저자는 프로토콜을 개발하고 시약을 제공하는 인물, N. Gudimchuk 및 P. Zakharov에 대한 이미지를 제공하기 위해, N. Dashkevich, N. Gudimchuk 및 A. Korbalev을 재사용 할 수있는 유량 용기를 디자인하고 제조하는 데 도움을 위해 FI Ataullakhanov에게 감사의 말씀을 전합니다 텍스트 편집 및 팁 및 토론을위한 Grishchuk 연구소의 다른 회원들과 도움을 제닌 표지 미세 소관 씨, A. Potapenko를 준비합니다. 이 작품은에 의해 부분적으로 지원되었다 NIH 부여 GM R01-098389과 킴멜 학자 ELG 펜실베니아 근육 연구소에서 파일럿 부여, RFBR 12 채로 04-00111-13-04-40190-H를 부여하고 (13) - 04-40188-H, 과학 상임위원회 보조금 러시아 아카데미 FI Ataullakhanov에 (분자 시스템 통합 및 분자 및 분자 세포 생물학 프로그램의 메커니즘), NIH는 JR 매킨토시에 GM R01 GM033787을 부여하고, 드미트리 Zimin 왕조 재단 박사의 교제 VAV

저자가 공개하는 게 없다.

대부분의 단일 분자 분석은 오늘날 일상적으로 크게 특이 단백질의 고집을 줄이기 위해 특수 처리 커버 슬립을 사용합니다. 우리가 여기에서 설명하는 절차는 하워드 랩 32에서 개발 된 원래의 프로토콜의 변형이며, 우리는 커버 슬립을 silanizing은도 형광을 사용하지 않는 DIC 기반 비드 분석과 노력의 가치는 사실을 알게 될 것입니다. 이러한 커버 슬립 조립 챔버 훨씬 청소기 표면을 보여?...

