The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H + 죽이는 선택 <ol><li> 세포주 <ol><li> 안정적 NHE 결핍 세포를 형질 감염 (예, CCL39 유래 PS120 세포주 16) 섬유 모세포 세포주에서 효율적인 형질 전환 수율 및 선택을 생산할 예정 포유 동물 발현 벡터 및 형질 전환 방법의 조합을 사용. 참고 : 여러 해 동안 인산 칼슘 침전 (19)가 좋은 형질 전환 수율로 사용되었다. 여기에는 리포 펙 타민 같은 상용 시약에 의해 최근 대체되었습니다, 형질은 제조업체의 지침에 따라 수행된다. </li></ol></li><li> 솔루션 <ol><li> 아래 설명 세 멸균 솔루션을 준비합니다. 여과 (0.22 μm의) 이러한 솔루션을 소독. <ol><li> 50 mM의 CL NH 4, 70 mM의 염화 콜린, 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를 5 밀리미터 GL 구성된 NH 4 + 로딩 용액을 준비 (PH 7.4)ucose 및 pH가 7.4에서 15 mM의 MOPS. </li><li> 120 mM의 염화 콜린 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 포도당하여 pH 7.0에서 15 mM의 HEPES 이루어지는 세정액 (PH 7.0)을 준비한다. </li><li> pH가 7.4에서 120 mM의 염화나트륨, 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 글루코오스, 15 mM의 HEPES 구성된 복구 솔루션 (PH 7.4)을 준비한다. </li></ol></li></ol></li><li> CO 2가 훼손 될 수 해당 버퍼링 될 것이다 5 %로서, - 선택을 시작하기 전에 ( &quot;무료&quot;인큐베이터 CO 2 라) 외부 탱크로부터 추가 CO 2, 37 ℃에서 사용될 수있다 세포 배양기를 얻기 산성화. 약 2 × 108 세포 (전형적으로 약 20 합류 100mm 플레이트)에 관심 업 NHE 발현 세포주를 증폭. </li><li> H는 절차를 죽이는 + : <ol><li> (3)에 로딩 용액에 1 시간 동안 관심 NHE 세포를 발현 세포를 부화CO 2 - 무료 인큐베이터에서 7 ° C. </li><li> 로딩 솔루션을 대기음 후 상기-세정액에 두 번 씻어 내야합니다. 산성화를 생성하는 가파른 NH 4 + 구배의 생성을 저해 할 잔여 세포 외 NH 4 (CL)을 제거하기 위해 효율적으로이 단계를 수행합니다. </li><li> 흡인 린스 매질을 제거하고 프리 CO 2 인큐베이터에서 37 ° C로 다시 복구 솔루션에서 한 시간 동안 세포를 배양한다. </li><li> 일반 배양 배지 (7.5 % FCS 전형적 DMEM)와 복구 매체를 장착하고 (5 % CO 2 및 95 %의 공기가 공급되는 습한 공기 조건에서 37 ° C)를 표준 배양 조건에서 세포를 성장한다. </li></ol></li><li> 안정적인 클론이 등장 할 때까지 일주일에 두 번 선택의이주기를 반복합니다. 참고 : 세포 배양 및 선택 조건에 관한 특별한주의하십시오. 작품의 달은 오 더 경향이 오염에 의해 파괴 될 수있다문화와 반복 세포 조작의 긴 기간 후 ccur. <ol><li> 여기서, 클론을 증폭하고 상기와 같은 개별적 2-3 절에서 설명한 이들의 특성, 또는 특성화 전에 세포 집단을 생성하기 위해 이들을 함께 풀로 여부를 선택한다. </li></ol></li><li> 카운터 선택 부모의 산에 민감한 표현형 (그림 1B)에 복귀 한 수 사람들을하여 산성화 방지 세포를 유지하기 위해 가끔 선택 (약 2 주에 1 회)를 적용합니다. </li></ol> 2. 