이 작품은 # DA15169 앙에 NIH 교부금에 의해 투자되었다

절차 개요 : 이 방법을 사용하여, 세포 표면 단백질은 covalently (그림 참조 1. 인신 매매 제한 조건 (즉, 낮은 온도)에서 막 impermeant, 이황화 결합 biotinylation 시약을 (sulfo - NHS - SS - 비오틴)를 사용하여 사용 가능한 세포 리신 잔류물에 비오틴과 레이블 아르 그림에 대한). 세포 중 하나 세트 매매 허용 조건 이동 (37 ° C)와 biotinylated 단백질 internalize입니다. 세포의 다른 집합 시간 = 0 1)의 전체 표면 단백질, 그리고 2) 스트립 컨트롤에 대한 컨트롤로 저온에서 보관됩니다. 국제화의 짧은 기간에 따라, 세포가 내면화을 막을 저온으로 이동하고, 잔여 표면 비오틴은 이황화 결합 비오틴을 클리브스 감소 요원과 세포를 치료에 의해 해제 벗겨지고 있습니다. 세포 표면에서 발생한 및 internalized되었습니다 Biotinylated 단백질은 스트립 단계에서 보호하고 있으며, 남아있는 유일한 biotinylated 단백질 것입니다. 세포 용해 후, biotinylated 단백질은 streptavidin 친화도 크로마 토그래피에 의해 고립되고 관심의 단백질은 양적 immunoblotting에 의해 감지됩니다. endocytic 속도를 확인하려면, internalized 단백질의 양은 시간이 = 0에 표시된 전체 표면 제어 비교됩니다. 우리는 성공적으로의 연결을 norepinephrine 1 도파민 1-4 전송기의 국제화 속도를 측정하는이 방법을 사용했습니다. 상세 프로토콜 : 1 일 : <ol><li> 그들은 일 2 ~ 80 %의 합류한다는 등 6 접시에 잘 플레이트 세포. 또는, transfected 세포를 사용하고있다면, 그들은 국제화 속도가 측정되는 시간 ~ 80 %의 합류됩니다 같은 밀도에서 transfect. 세포가 강력하게 자기편하지 않는 경우, 조직 문화 도자기는 광범위한 세척 단계 동안 세포 손실을 방지하기 위해 세포 부착 기판 (예 : 폴리 - D - 라이신)와 함께 치료를해야합니다. </li><li> 각각의 단백질이 테스트되고 들어, 하나의 접시에 번호판이 우물은 전체 표면 단백질 (T = 0)와 스트립 컨트롤로 사용할 수 있습니다. 두 번째 접시에, 테스트중인 각각의 국제화 조건에 대해 잘 플레이트 한 (즉, 기초 endocytic 속도 대 약물 치료). </li><li> 일 2 사용하기 위해 지정된 온도에서 다음과 같은 솔루션 및 상점 준비 : <ul><li> PBS 2 + : 인산 1.5 MM MgCl 2, 0.2 MM CaCl 2, (4 ° C)과 보충 (산도 7.4) 살린 버퍼 </li><li> Biotinylation는 솔루션 끄지 : PBS 2 + 100 MM 글리신 (4 ° C)와 보충 </li><li> NT 버퍼 : 150 MM NaCl, 1.0 MM EDTA (에틸렌 다이아 민 테트라 초산), 0.2 % BSA, 20 MM 트리스, 산도 8.6 (4 ° C) </li><li> 리파 버퍼 : 10 MM 트리스, 산도 7.4, 150 MM NaCl, 1.0 MM EDTA (에틸렌 다이아 민 테트라 초산), 0.1 % SDS, 1.0 % 트리톤 X 100, 1.0 %의 나트륨 deoxycholate (4 ° C) </li><li> Sulfo - NHS - SS - 비오틴 주식 솔루션 : 200 MG / ML (-20 ° C)에 (DMSO) dimethylsulfoxide에서 디졸브 </li><li> 트리스 (2 - Carboxyethyl) phosphine 하이드로 클로라이드 (TCEP) 재고 솔루션 : H 2 O 500 MM (-20 ° C, 빛을 차단하는 호일로 덮여) </li></ul></li></ol> 일 2 : <ol><li> PBS 2 + 0.18g/ml 포도당과 보충, 0.2 % IgG / 프로 테아제 무료 소 혈청 알부민 (PBS 2 + / g / BSA). 