미세 소관은 높은 세포 구조, 세포 운동성, 세포 분열, 세포 내 수송 1에 중요한 골격 구조를 보존하고 있습니다. 이러한 동적 중합체는 GTP의 존재 튜 불린에서 조립 이들은 자발적 성장이 단축 전환. 미세 소관은 (직경 25 nm의) 콘트라스트가 광학 현미경으로 미세 소관을 관찰하는 데 사용되어야합니다 강화하기 때문에 특별한 광학 기술 매우 얇은입니다. 이러한 폴리머와 이전 작업은 미분 간섭 대비 (DIC)을 3 사용하여 동적 동작을 살펴 보았다. 체외에서 이것과 유사한 연구는 일반적인 실험 조건에서 미세 소관은, 드물게 해중합에 재앙과 스위치를 거쳐야 밝혀마다 5 ~ 15 분 (이 주파수는 28 ~ 32 ° C에서 검사 7-15 밀리미터 용해 튜 불린의 농도입니다 ) 4. 다른 기술은 따라서 인덕턴스에 제안되었다제어 방식으로 전자 미세 소관 해중합. 미세 소관 쇼트닝 여기서 설명한 바와 같이, 거리 수용성 튜 불린 5,6 세척 레이저 광 (7)로 미세 소관을 절단 또는 분할 된 미세 소관 8을 사용하여 발생 될 수있다. 분할 된 미세 소관뿐만 아니라, 확률 론적으로 전환 폴리머를 사용하여 이전 작업은 이러한 염색체, 소포, 단백질 코팅 비즈 작은 세포 내화물은, 단축 미세 소관 9-13의 끝에서 이동할 수 있다는 것을 발견했다. 이러한 현상은 유사 분열 세포의 염색체 운동에 대한 직접적인 의미를 생각하고, 기본 메커니즘이 활성화 연구 14-16에서 현재됩니다. 최근, 총 내부 반사 형광 (TIRF) 현미경 등의 형광 기반 기술, 동적 인 미세 소관과 운동성을 연구하기 위해 사용되었다가 17-24로 끝났습니다. 이 방법의 장점은 허용한다는 것이다 상호 작용의 검토다른 형광 물질로 표지 단백질을 사용하여 실시간으로 미세 소관과 미세 소관 결합 단백질 사이의. 몇몇 단백질 복합체는 미세 소관 단부를 연신 및 / 또는 단축으로 processively 이동 발견되었다. 그들은 미세 소관 관련 단백질 Dam1 10,12,18, Ska1 19 일 및 XMAP215 (20)뿐만 아니라, 키네신 모터 Kif18A 21, 22, MCAK 23 CENP-E (24)이 (가) 있습니다. 이 단백질은 EB1 (25)와 같은 고전적인 팁 추적 단백질의 그것과 근본적으로 다른 processive 팁 추적을 나타낸다. EB1 분자 및 관련 파트너는 안정적으로 동적 미세 소관은 개별 분자가 빠르게 용해 수영장 (26)와 교환 만 ~ 0.8 초 미세 소관 팁에 바인딩 유지, 종료와 관련된 남아있는 것처럼 보이지만. 미세 소관이 많은 마이크론에 끝 반면, processive 팁 추적기, Dam1처럼, 여행, 및 미세 소관 조언과의 관계는 M 지속될 수모든 초. 팁 연관 시간뿐만 아니라, 추적 결과 속도는, 팁 추적 복합체를 형성하는 분자 (27)의 수에 크게 의존한다. 큰 단백질 앙상블은 일반적으로 훨씬 더 팁 추적기입니다. 예를 들어, 분리 된 효모 kinetochores와 같은 복잡한 어셈블리는 미세 소관에 결합 된 상태를 유지할 수는 시간 28 일 끝납니다. 일부 미세 소관 결합 단백질은, 예를 들어 Ndc80 동원체의 단백질 복합체, 단일 분자 수준에서 종료 미세 소관으로 추적 할 수없는 것으로, 아직 Ndc80은 구슬화물 19,29-31의 움직임을 결합에 매우 효율적입니다되었습니다. 따라서, 다른 단백질 복합체뿐만 아니라 생물학적 역할에 의한 팁 추적 메커니즘을 이해하기 위해, 그 팁 추적 복잡한 분자의 개수의 함수로서 팁 추적을 검사 할뿐만 아니라, 결정하는 것이 중요하다 구슬화물의 표면에 집단 운동을 전시하는이 단지의 기능을 제공합니다. (그림 1A)와 함께 실험을 준비하고 수행 할 수있는 세부적인 프로토콜을 제공하는 아래. 첫째, 상업적으로 이용 가능한 유리 슬라이드 짧은 폴리에틸렌 관 (프로토콜 1)를 첨부하도록 수정됩니다. 재사용 할 수있는 현미경 흐름 챔버는 다음과 같은 슬라이드에서 조립 및 화학적 또는 플라즈마 세정 및 실란 커버 슬립 (프로토콜 2) 32 ~ 34됩니다. 얻어진 챔버 체적은 유입 배관의 용적 등 만 20-25 μL (또는 15 μL 한 작게, 프로토콜 1 주 3 참조)이다. 시판 유동 챔버도 사용될 수 있지만, 그 양이 단백질의 불필요한 낭비로 이어지는, 보통 크다. 큰 챔버가 사용되는 경우, 아래의 프로토콜의 모든 솔루션의 볼륨이 비례 적으로 조정해야한다. 미세 소관의 씨앗이 서서히 가수 분해 GTP 아날로그, GMPCPP (구아노 신-5&#39;-[(α, β) methyleno] 트리 포스페이트) (프로토콜 3 등을 사용하여, 준비, 하이 먼 &lt;도 참조EM&gt; 등. 35). 씨앗 청소 커버 슬립에 고정하고 표면은 이후에 다른 단백질의 비특이적 흡착을 방지하기 위해 차단 32 (프로토콜 4 제닌 사용 씨의 고정화를 설명합니다). 분단 미세 소관은 5 프로토콜을 사용하여 제조 될 수있다. 이 방법의 주된 이론적 근거는 GTP의 존재하에 동적 미세 소관 형성 중합체, GMPCPP 포함될 안정 튜 불린 세그먼트의 짧은 &quot;캡&quot;을 추가하여 일시적으로 안정화 될 수 있다는 것이다. 이 모자는 로다 민 표지 튜 불린을 포함, 그래서 그들은 530-550 nm의 레이저 또는 수은 아크 램프 (프로토콜 6) (36)의 시야를 비추는 것만으로 간단하게 제거 할 수 있습니다. 팁 트래킹 신호의 형광 강도는 고려 현미경 야 조사 (프로토콜 12)의 요철을 가지고, 분해 미세 소관 단부로 여행 분자의 수를 추정하기 위해 사용될 수있다. 유사한 접근법이 사용될 수있다27 (프로토콜 8)에 기술 된 바와 같이 제조 된 해중합의 미세 소관 단백질 코팅 구슬 사이의 상호 작용을 연구합니다. 일부 단백질은 쉽게 분할 된 미세 소관의 벽에 결합하지만, 레이저 핀셋도하여 그 바인딩 홍보, 미세 소관 벽 근처 비드를 보유 할 수 있습니다.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Table 1. Microscopy and other equipment.&lt;/strong&gt;</td></tr><tr><td>Microscope</td><td>Zeiss&lt;br/&gt; Nikon</td><td>Axio Imager 2&lt;br/&gt; Eclipse Ti</td><td>other microscope models capable of DIC and epifluorescence-imaging can be used</td></tr><tr><td>Objective</td><td>Zeiss&lt;br/&gt; Nikon</td><td>420490-9900-000&lt;br/&gt; CFI Apo 100x Oil 1.49</td><td>100X, DIC, 1.3-1.49 NA</td></tr><tr><td>Objective heater</td><td>Bioptechs</td><td>150803, 150819-19</td><td></td></tr><tr><td>Fluorescent filter cube</td><td>Chroma</td><td>49004 or 49008&lt;br/&gt; 41017 or 49020</td><td>optimized for Rhodamine fluorescence&amp;nbsp;&lt;br/&gt; optimized for GFP fluorescence</td></tr><tr><td>Acquisition software</td><td>freeware MicroManager&lt;br/&gt; Molecular Devices</td><td>not applicable&lt;br/&gt; MetaMorph 7.5</td><td>http://valelab.ucsf.edu/~MM/MMwiki/&lt;br/&gt; other software can be used to acquire images and for a particle tracking</td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon3, DU-897E-cs0-#BV</td><td>Highly sensitive EMCCD camera</td></tr><tr><td>Trapping laser</td><td>IPG Photonics</td><td>YLR-10-1064-LP</td><td>1,064 nm laser, 10 W</td></tr><tr><td>Fluorescence excitation lasers</td><td>Coherent, Inc.