세포 내 pH 측정 2.1) 형광의 pH 이미징 <ol><li> 490 nm에서 흥분 할 때의 pH 민감하고 제조 업체의 프로토콜 다음 445 nm의 isosbestic 점을, 소유 비율 계량 pH 민감성 형광 염료 BCECF / AM을 사용합니다. <ol><li> 또한, 제조 업체의 프로토콜 다음 다른 pH 민감성 프로브를 사용합니다. </li></ol></li><li> 영상 설정 콘을 사용하여고감도 비디오 카메라에 결합 도립 현미경 sisting. 450 nm의 여기에 적합한 석영 중립 밀도 필터와 결합 490 nm의 협 대역 간섭 필터 세트를 착용. <ol><li> 가능하면 비도 모두 λ1 및 / 또는 λ2를 들면, 어느 포화 또는 너무 낮게 설정된 기저 형광 값을 피하기 1에 가까워 지도록, λ1과 λ2 위해 설치 필적 형광 값 필터 및 조명 조건의 적절한 세트를 사용. 이 세포 내 pH가 변화하고 있지만 비율이 검출되지 수 있습니다으로 다음 중요한 유물을 얻을 수 있습니다. 참고 :이 시스템에 적절한 발광 파장 (BCECF 535 ㎚)에서 빠른 관류, 시간 경과에 두 개의 상기 파장에서 여기 및 인수를 사용하도록 설정해야합니다. 소프트웨어는 자동 데이터 수집 및 저장을 가능하게하여 시스템을 장비. </li></ol></li></ol> NHE 앞으로 활동 2.2) 측정 (압출 성형세포질에서 양성자) <ol><li> NH 4 + 로딩 용액에 1 시간 배양 세포 산성화 CO 2의 부재 (단계 1.4 참조). </li><li> 마지막 5 분 동안 용액에 BCECF-AM (5 μM 최종 농도)를 첨가하고 세포 외 프로브를 제거 NH 4 + 로딩 용액을 헹군다. </li><li> 현미경 세포를 마운트하고 안정적​​인 바탕을 기록하는 여러 이미지를 가져 가라. 세정액 (위 참조) 관류. 이 산성화에 대응 형광의 드롭을 초래한다. </li><li> pH가 안정화되면, 리 + / H +, 나트륨 + / H의 + 또는 K + / H +로 교환하여 pH 매개 회복율을 측정하기 위해 아래에 주어진 임의의 솔루션을 관류. 각 솔루션은 잠재적 인 커플 링 양이온 H + 교환을 테스트 할 포함되어 있습니다. 그 중 하나가 이동하는 경우, 이는 corresp 것이다 495 nm에서의 형광 증가를 초래한다번째 세포 내 pH를 높이려면 : pH가 7.4에서 120 mM의 LiCl을, 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 글루코오스, 15 mM의 HEPES pH가 7.4에서 120 mM의 염화나트륨, 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 글루코오스, 15 mM의 HEPES pH가 7.4에서 125 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 글루코오스, 15 mM의 HEPES </li></ol> NHE 역 활동의 2.3) 측정 참고 : 여기 원리는 횡단 이온 그라디언트를 반전하는 것입니다. <ol><li> 측정에 앞서, 관심있는 세포 내 양이온과로드 셀 (아래 참조). </li><li> BCECF / AM (단계 참조 2.2.2)와 5 분 동안 그들을 품어; 그들을 씻어 비디오 현미경을 탑재합니다. </li><li> 로딩 솔루션을 관류하고 안정적​​인 바탕을 기록하는 여러 이미지를 가져 가라. </li><li> 베이스 라인 안정화, 이미지 세포가 세포 외 산성 매체가 120m를 포함 관류하는 pH를 변화 후M의 염화 콜린, 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 포도당하여 pH 6.5에서 15 mM의 MES에. </li><li> LiCl을 로딩, pH를 7.4에서 120 mM의 LiCl을, 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 글루코오스, 15 mM의 HEPES 이루어지는 용액에서 2 시간 동안 세포를 배양한다. 