A.A.J.와 M.A.가 원고를 공동 집필했으며 모든 공동 저자가 원고에 대해 논평했습니다. M.A.는 CNRS와 "Projet Fondation ARC"(PJA2022070005322)의 지원을 받습니다.A.A.J.는 Fondation des Treilles, Fonds Saint-Michel 및 Fondation du Collège de France의 지원을 받습니다.

Impact of cytoskeleton on nuclear shape and condensate dynamics in mouse oocytes(생쥐 난모세포의 핵 형태 및 응축물 역학에 대한 세포골격의 영향)

<ol>
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J., Maro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+and+chromatin+behavior+follow+MAP+kinase+activity+but+not+MPF+activity+during+meiosis+in+mouse+oocytes.">Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes.</a> Development. 120 (4), 1017-1025 (1994).</li><li>Reis, A., Chang, H. Y., Levasseur, M., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APCcdh1+activity+in+mouse+oocytes+prevents+entry+into+the+first+meiotic+division.">APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division.</a> Nat Cell Biol. 8 (5), 539-540 (2006).</li><li>El-Hayek, S., Clarke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+oocyte+growth+and+development+by+intercellular+communication+within+the+follicular+niche.">Control of oocyte growth and development by intercellular communication within the follicular niche.</a> Results Probl Cell Differ. 58, 191-224 (2016).</li><li>Dumont, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+centriole-+and+RanGTP-independent+spindle+assembly+pathway+in+meiosis+I+of+vertebrate+oocytes.">A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes.</a> J Cell Biol. 176 (3), 295-305 (2007).</li><li>Kal&#225;b, P., Pralle, A., Isacoff, E. 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ImageJ Available from: <a target="_blank" href="https://imagej.github.io/update-sites/big-epfl">https://imagej.github.io/update-sites/big-epfl</a> (2023)</li><li>Hebert, B., Costantino, S., Wiseman, P. 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Stowers ImageJ Plugins Available from: <a target="_blank" href="https://research.stowers.org/imagejplugins/">https://research.stowers.org/imagejplugins/</a> (2023)</li><li>Luisier, F., Vonesch, C., Blu, T., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+interscale+wavelet+denoising+of+Poisson-corrupted+images.">Fast interscale wavelet denoising of Poisson-corrupted images.</a> Signal Process. 90 (2), 415-427 (2010).</li><li>. Ovocyte_Nucleus Available from: <a target="_blank" href="https://github.com/orion-cirb/Ovocyte_Nucleus">https://github.com/orion-cirb/Ovocyte_Nucleus</a> (2023)</li><li>Letort, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> gletort/ImageJFiles: GLijplugs_v0.2.0. , (2022).</li><li>. ImageJFiles Available from: <a target="_blank" href="https://github.com/gletort/ImageJFiles/tree/master/radioak">https://github.com/gletort/ImageJFiles/tree/master/radioak</a> (2023)</li><li>Chu, F. Y., Haley, S. C., Zidovska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+shape+fluctuations+of+the+cell+nucleus.">On the origin of shape fluctuations of the cell nucleus.</a> Proc Natl Acad Sci U S A. 114 (39), 10338-10343 (2017).</li><li>Caragine, C. M., Haley, S. C., Zidovska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+fluctuations+and+coalescence+of+nucleolar+droplets+in+the+human+cell+nucleus.">Surface fluctuations and coalescence of nucleolar droplets in the human cell nucleus.</a> Phys Rev Lett. 121 (14), 148101 (2018).</li><li>Introini, V., Kidiyoor, G. R., Porcella, G., Cicuta, P., Cosentino Lagomarsino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centripetal+nuclear+shape+fluctuations+associate+with+chromatin+condensation+in+early+prophase.">Centripetal nuclear shape fluctuations associate with chromatin condensation in early prophase.</a> Commun Biol. 6 (1), 1-11 (2023).</li><li>Boija, A., Klein, I. A., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+condensates+and+cancer.">Biomolecular condensates and cancer.</a> Cancer Cell. 39 (2), 174-192 (2021).</li></ol>

저자는 상충되는 이해관계가 없음을 선언합니다.