준비 ° C 물 목욕에서 37 미리 따뜻한이 솔루션입니다. </li><li> DMSO을 녹일 벤치 위에 sulfo - NHS - SS - 비오틴 주식 솔루션을 누그 러 뜨 리다. 사용하기 바로 전에, 신선한 sulfo - NHS - SS - 비오틴 솔루션 (2.5 얼음 차가운 PBS 2 +, 0.75에 대한 충분한 ML / 음에 MG / ML) 준비합니다. DMSO를 solubilize하기 위해 적극적으로 소용돌이 솔루션입니다. NHS - 비오틴 시약이 수용액에서 쉽게 분해됩니다. 따라서, 모든 솔루션은 사용하기 전에 바로 준비를해야합니다. </li><li> Biotinylation : 플레이스 추운 방에 얼음 욕조에 접시와 얼음 차가운 PBS 2 3 X 2 ML을 씻어 +. 접시가 약간 완전한 배수 및 버퍼 솔루션의 제거를 허용하기 위해 직각 것을 확신한다. / 잘 각 잘 수있는 신선한 sulfo - NHS - SS - 비오틴 솔루션의 0.75 ML을 추가합니다. 부화 X 15 &#39;, 4 명이 활발한 흔들림으로 얼음 욕조에 C. 배양이 완료되면, 또 다른 신선한 sulfo - NHS - SS - 비오틴 솔루션을 준비합니다. 신선한 솔루션 및 부화 X 15 &#39;, 4 ° C.와 함께 기존 솔루션을 대체 </li><li> 담금질 : 그들이 함께 반응하고 세포 lysed되면 세포내 단백질을 biotinylate 않도록 모든 비 반응 NHS - 비오틴 분자가 침묵하는 중요한 IT합니다. 칭 솔루션 세포에게 3 X 2 ML 씻으십시오 2 ML의 담금질 솔루션 X 15 &#39;, 4 명이 부드러운 잡고 C 두 번 품어. </li><li> 국제화 : 약물 치료가 테스트되고있다면, PBS 2 + / g / BSA 적절한 약물 농도를 추가합니다. 4 ° C에서 컨트롤 플레이트를 유지하고 국제화 플레이트 차가운 방에서 가지고. 사전 예열 PBS 2 3 X 2 ML 씻으 + / g / BSA (+ / - 박사동일한 솔루션 (2 ML / 음)에 UGS)와 두십시오. 접시에 걸쳐도 온도를 보장하기 위해 미리 예열 솔루션으로 모든 빈 우물을 입력합니다. 10 &#39;에 대한 37 ° C 배양기에 세포를 전송합니다. 37 종료 즉시 이전 ° C 부화, 얼음 스트립 단계 및 저장을 위해 사용되는 NT 버퍼의 신선한 50 MM의 TCEP 솔루션을 준비합니다. 1.0 ML / 음에 대한 충분한 양의를 준비합니다. </li><li> 스트립 : 얼음 욕조와 차가운 방으로 다시 즉시 전송 플레이트 (S). 신속 얼음 차가운 NT 버퍼, 3 세포를 씻어 endocytosis를 중지 X 2 ML. 또한 NT 버퍼와 스트립 제어 우물 3 X 2 ML 씻는다. 각 물론 1.0 ML 신선한 잎을 솔루션을 추가합니다. 부드러운 잡고, 얼음 15 &#39;, 4 ° C를 품어. 신선한 스트립 솔루션 우물을 교체하고 얼음에 추가 15 &#39;, 4 ° C를 품어. </li><li> 용해 : 스트립 솔루션, 3 X 2 ML NT 버퍼에 노출된 모든 우물을 씻으십시오. 다음 3 * 2 ML PBS 2 + 모든 우물 (총 컨트롤 포함) 씻으십시오. 신선한 테아제 억제제 (1.0 MM PMSF, 1.0 μg / ML 각 류펩틴, aprotinin과 pepstatin)이있는 300μl/well 리파 버퍼 (또는 관심의 단백질에 대한 호환 가능한 다른 용해 버퍼)에 Lyse. X 20 &#39;, 4 ° C.