원자 흡광 법에 의해 측정 된 세포 내 리튬은 약 60 mm의 값을 산출한다. </li><li> 나트륨 + 로딩, 1 밀리미터의 흡수를의 존재 하에서 pH가 7.4에서 120 mM의 염화나트륨, 5 mM의 KCl을, 1 ㎜의 MgCl 2, 2 mM의 CaCl2를, 5 mM의 글루코오스, 15 mM의 HEPES 이루어진 용액에 2 시간 동안 세포를 배양한다 나 / K ATPase의 차단합니다. 이러한 조건에서, 세포 내 나트륨 + 농도는 40 mM의 범위이다. 주 : 세포질 K + 농도가 140 mM이 범위이고 외향 K + - 지향 구배 달성하는 것이 쉽기 때문에 K + 로딩이 필요하지 않다. </li></ol> 2.4) 데이터 처리 및 교정: 주 :이 단계는 순방향 및 역방향 측정 모두에 적용된다. <ol><li> 각 실험의 끝에, 6.5와 7.4 사이의 pH 값으로 조정 된 140 mM의 KCl을 20 mM의 HEPES 및 5 μM의 nigericin의 용액으로 세포를 관류. </li><li> 텍스트 형식으로 아래의 이미지에서 형광 측정과 같은 데이터 (회색 수준의 강도 수준을 나타냄) 및 수출을 수집하고 같은 스프레드 시트 프로그램을 사용하여 아래에 설명 취급합니다. </li><li> 각각의 개별 셀, 또는 다음의 식을 이용하여 관심 영역에 대한 세포 내 pH 값을 계산한다 : pH가의 pKa + (로그 (R이-Rmin을) / (R 최대 -R가)) × F의 분 (λ2) / F 최대 (λ2) </li><li> F의 분 (λ2) / F 엄마 X (λ2) 실험에 걸쳐 450 nm의 형광의 감소의 결과로, 프로브 표백 또는 누설이있는 경우 비율을 사용합니다. 그러나 이것은 매우 드문 데에 사용 된 조건에서 관찰 없습니다cribed 실험. </li><li> 교정 데이터는 각각 pH 측정의 끝에 존재하는 바와 같이 계산 된 값에 대응하는 캘리브레이션에 사용되는 pH 값에서 다른지를 확인한다. 이 경우라면, 다음과 같은 절차를 사용하여 교정에 모두 실험적으로 결정된 pH 값 조정 : <ol><li> 다음 몫 (Q, 보정 값의 측정 값 / 차이의 차이)를 계산. 계산 된 값이 대응하는 캘리브레이션 값을 같게되도록 Q.함으​​로써 전체 데이터 세트가 부가 또는 감산함으로써 최종 조정을 곱한다. </li></ol></li></ol> 빠른 리 + 통풍 관에 의해 NHE7의 초기 요금 3. 측정 <ol><li> 멀티 웰 플레이트 상에 종자 세포 (6- 웰-24) 또는 20 이상에서 설명한 교대 nigericin / 소 혈청 알부민 (BSA) 산성화 바와 NH 4 + 로딩 기술을 사용하여이를 산성화. </li><l난&gt; 린스 플레이트 배 빠르게 상술 세정액을 이용하고, 원하는 양 (약간 km 값 이하) 1-10 mM의 리튬을 함유하는 흡수 용액의 세포와 양이온의 목적하는 농도 및 / 또는 관심의 억제제를 부화 시간. </li><li> 해당 전송 초기 속도 조건에서 작동하기 위해 짧고 일관된 흡수 기간 (일반적으로 1 분 이하)를 유지한다. 또한 흡수에 사용되는 모든 솔루션이 등장 있는지 확인하십시오. </li><li> 흡수 시간의 끝에서, 신중하게 흡수 매질을 제거하고 얼음 - 냉각 된 인산 완충 생리 식염수 (PBS)으로 세포를 네 번 세척 하였다. (그 중 네 적합 미만 10 초) 리튬 유출을 막기 위해 가능한 한 빨리 이러한 세척을 수행한다. </li><li> 를 Lyse 25 % 질산에 세포. 25 % 질산 웰 당 250 μL를 적용하고 그 후, 피펫 팁의 단부와 각 웰 스크랩 새로운 미시간 전체 웰 현탁액을 전송하고, 적어도 1 시간 방치crocentrifuge 튜브. </li><li> 세포 파편을 제거하기 위해 15,000 XG (실온)에서 5 분간 원심 분리하여이를 1.5 ml의 마이크로 원심 분리 튜브에 이동 분해물. </li><li> 원자 흡수 분광법 (21)에 의해 상층 액의 리튬 함량을 측정. 참고 : 다른 기업들이 리튬 중공 음극 램프를 장착 할 필요가 원자 흡수 분광기를 제공합니다. 이 기계는 매우 정밀한뿐만 아니라 매우 섬세으로 제조업체의 지침을 따르십시오. </li></ol>

