coronin는 &#39;텐덤&#39;, 12,15 공동 발현 될 수있다. Coronin 1은 가장 광범위하게 연구 가족 구성원이며, 병원균 파괴, T 세포 및 신경 세포의 생존 시그널링에 관여하는 것으로 나타났다. 어떻게, 정확히, coronin 1은 이러한 활동을 수행하는 것은 확실하지 않다. 1 coronin하는 칼슘 및 캠프 종속 신호뿐만 아니라 F - 굴지으로 세포 골격 변조 16-18, 잠재적 인 협력을 조절하는 것으로 나타났다 동안포유 동물에서 최대 7 가족의 -expression는 도전으로 인해 잠재적 중복에, 이러한 시스템에 coronins의 분자 기능을 공부했다. 포유 동물의 생물과는 달리, 낮은 진핵 생물의 Dictyostelium의 discoideum은 분명히 비 중복 기능 15,19,20 만이 coronin 가족 (coronin의 A, 포유 동물 coronin 1 coronin B의 ortholog, 포유 동물 coronin 7의 ortholog)을 표현한다. 이 사실은 Dictyostelium의 discoideum에게 coronins의 기능을 연구하는 강력한 모델을 만든다. Dictyostelium의 discoideum에서 coronin A의 역할을 연구하기 위해, 우리는 10 coronin 결여 야생형 세포 또는 세포를 사용하여 평형 염 용액 (BSS) 버퍼를 포함하는 조직 배양 플레이트에 기아로 발달주기를 유도. 우리는 coronin 부족한 세포가 기아에 따라 다세포 집계를 형성 할 수없는 것으로 나타났다. 이 본 표현형의 정확한 정량 평가이 프로토콜에 설명 된 자동화 라이브 세포 이미징은 중요한 도구입니다. 을 coronin 부족한 세포에서 초기 기아 응답의 시작에 결함이 캠프 폭포의 상류 행위를 coronin 것을 제안, 캠프의 펄스를 공급하여 구조 할 수 있습니다. 발전 개시를 시뮬레이트 된 cAMP 펄스 외인성 출원은 과거 8,9 여러 실험실에서 이용되고있다. 그러나,이 절차는 세포 밀도 및 타이밍에 크게 의존하는 것으로 알려져있다. 따라서, 여기에 설명 된 프로토콜 재현성의 높은 수준을 보장하기 위해 이러한 가변성을 감소시키는 것을 목적으로한다. 종합하면, 이들 연구에 이용 된 기술은 Dictyostelium의 discoideum의 개발주기의 초기 단계에서의 단백질의 기능을 조사하기 위하여 강력한 도구를 제공하고, 업 식별뿐만 아니라 기능 coronin 하류 이펙터 할 가능성이있다.

<table><tbody><tr><td>HL-5 media (for 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O, 0.35 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)</td><td></td><td></td><td></td></tr><tr><td>Proteose peptone</td><td>BD Bioscience</td><td>211693</td><td></td></tr><tr><td>Thiotone E peptone</td><td>BD Bioscience</td><td>211684</td><td></td></tr><tr><td>Yeast extract</td><td>BD Bioscience</td><td>212750</td><td></td></tr><tr><td>Glucose</td><td>AppliChem</td><td>A3666</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fluka</td><td>71643</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1043</td><td></td></tr><tr><td>dihydrostreptomycin-sulfate</td><td>Sigma-Aldrich</td><td>D1954000</td><td></td></tr><tr><td>PBM (0.02 M potassium phosphate, 10 &amp;mu;M CaCl&lt;sub&gt;2&lt;/sub&gt;, and l mM MgCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.1)</td><td></td><td></td><td>self made</td></tr><tr><td>BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.5)</td><td></td><td></td><td>self made</td></tr><tr><td>0.45-&amp;mu;m Filtropure S filter</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>Falcon 24-well Tissue culture plate</td><td>Fisher Scientific</td><td>08-772-1H</td><td></td></tr><tr><td>Cellobserver microscope</td><td>Zeiss</td><td></td><td>custom built</td></tr><tr><td>AxioVision software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>IPC Microprocessor&amp;ndash;controlled dispensing pump</td><td>ISMATEC</td><td>ISM 931</td><td></td></tr><tr><td>Axiovert 135M microscope</td><td>Zeiss</td><td>491237-0001-000</td><td></td></tr><tr><td>Incubation Shaker</td><td>Inforst HT Minitron</td><td></td><td></td></tr></tbody></table>

investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays

Dictyostelium의 discoideum의 아메바는 박테리아에 먹이 토양에서 발견된다. 음식 공급원이 부족해질 때, 그들은 단일 세포 집계 센터 1-4 향해 chemotax 동안 다세포 개발 프로그램을 시작하는 인자를 분비. 이 과정은 사이 클릭 아데노신 모노 포스페이트 (cAMP) (5)의 분리에 의존한다. 캠프는 아데 닐 레이트 사이 클라 제와 포스 포의 공동 작업을 통해 파도에서 생산, 및 G 단백질 결합 된 cAMP 수용체 6,7 결합된다. 널리 사용되는 분석은 잠긴 상태에서 8,9 세포 응집의 관찰에 기초 하부의 진핵 Dictyostelium discoideum의 개발주기와 관련된 메커니즘을 분석한다. 이 프로토콜은 평형 염 용액 (BSS) (10)에 잠긴 조직 배양 플레이트에 기아로 발달주기 coronin A의 역할의 분석을 설명한다. Coronin A는 광범위하게 보존 된 PR의 구성원활동 (11, 12)의 다양한 연루되어 coronins의 otein 가족.를 coronin 부족한 Dictyostelium 세포는 다세포 응집체를 형성 할 수없는, 이러한 결함은 제안 된 cAMP의 펄스를 공급하여 구출 될 수의 상류 작용 coronin 캠프는 10 계단식. 이러한 연구에서 설명 된 기술은 캠프 캐스케이드의 상류 Dictyostelium discoideum의 개발주기의 초기 단계에서의 단백질의 기능을 조사하기 위하여 강력한 도구를 제공한다. 따라서이 집계 분석을 이용하여 함수를 coronin의 추가 연구를 허용하고 coronin 생물학에 대한 우리의 이해를 진행할 수있다.

초기 개발에 쇼에게 결함을 coronin 결핍 세포 (그림 2). 세포 coronin의 부재 Dictyostelium의 discoideum의 발달주기 동안의 초기 단계 다세포 응집체를 형성 할 수 없다. 따라서, coronin A는 초기 기아 응답 및 / 또는 캠프 신호 동안 역할을 나타납니다. 실제로 coronin A의 부재 하에서 다세포 골재 형성의 결여는 감소 캠프 10 시그널링을 수반한다. 그러나, 외?...

Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

의 조기 기아 응답에 Coronin (A)의 기능 조사<em&gt; Dictyostelium의 discoideum</em&gt; 집계 분석 실험에 의해

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

investigating-function-coronin-early-starvation-response

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Biology

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

7.2K 조회수.  University of Basel. The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

비디오: Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum 셀 선택에 따라 성장과 박테리아에서의 유전 공학

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

연구

유전학

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Caenorhabditis elegans에서 자동 포함 계수를 사용하여 생체 내에서 단백질 응집 동역학 모니터링

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

생화학

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

이미징 G - 단백질 결합 수용체 (GPCR) - 중재 신호 행사 그 제어 Chemotaxis Dictyostelium Discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

생물학

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

이종 세포 시스템에서 단백질 응집을 통한 세포 간 상호 작용 측정

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

신경과학

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.

단백질의 품질 관리 시스템을 공부<em&gt; D. discoideum</em&gt; 온도 제어 라이브 셀 이미징을 사용하여

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

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Dictyostelium discoideum 응답 심각한 기계적인 자극의 평가

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

꼬마 선 충 나이 연구 변화 내재 단백질 집계에 방법

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Flow Cytometry 여 Dictyostelium discoideum Macropinocytosis의 높은 처리량 측정

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

형광 상관 분광법을 사용하여 단백질 응집 감지

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

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