를 흔들어하여 Lyse 18,000 XG, 10 &#39;, 4 ° C.를 centrifuging하여 microfuge 튜브 맑은 세포 부스러기로 전송 </li><li> 단백질 농도 결정 : 표준 BSA 곡선에 비해 lysates의 단백질 농도를 결정하여 용해 조건 (예 : DC 단백질 분석, 바이오 래드)와 호환되는 단백질 분석을 사용합니다. </li><li> Streptavidin 친화도 크로마 토그래피 : 각 시료에 대해 동일한 양의 단백질과 microfuge 튜브를 준비합니다. 200μl 각 샘플의 최종 볼륨을 가지고 용해 버퍼를 추가합니다. 소용돌이 streptavidin 아가로 오스 비즈 최선을 다해도 중지 가지고 있습니다. 끝에있는 200μl pipettor을 사용하면 각각의 튜브에, 피펫 비즈를 잘라. 20μl beads/50 μg의 lysate를 추천. 튜브 회전에서 하룻밤, 4 ° C를 품어. </li></ol> 3 일 : <ol><li> 비드 세척 : 원심 분리기 샘플, 18000 XG 2 &#39;구슬을 수집합니다. 흡인기로 비드 펠렛 접근하지 않도록주의하고, lysate를 대기음. 더 나은 컨트롤에 대한 흡인기의 끝에 플라스틱 피펫 팁을 사용합니다. 0.75 ML의 용해 각각의 튜브에 버퍼, 그리고 구슬을 씻어 와동을 추가합니다. 원심 분리기 샘플, 18000 XG 2 &#39;구슬과 반복 뜻을 수집하고 (총 세 세척) 두 번 세탁합니다. 최종 세척 후, 비드 펠렛을 방해하지 않고 가능한 버퍼만큼 대기음. 흡인기쪽으로 튜브를 티핑하면이 단계 지원합니다. </li><li> 샘플 용출 : 각 튜브 20 - 25μl 2X Laemmli 샘플 버퍼 (감소)를 추가합니다. 감소 에이전트 솔루션으로 분리된 단백질을 방출 클리브는 NHS - SS - 비오틴의 이황화 결합합니다. 대부분의 멤브레인 단백질이 삶은 때 집계에 매우 취약하며, SDS - PAGE를위한 젤에 로딩하기 전에 가열해서는 안됩니다. 이러한 실험을 수행하기 전에 관심의 단백질의 열 감도를 확인합니다. 단백질 / 끓는 난방을 용인할 수 없다면, SDS - PAGE로 분석하기 전에 회전, 30 &#39;, 실온에서 알을 품다. 이것은 클리브 이황화 결합에 충분하고 단백질을 elute. </li><li> SDS - PAGE과 immunoblotting : SDS - PAGE에 의해 분리 단백질. 각 샘플은 다음 순서로 샘플을 실행하는 가장 쉬운 방법을입니다 단백질에 적합한 항체와 함께 총 표면 (시간 = 0), 스트립, 국제화 조건 # 1, 조건 # 2, 등등 immunoblotting을 위해 막에 전송하고 얼룩 관심. 더 포화 픽셀이 없다는 것을 확신하고, CCD 카메라 겔 문서 시스템을 사용 immunoreactive 밴드를 캡처합니다. 겔 분석 / densitometry 소프트웨어를 사용하여 각 밴드 밀도를 계량. </li></ol> 대표 결과 : 대표 immunoblot 결과는 그림 2A에 표시됩니다. 강한 신호는 이전에 내면화하는 표면 단백질의 총 금액은 &quot;총&quot;레인 (T)에 있습니다. &quot;스트립&quot;제어 (S)는 이상적인 스트립이 실험을 위해 효율적인 것을 보여줍니다 공백에 가까이해야합니다. 스트립 효율성은 &quot;총&quot;차선의에있는 &#39;스트립&#39;차선의 밀도를 비교하여 계산 (스트립과 병렬로 biotinylated되었다 예 : 단백질,하지만 둘 다 37 ° C로 예열하지 않고 스트립 솔루션에 노출되었습니다.) 