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The authors have nothing to disclose.

이 프로토콜은 부위 특이 적 변이에 의해 자신의 기본 순서를 변경하지 않고 세포막에서 세포 나 + / H + 교환기를 발현하는 세포를 선택하는 방법에 대해 설명합니다. 이러한 교환기 이제 특성화 될 수있다. 이 방법은 세포막에 소포 나 + / H + 교환기를 표현한다 세포주를 선택하는 양성자 세포의 세포 독성에 기초한다. 여기에는 리튬 +,</su...

대부분의 세포 내 구획 성숙, 인신 매매, 단백질이나 호르몬과로드 신경 전달 물질의 재활용을위한 핵심 매개 변수입니다 산성 내강의 pH를 표시합니다. 그것은 세포질 포성 내용 사이의 pH 구배 소포 CLC 클로라이드 컨베이어 (2)에 결합 된 액포 H + -ATPase를 (1)에 의해 생성되는 것으로 밝혀졌다. 두 녹아웃 (KO) 마우스 및 인간 환자에서, 이들 컨베이어의 중요성 3-6 유전자 돌연변이로 인한 무거운 표현형에 의해 강조되었다. 나트륨 수소 교환기 SLC9A 가족의 구성원, 또한 나트륨 + / H + 교환기 NHEs 불리는 세포 내 pH 및 세포 볼륨 조절 키 이펙터에서뿐만 아니라 상피 걸쳐 산 - 염기 당량의 vectorial 수송 것으로 나타났다 . 세포막 NHEs 외에도 세 고도로 + / H + 기, NHE 6, 7은 Na 보존9는 트랜스 - 골지 네트워크에서 초기 엔도 좀 (7)로 표현된다. 유전자의 돌연변이는 엔젤 형 또는 크리스찬 증후군 8-9, 가족 기반의 자폐증 (10)와 주의력 결핍 과잉 행동 장애 11-12로 연결되어있다. 이 교환기는 또한 알츠하이머 병의 감수성 (13)과 X-링크 된 정신 지체 연속 유전자와 같은 신경 퇴행성 문제 (14) 증후군에 참여했습니다. 함께 찍은, 이러한 연구는 두뇌 발달 및 / 또는 기능에서 이러한 세포 내 NHEs의 중요성을 강조 표시합니다. 이러한 기의 세포 내 현지화 정확한 자신의 이온 선택성 측정, 전송 방향, 운동 매개 변수 및 규제를 방지 할 수 있습니다. 세포 내 구획에서 발현 모든 컨베이어의 경우와 같이, 자신의 생화학 활동을 평가하기 때문에 완전히 생리적 역할과 mechan을 이해하는 것은 매우 어렵다ISMS는 병적 인 의미를 기본. 높은 세포질 K + 농도에 따라, 가장 일반적으로 받아 들여지는 가설은 K + 결합 된 양성자 유출 컨베이어로 작업하는 것이 었습니다. 이 정상 상태 포성 산도를 유지하기 위해 V--ATPase를 펌핑하여 양성자를 상쇄 할 수있다 등의 양자 누출의 존재가 가정되었다. 이 시각 문서의 목적은 세포막에서 이러한 소포 ​​수송차를 표현하고, (ⅱ) 이러한 수송의 기능을 측정하는 두 개의 독립적 인 방법을 보여 세포주의 유전을 선택할 수 있도록하는 방법을 설명하기 (I)이다. 세 년 전, Pouysségur과 프란은 NHE 가족 (15)의 구성원의 분자 클로닝 및 특성을 활성화 유전 적 접근 방식을 개척했다. 이 스크리닝 방법으로 세포 독성 양성자에 기초 하였다. 첫 번째 단계는 결핍 세포주를 수득 하였다어떤 나 + / H +의 교환이 전달체 가역성을 이용 세포막에서 발현. 섬유 아세포 (CCL39 세포주) 또는 리튬 나트륨 + + 미리로드 한 후 2 시간 동안 세포 외 산성 매질 (PH 6.5)에 넣었다. 이것은 기능적 나 + / H + 교환을 발현하는 세포의 죽음과 antiporter 결핍 세포 (PS120 세포주) (16)의 선택되었다. 중탄산없는 배지에서 배양하면, 이들 세포는 급성 세포 내 산성화에 매우 민감하다. 이러한 세포는 세포 급성 acidifications에 제출하는 경우 결과적으로, 세포막에서 기능적 양성자 유출기구의 발현 양 (도 17 참조)를 선택한다. 이러한 기술은 산성화 세포막에서 WT 세포 NHEs의 강제 발현을 활성화 결함 트래 피킹 세포주를 분리 할 수​​있다. 진핵 세포 나 + / H로교환기 electroneutral 있습니다 +, 그들은 채널을 측정하기 위해 큰 성공과 함께 사용 된 전기 생리 학적 방법에 의해 측정 할 수 없습니다. 이 원고 따라서 세포 내 pH 측정과 리튬의 빠른 흡수 동력학하여 교환기의 활성을 측정하는 방법을 설명한다. 기본 개념은 동일한하는, 그 선택 부 위해 개발 된 많은 프로세스는 또한 작용 성 측정에 사용되는 직접 통지 흥미 롭다. 흥미롭게도,이 논문에 설명 된 접근법을 사용하여 선택된 세포주에 존재 매매 결함은 소낭 칼륨 채널 TWIK1 18과 같이 세포막에 다른 소포의 단백질보다 발현에 이르게 것을 관찰했다. 이것은 소포 막 관통 단백질 일반적인 고정 결함기구의 선택을 향해 지적한다. 그러므로 이러한 선택 절차 및 5 월의 생성 세포세포 내 구획의 막 단백질에 근무하는 과학적인 지역 사회를위한 유망한 도구를 구성한다. 뿐만 아니라 여기에 제시된 측정 기술은 다른 비 electrogenic 운송을 연구 적용 할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard cell culture equipement</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td>Used for H+ killing selection composed of many devices with different catalog numbers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator with CO2</td>
			<td style="width:119px;">Sanyo</td>
			<td style="width:119px;">MCO15A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incubator without CO2</td>
			<td style="width:119px;">Heraeus instrument</td>
			<td style="width:119px;">BB6220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laminar flow hood PSM1200NF</td>
			<td style="width:119px;">Fisher&#160;</td>
			<td style="width:119px;">52010120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM medium</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">D5796</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS gold</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:119px;">A15-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicilin/streptomycin</td>
			<td style="width:119px;">PAA</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 10X</td>
			<td style="width:119px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Atomic Absorption spectrometer with Zeeman furnace system</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:119px;">ICE 3500 GFZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LiCl</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">L4408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitric Acid</td>
			<td style="width:119px;">sigma</td>
			<td align="right" style="width:119px;">438073</td>
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			<td style="width:119px;">Leica</td>
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			<td>Composed of many devices with different catalog numbers</td>
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			<td style="width:119px;">leica</td>
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			<td height="21" style="height:21px;width:216px;">camera output</td>
			<td style="width:119px;">leica</td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888393</td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
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			<td height="42" style="height:42px;width:216px;">images acquisition/analysis software</td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">fluorescence Xenon lamp</td>
			<td style="width:119px;">leica</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH sensitive probe, BCECF-AM</td>
			<td style="width:119px;">life technologies</td>
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			<td style="width:119px;">N7143</td>
			<td></td>
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	</tbody>
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functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