이 프로토콜의 주요 단계에는 생존 또는 정상 기능에 영향을 미치지 않는 난모세포의 적절한 미세주입(9,10,11)과 핵 반점과 같은 관련 구조의 정확한 시각화를 가능하게 하는 사전 정의된 양의 cRNA를 미세주입하는 것이 포함됩니다.
세포질과 (내)핵 역학 사이의 연관성을 확립하는 것은 세포골격이 핵 또는 ...

핵 포지셔닝은 여러 세포 및 발달 기능에 필수적이다 1,2,3,4,5. 난모세포(oocyte)라고 불리는 포유류 암컷 생식세포는 크기가 비대칭적으로 분할됨에도 불구하고 세포질을 리모델링하여 세포중심에 핵을 위치시키는데, 이는 후속 염색체의 중심에서 벗어난 염색체에 의존합니다6(그림 1). 이러한 핵의 중심화는 생쥐와 인간에서 성공적인 난모세포 발달을 예측하며, 7,8 따라서 배아 형성 잠재력을 예측합니다(그림 1).
마우스 난모세포의 세포질 리모델링은 주로 악토미오신 세포골격(actomyosin cytoskeleton)9에 의해 주도됩니다(그림 2). 그 활동은 핵(10)을 교반하고, 재배치하고, 관통하는 기계적 힘을 발생시킨다(그림 2). 결과적으로, 이러한 세포질-핵질 힘 전달은 생체 분자 응축물 12,13,14,15,16으로 알려진 핵의 여러 막이 없는 소기관 중 하나인 핵 스페클 11이라고 불리는 핵 메신저 RNA 처리 소기관의 역학을 조정합니다(그림 2).
실시간 이미징은 핵 교반의 기능적 의미를 해독하는 데 결정적인 역할을 했습니다. 액틴 메쉬(actin mesh)와 벌크 세포질(bulk cytoplasm)에 대한 시간적 해상도가 높은 영상뿐만 아니라, 몇 시간에 걸친 핵 이동에 관한 영상은 서로 다른 시간 척도를 연결하는 핵 위치 결정(nuclear positioning)에 대한 이론적 모델을 정교화하는 데 크게 기여했다9. 또한, 세포질, 핵 윤곽 및 염색질 및 핵 응축물과 같은 핵 구성 요소의 높은 시간 해상도 동영상은 쥐 난모세포에서 RNA 처리 및 유전자 발현에 대한 핵의 세포골격 기반 교반의 역할을 강조하여 세포 내의 다양한 시공간 규모를 연결했습니다10,11. 전체적으로, 실시간 이미징을 기반으로 한 이러한 스케일 교차 접근 방식은 핵의 세포골격 교반과 난모세포의 발달 성공을 연결하는 첫 번째 이론적 근거를 제공했습니다.
이 프로토콜은 생쥐 난모세포의 핵 및 내부 구성 요소로의 세포질 힘(주로 F-액틴에 의해 생성되고 부분적으로 미세소관에 의해 생성됨)의 전달을 연구하는 데 사용되는 이미징 및 이미지 분석 파이프라인을 제공합니다. 이러한 실험의 결과는 최근 두 가지 연구10,11에서 볼 수 있듯이 세포질의 세포골격에서 핵 내부에 이르기까지 공간적 규모에 걸쳐 힘의 연속체를 포착하는 것이며, 이는 세포질 활성 운동, 핵 윤곽의 변동, 단일 유형의 핵 생체 분자 응축물의 움직임 및 표면 변동 사이의 연관성을 확립했습니다. 핵 얼룩. 동일한 접근법이 악성 암세포(17)의 맥락에서와 같이 세포질 힘이 변화할 것으로 예상되는 다른 모델 시스템에도 적용될 수 있다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma&#160;</td>
			<td>A3311</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-X1-M1 spinning disk</td>
			<td>Yokogawa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMI6000B microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet microinjector</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter wheel</td>
			<td>Sutter Instruments Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorodish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Metamorph software&#160;</td>
			<td>Universal Imaging,&#160;</td>
			<td></td>
			<td>version 7.7.9.0</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M8410-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OF1 and C57BL/6 mice&#160;</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tailing kit&#160;</td>
			<td>Thermo Fisher</td>
			<td>AM1350</td>
			<td></td>
		</tr>
		<tr>
			<td>Retiga 3 CCD camera&#160;</td>
			<td>QImaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNAeasy kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>SC35 antibody</td>
			<td>Abcam</td>
			<td>ab11826</td>
			<td>Nuclear speckle antibody; mouse IgG1 anti-SRSF2/SC35 (1:400)</td>
		</tr>
		<tr>
			<td>SRSF2-GFP plasmid&#160;</td>
			<td>&#160;OriGene Technologies</td>
			<td>MG202528</td>
			<td>NM_011358</td>
		</tr>
		<tr>
			<td>Stripper Micropipette</td>
			<td>&#160;XLAB Solutions</td>
			<td></td>
			<td>specialized for oocyte collection</td>
		</tr>
		<tr>
			<td>T3 mMessage mMachine</td>
			<td>Thermo Fisher</td>
			<td>AM1384&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 mMessage mMachine&#160;</td>
			<td>Thermo Fisher</td>
			<td>AM13344</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic chamber</td>
			<td>Life Imaging Service</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Windows Excel</td>
			<td>Windows</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing cytoskeleton-based agitation of the mouse oocyte nucleus across spatial scales