다음과 같은 공식이 사용됩니다 : [1 - (스트립 / 총)] * 백퍼센트 이 수식을 사용하여, 그림 2의 스트립은 99.8 % 효율되었다. 마지막으로, 당신은 국제화 차선의 총 (S) (I)보다 낮은 강도의 밴드를 볼 수 있습니다. 예를 들어 (그림 2)에서 치료 세포가 차량 또는 10 중에 1μM phorbol myristate 아세테이트 (PMA) 중 하나와 함께 치료를했다 초기 국제화 시대 &#39;국제화와 도파민 전송 국제화 요금은 10 측정되었다. 국제화 속도로 계산됩니다다음 : 합계 / internalized * 100 그림 2B에서 본, 10.4 % 표면 DAT는 차량 처리 조건에서 10 &#39;이상 internalized. PMA 치료 총 표면 DAT의 23.2 %에 DAT의 국제화 속도를 향상시켰습니다. <img src="/files/ftp_upload/1669/1669fig1.jpg" alt="그림 1" /> 그림 1. 프로토콜 일러스트 레이션. 전지 4 biotinylated 아르 ° C가 독점적으로 표면 인구 라벨, 37으로 이동 아르 ° C 국제화를 시작합니다. 국제화에 따라, 세포가 빠르게 endocytic 프로세스와 잔여 표면 비오틴를 중지 추위 것은 줄이고 요원과 세포를 치료에 의해 분해까지입니다. 남아있는 유일한 biotinylated 단백질은 t = 0의 표면에서 발생한 및 internalized 있었다 따라서 스트립 치료에서 그들을 보호하시는 분들입니다. Biotinylated 단백질은 streptavidin 비즈와 함께 배치 친화도 크로마 토그래피에 의해 고립되고 관심의 단백질은 immunoblotting에 의해 감지됩니다. <img src="/files/ftp_upload/1669/1669fig2.jpg" alt="그림 2" /> 그림 2. PKC의 활성화는 DAT endocytic 속도를 증가시킵니다. 내면화 분석은. 안정 DAT 표현 PC12 세포는 biotinylated, 4 ° C로 &quot;상세 프로토콜&quot;에 설명되어 하였다. 전지는 급속하게 37 기까지했다 ° C ± 1 μm의 PMA와 incubated 10 &#39;37 ° C. 나머지 비오틴은 줄임으로써 뺏겼어요, streptavidin 친화도 크로마 토그래피에 의해 세포 lysed되었고 biotinylated 단백질은 고립되었다. (A) 대표 immunoblot 총 표면 DAT를 보여주는 t에서 = 0 (T), 스트립 제어 (S), 그리고 지정된 조건 하에서 internalized DAT (I). (B) 밴드는 CCD 카메라로 캡처한 및 수량 데이터 소프트웨어 (바이오 래드)와 계량되었습니다. 데이터는 %의 총 DAT internalized/10의 분으로 표현됩니다. <img src="/files/ftp_upload/1669/1669fig3.jpg" alt="그림 3" /> 그림 3. 예 immunoblot는 가난한 비오틴의 스트립. 내면화 분석을 묘사한. 안정 DAT 표현 PC12 세포는 biotinylated, 4 ° C로 &quot;상세 프로토콜&quot;에 설명되어 하였다. 전지는 급속하게 37 기까지했다 ° C와 incubated 10 &#39;37 ° C. 나머지 비오틴은 줄임으로써 뺏겼어요, streptavidin 친화도 크로마 토그래피에 의해 세포 lysed되었고 biotinylated 단백질은 고립되었다. immunoblot 총 표면 DAT를 보여줍니다 t = 0에서 (T), 스트립 제어 (S), 그리고 internalized DAT (I). 가난한 스트립 효율을 나타내는 스트립 제어 차선에서 보이는 밴드를합니다.