&lt;br/&gt; Coherent, Inc.</td><td>Sapphire 488 LP&lt;br/&gt; Sapphire 552 LP</td><td>excitation of green fluorophores&lt;br/&gt; excitation of red fluorophores</td></tr><tr><td>Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC-001</td><td></td></tr><tr><td>Commercial flow chambers</td><td>Warner Instruments</td><td>RC-20 or RC-30</td><td></td></tr><tr><td>Perfusion pump</td><td>Cole Palmer&lt;br/&gt; Harvard Apparatus</td><td>Masterflex 77120-52&lt;br/&gt; Pico Plus</td><td>Both pumps provide the required rate of liquid flow but a peristaltic pump may pulse at very slow speed. The flow with a syringe pump is more consistent for a wide range of rates but this pump has inertia.&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;Table 2. Microscopy chamber preparation.&lt;/strong&gt;</td></tr><tr><td>Modified microscope slides for reusable chambers</td><td>Precision Glassblowing of Colorado</td><td>Custom order www.precisionglassblowing.com</td><td>Sonic slots in slides using schematics in &lt;strong&gt;Figure 1&lt;/strong&gt;</td></tr><tr><td>Polyethylene tubing</td><td>Intramedic</td><td>427410</td><td>I.D. 0.58 mm, O.D. 0.965 mm; use these tubes to connect assembled chamber to the pump and waste container</td></tr><tr><td>Polyethylene tubing</td><td>Intramedic</td><td>427400</td><td>I.D. 0.28 mm, O.D. 0.61 mm; use these tubes to make the reusable chamber</td></tr><tr><td>Regular microscope slides</td><td>VWR</td><td>48312-003</td><td>Other similar slides can be used</td></tr><tr><td>Coverslips</td><td>VWR</td><td>48393-150, 48366-067</td><td>Other similar coverslips can be used</td></tr><tr><td>Silicon sealant</td><td>World Precision Instruments</td><td>KIT, SILICON SEALANT 5 MIN CURE</td><td></td></tr><tr><td>Epoxy glue</td><td>Loctite</td><td>83082</td><td></td></tr><tr><td>Cyanoacrylate adhesive</td><td>Scotch 3M</td><td>AD114</td><td>Or cyanoacrylate adhesive from other manufacturers</td></tr><tr><td colspan="4">&lt;strong&gt;Table 3. Coverslips cleaning and coating.&lt;/strong&gt;</td></tr><tr><td>Molecular Sieves, Grade 564</td><td>Macron</td><td>4490-04</td><td></td></tr><tr><td>Coverglass Staining Jar</td><td>Ted Pella, Inc.</td><td>21036</td><td></td></tr><tr><td>Coverslip Ceramic Holder</td><td>Thomas Scientific</td><td>8542e40</td><td></td></tr><tr><td>PlusOne Repel Silane</td><td>GE Healthcare Biosciences</td><td>17-1332-01</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>Anti-digoxigenin AB</td><td>Roche Applied Science</td><td>11093274910</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 4. Preparation of seeds and segmented microtubules.&lt;/strong&gt;</td></tr><tr><td>Tubulin</td><td>purified from cow brains&lt;br/&gt; Cytoskeleton, Inc</td><td>&lt;br/&gt; T238P</td><td>For purification protocols see &lt;sup&gt;49&amp;ndash;51&lt;/sup&gt;&lt;br/&gt; Unlabeled porcine tubulin</td></tr><tr><td>Labeled tubulin</td><td>Cytoskeleton, Inc&lt;br/&gt; Invitrogen&lt;br/&gt; Invitrogen</td><td>TL590M&lt;br/&gt; C1171 (Rhodamine)&lt;br/&gt; A-2952 (Digoxigenin)</td><td>Rhodamine-labeled porcine tubulin&lt;br/&gt; Tubulin can be labeled with any amine-reactive dye as in reference&lt;sup&gt;52&lt;/sup&gt;.</td></tr><tr><td>GMPCPP</td><td>Jena Biosciences</td><td>NU-405</td><td>Aliquot and store at -70 &amp;deg;C</td></tr><tr><td>VALAP</td><td>Vaseline, lanolin, and paraffin at 1:1:2 by mass</td><td></td><td>see reference&lt;sup&gt;9&lt;/sup&gt;</td></tr></tbody></table>