엔도 솜 산성화는 시냅스 소포의 단백질 분해 및 재활용, 둔감 수용체 및 신경 전달 물질의 로딩 등의 처리, 다양한 중요하다. 이 산성화는 CLC 염화 컨베이어에 결합 된 양성자 -ATPase를,에 의해 매개되는 기술되어있다. electroneutral 양성자 수송을 고도로 보존, ​​나 + / H + 교환기 (NHE) 6, 7, 9는 이러한 구획에서 표현된다. 유전자의 돌연변이는 인간의인지 적, 신경 퇴행성 질환과 연결되어있다. 자신의 세포 내 현지화 상세한 기능 특성을 방지 것처럼 역설적으로 자신의 역할은 애매 남아있다. 이 논문은이 문제를 해결하는 방법을 나타내고있다. 이것은 세포막에서 세포를 유지하여 NHEs 급성 세포질 산성화 생존 가능한 돌연변이 세포주의 선택으로 구성. 그러므로 이온 선택성 및 활성을 측정하기 위해 두 개의 상보적인 프로토콜을 도시(ⅰ) 하나의 비디오 형광 현미경을 사용하여 세포 내 pH 측정에 기초하고, (ⅱ) 하나의 리튬의 빠른 흡수 동력학에 기초한 이러한 교환기. 이러한 프로토콜은 다른 비 electrogenic 수송차를 측정하도록 외삽 될 수있다. 또한, 여기에 제시된 선택 절차는 세포 표현형으로 고정 불량 셀을 생성한다. 따라서, 이러한 세포는 세포막에서 다른 소포 막 단백질을 발현한다. 여기에 묘사 된 실험 전략은 따라서 다음 소낭 + / H + 선택에 사용 교환기 나트륨과 함께 ​​세포막에서 발현된다 다른 세포 내 단백질을 연구하기 위해 잠재적으로 강력한 도구를 구성 할 수있다.