<ol>
	<li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat Neurosci. 8, 881-888 (2005).</li><li>Boudanova, E., Navaroli, D. M., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+decreases+in+dopamine+transporter+surface+expression+are+protein+kinase+C-independent.">Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent.</a> Neuropharmacology. 54, 605-612 (2008).</li><li>Boudanova, E., Navaroli, D. M., Stevens, Z., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+transporter+endocytic+determinants:+carboxy+terminal+residues+critical+for+basal+and+PKC-stimulated+internalization.">Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization.</a> Mol Cell Neurosci. 39, 211-217 (2008).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J Biol Chem. 278, 22168-22174 (2003).</li></ol>

일반적인 문제 :이 실험에서 발생하는 가장 일반적인 문제는 가난한 스트립의 효율성이다. 스트립의 효율 결과를 해석하는 수있는에 중요합니다. 스트립은 매우 효율적인 게 아니라면, 그것은 국제화 차선에 biotinylated 단백질의 표면에서 internalized 사실에 있다고 결론하는 것은 불가능합니다. 스트립 ≥ 90 % 효율이 최적이며, 스트립이 수준 아래로 떨어지면면 우리는 어떤 결과를 폐기하십시...

measuring plasma membrane protein endocytic rates by reversible biotinylation

플라즈마 막 단백질은 수용체, 이온 채널, 전송기 및 펌프로 구성된 단백질 많은 다양한 그룹입니다. 이 단백질의 활동이 양분 전달, 세포 흥분, 화학 신호를 포함한 주요 세포 행사의 다양한 책임이 있습니다. 대부분의 플라스마 막 단백질은 동적 단백질 표면 표현을 변경하여 단백질 기능을 modulates endocytic 인신 매매에 의해 규제됩니다. 단백질 endocytosis를 촉진 메커니즘은 복잡하며 충분히 많은 막 단백질에 대한 이해되지 않습니다. 완전히 주어진 단백질의 endocytic 밀매를 제어하는​​ 메커니즘을 이해하기 위해서는 단백질의 endocytic 속도가 정확하게 측정하는 것이 중요합니다. 많은 수용체에 대한 직접 endocytic 속도 측정이 자주 표시 수용체 리간드를 활용 달성하고 있습니다. 그러나, 전송기, 펌프와 이온 채널과 같은 막 단백질의 여러 클래스에 대한, endocytic 속도를 측정하는 데 사용할 수있는 편리 리간드가 없습니다. 현재 보고서에서, 우리는 도파민 수송 (DAT) endocytic 속도를 측정하기 위하여 고용 가역 biotinylation 방법을 설명합니다. 이 방법은 국제화 속도를 측정하는 간단한 방법을 제공하며, 쉽게 대부분의 막 단백질의 밀매 연구에 대한 고용 수 있습니다.

Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

규제 endocytosis는 막 단백질의 대부분의 세포 표면 발현 수준을 관리합니다. 여기서 우리는 도파민 전송 (DAT), polytopic 멤브레인 단백질의 endocytic 속도를 측정하는 줄이할 수있는, 멤브레인 impermeant biotinylation의 시약을 사용합니다. 방법은 대부분의 플라즈마 막 단백질의 endocytic 속도를 측정하는 직접적인 접근을 용이하게합니다.

가역 Biotinylation에 의해 플라즈마 막 단백질 Endocytic 요금 측정

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

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Biology

16.9K 조회수.  University of Massachusetts Medical School. 규제 endocytosis는 막 단백질의 대부분의 세포 표면 발현 수준을 관리합니다. 여기서 우리는 도파민 전송 (DAT), polytopic 멤브레인 단백질의 endocytic 속도를 측정하는 줄이할 수있는, 멤브레인 impermeant biotinylation의 시약을 사용합니다. 방법은 대부분의 플라즈마 막 단백질의 endocytic 속도를 측정하는 직접적인 접근을 용이하게합니다.

비디오: Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Astrocytes의 플라즈마 막 글로벌 세포내 칼슘 역학 니어 측정

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

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Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

디 - 8 - ANEPPS으로 유도된 막 전압 측정

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

유동세포계측법에 의해 플라즈마 막에 GLUT4 Translocation의 양적 측정

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

다중 타겟 추적 (MTT)에 의해 플라즈마 막의 분자 확산을 매핑

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Macromolecular의 결정학에 의해 구조 결정에 대한 Lipidic Mesophases에서 재배 막 단백질의 수확 및 Cryo 냉각 크리스털

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

플라즈마 레티놀 바인딩 단백질의 막 수용체의 레티놀 전송의 실시간 분석

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

pTIRFM와 이미징 플라즈마 막 변형

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

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

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

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

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

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

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

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

멤브레인 운송은 단일 단백질 해상도에서 고도의 병렬 나노 기공 칩 시스템에 의해 분석 처리합니다

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

원형질 막 주변 관련 된 단백질에 대 한 분할 결정

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...