preparation of segmented microtubules to study motions driven by the disassembling microtubule ends

미세 소관 해중합은 체외에서 다른 단백질 복합체 단백질 코팅 비즈를 수송하는 힘을 제공 할 수 있습니다. 기본 메커니즘은 세포 분열 중에 미세 소관 의존 염색체 운동에 중요한 역할을하는 것으로 생각되지만, 관련 단백질과 자신의 정확한 역할은 불분명합니다. 따라서, 정제 된 구성 요소와 정의 생화학 적 환경을 사용하여 체외에서 이러한 운동을 연구하는있는 분석법을 개발하는 요구가 증가하고 있습니다. 미세 소관은, 그러나, 본질적으로 불안정 중합체이다, 성장과 단축 사이에 자신의 전환 확률 통제하기가 어렵습니다. 우리가 여기에서 설명하는 프로토콜은 photoablatable는 캡을 안정으로 만든 분할 된 미세 소관을 활용. 이러한 분할 된 미세 소관 해중합함으로써 분해 미세 소관의 끝에서 운동의 연구를 지원, 높은 공간적 해상도로 트리거 될 수 있습니다. 이 기술은 CA에 사용될 수있다동적 미세 소관은 끝 processively 이동 형광 표지 된 단백질 복합체, 분자의 수의 정량적 인 분석을 RRY. 이것으로 신호 대 잡음 비율 및 기타 정량적 형광 분석법을 최적화하려면, 커버 슬립은 수용성 형광 표지 된 단백질의 비특이적 흡착을 감소하도록 처리한다. 상세한 프로토콜은 고려 형광 조명의 불균일을 취해, 등거리 가우시안 적합하여 하나의 형광의 강도를 결정하기 위해 제공된다. 마지막으로, 우리는 processive화물 운동에 몇 미세 소관 해중합에 다른 모터와 nonmotor 단백질의 기능에 대한 통찰력을 제공하는 단백질 코팅 마이크로 비즈의 미세 소관에 의존하는 동작을 연구하는 분할 된 미세 소관의 사용을 설명합니다.

미세 소관을 해중합과 추적 단백질이 끝납니다. 효모 동원체 구성 요소 Dam1는 지금까지 해중합 미세 소관의 최고의 팁 트래커 (14)를 종료한다. GFP로 ​​표지이 10-서브 유닛의 복잡한 쉽게 표현 박테리아 세포 18,38에서 정제, 그래서 우리는 끝 추적 분석에 대한 긍정적 인 제어로 사용하는 것을 권장 할 수 있습니다. 미세 소관의 해중합 끝으로 추적 형광 단백질은 꾸?...

Watch this Scientific Journal Video about Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends at JoVE.com

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

미세 소관은 본질적으로 불안정 폴리머, 그리고 성장과 단축 사이에 자신의 전환 확률 통제하기가 어렵습니다. 여기에서 우리는 photoablatable 안정 캡으로 분할 미세 소관을 사용하여 프로토콜을 설명합니다. 분할 된 미세 소관 해중합함으로써 분해 미세 소관의 끝 동작의 분석을 지원, 높은 공간적 해상도로 트리거 될 수 있습니다.