선택 : 도 1a에 도시 된 바와 같이 H + 죽이는 선택은, 암모늄 약 염기의 확산에 기초한다. 약염기의 pH 및 세포 내에서 산의 확산 효과는 월터 붕소 및 공동 (22)에 의해 개척되었다. 우아한 아이디어는 긍정적 인 유전자 선택을위한 치명적인 산성화가 다음 자크 Pouysségur (17)에 의해 개발 된 생산하는이 현상을 사용할 수 있습니다. 이러한 프...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

나의 기능적 특성<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; 선택을 양자가 살해 사용하여 세포 내 구획의 교환기는 플라즈마 막에서 그들을 표현하는

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

functional-characterization-na-h-exchangers-intracellular

Research

JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K 조회수.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

비디오: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

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, ...

연구

생물학

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidic Mesophases를 사용하여 구조 결정을위한 막 단백질 Crystallizing

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

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 ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

내생 와일드 타입 BRCA1을 표현 세포를 사용하는 동종 재조합에 BRCA1 변이의 효과를 확인

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidic Mesophases의 막 단백질의 높은 처리량을 결정하기위한 로봇의 사용

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

형광 단백질을 표현 라이브 마우스의 이미징 세포 이하의 구조물의 intravital 현미경

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

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 ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

해로운 효과의 낮은 압력 플라즈마 살 균 새 균의 subtilis 포자를 사용 하 여 라이브 셀 현미경의 생존에 조사

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...