분해 미세 소관 엔드에 힘 입어 모션을 연구하기 위해 분단 미세 소관의 준비

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

preparation-segmented-microtubules-to-study-motions-driven

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.3K 조회수.  Russian Academy of Sciences.  미세 소관은 본질적으로 불안정 폴리머, 그리고 성장과 단축 사이에 자신의 전환 확률 통제하기가 어렵습니다. 여기에서 우리는 photoablatable 안정 캡으로 분할 미세 소관을 사용하여 프로토콜을 설명합니다. 분할 된 미세 소관 해중합함으로써 분해 미세 소관의 끝 동작의 분석을 지원, 높은 공간적 해상도로 트리거 될 수 있습니다. 

비디오: Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

TIRF 현미경에 의한 시험관 내에서 가교 및 단일 미세 소 관 의 역학의 동시 시각화

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

연구

생화학

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9 정제 초공정 키네신-3 제품군 모터의 단일 분자 분석

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

미세 소관 선인장체의 자체 조립

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the posttranslational modifications, microtubules can form complexes with various interacting proteins. These microtubule-protein complexes are often implicated in human diseases. Understanding the structure of such complexes is useful for elucidating their mechanisms of action and can be studied by cryo-electron microscopy (cryo-EM). To obtain such complexes for structural studies, it is important to extract microtubules containing or lacking specific posttranslational modifications. Here, we describe a simplified protocol to extract endogenous tubulin from genetically modified mammalian cells, involving microtubule polymerization, followed by sedimentation using ultracentrifugation. The extracted tubulin can then be used to prepare cryo-electron microscope grids with microtubules that are bound to a purified microtubule-binding protein of interest. As an example, we demonstrate the extraction of fully tyrosinated microtubules from cell lines engineered to lack the three known tubulin-detyrosinating enzymes. These microtubules are then used to make a protein complex with enzymatically inactive microtubule-associated tubulin detyrosinase on cryo-EM grids.

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

초저온 전자 현미경으로 미세소관-단백질 복합체를 연구하기 위해 포유류 세포에서 변형된 미세소관 추출

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects.
Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues.
The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 &#215; g) to remove debris. The first ultracentrifugation step (100,000 &#215; g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 &#215; g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Microtubules, which are tubulin polymers, play a crucial role as a cytoskeleton component in eukaryotic cells and are known for their dynamic instability. This study developed a method for fractionating microtubules to separate them into stable microtubules, labile microtubules, and free tubulin to evaluate the stability of microtubules in various mouse tissues.

마우스 조직에서 안정적인 미세소관, 불안정한 미세소관 및 유리 튜불린을 분리하기 위한 정량적 미세소관 분획 기술

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

고해상도 이미징 및 개인 별이 미세 소관 역학의 분석 신진 효모에서

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

생물학

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

단극성 미토틱 스핀들에서 디스크 현미경 검사법에 의한 미세소관 역학 측정

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

재구성된 활성 미세소관 다발 내의 힘을 직접 측정

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

조정 가능한 모터 구동 역학 및 역학을 사용한 Actin-Microtubule 복합재 재구성 및 특성화

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

Forming, Confining, and Observing Microtubule-Based Active Nematics

The formation of biopolymer-based active phases has become an important technique for researchers interested in exploring the emerging field of active liquid crystals and their possible roles in cell biology. These novel systems consist of self-driven sub-units that consume energy locally, producing an out-of-equilibrium dynamic fluid. To form the active liquid crystal phase described in this report, purified protein components including biopolymers and molecular motors are combined, and the active nematic phase spontaneously forms in the presence of adenosine triphosphate (ATP). To observe the nematic state, the material must be confined in a suitable geometry for microscopy at a high enough density. This article describes two different methods for the formation of an active nematic phase using microtubules and kinesin motors: assembly of a two-dimensional active layer at an oil and water interface and assembly under an oil layer using an elastomeric well. Techniques to insert the active material into small wells of different shapes are also described.

Presented here are methods for preparing active nematics from microtubules and kinesin motors, including protein preparation and construction and the use of wells for active nematic confinement.

미세소관 기반 활성 네마틱스의 형성, 제한 및 관찰

The formation of biopolymer-based active phases has become an important technique for researchers interested in exploring the emerging field of active ...