여성 불임의 원인을 이해하는 데 있어 가장 큰 어려움은 난모세포라고 하는 여성 생식세포의 발달을 관장하는 메커니즘을 밝히는 것입니다. 난모세포의 발달은 세포 성장과 후속 분열로 특징지어지며, 난모세포가 정자와 융합하여 배아 발생을 시작할 수 있도록 준비하는 두 가지 중요한 단계입니다. 성장하는 동안 난모세포는 세포질을 재조직하여 세포중심에 핵을 위치시키는데, 이는 생쥐와 인간의 성공적인 난모세포 발달을 예측하여 배아 생성 가능성을 예측하는 사건입니다. 쥐 난모세포에서 이러한 세포질 재편성은 세포골격에 의해 주도되는 것으로 나타났으며, 세포골격의 활동은 핵을 교반하고, 재배치하고, 관통하는 기계적 힘을 생성합니다. 결과적으로, 이 세포질에서 핵질로의 힘 전달은 생체 분자 응축물로 알려진 핵 RNA 처리 소기관의 역학을 조정합니다. 이 프로토콜은 높은 시간적 해상도로 생쥐 난모세포의 공간 규모에 걸쳐 세포골격이 핵에 미치는 영향을 문서화할 수 있는 실험적 프레임워크를 제공합니다. i) 난모세포 세포질의 세포골격 활성, ii) 난모세포핵의 세포골격 기반 교반, iii) 난모세포 핵의 생체 분자 응축물 역학에 미치는 영향을 평가하는 데 필요한 이미징 및 이미지 분석 단계와 도구를 자세히 설명합니다. 난모세포 생물학 외에도, 여기에 설명된 방법은 체세포에서 사용하여 규모 전반에 걸쳐 핵 역학의 세포골격 기반 조정을 유사하게 해결할 수 있습니다.

Nuclear positioning is essential for multiple cellular and developmental functions1,2,3,4,5. Mammalian female germ cells named oocytes remodel their cytoplasm to position the nucleus at the cell center despite undergoing an asymmetric division in size, which relies on subsequent chromosome off-centering6 (Figure 1). This centering of the nucleus predicts successful oocyte development in mice and humans7, 8, and thus, their embryogenic potential (Figure 1).
Cytoplasmic remodeling in mouse oocytes is driven primarily by the actomyosin cytoskeleton9 (Figure 2). Its activity generates mechanical forces that agitate, reposition, and penetrate the nucleus10 (Figure 2). Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear messenger RNA-processing organelles named nuclear speckles11, one of several membrane-less organelles in the nucleus known as biomolecular condensates12,13,14,15,16 (Figure 2).
Live imaging has been decisive in deciphering the functional implications of nuclear agitation. Movies of nuclear migration over hours, as well as high-temporal resolution movies of the actin mesh and the bulk cytoplasm, largely contributed to the elaboration of a theoretical model for nuclear positioning, linking different timescales9. Also, high temporal resolution movies of the cytoplasm, nuclear outline and nuclear components such as chromatin and nuclear condensates, highlighted the role of cytoskeleton-based agitation of the nucleus on RNA-processing and gene expression in mouse oocytes, bridging different spatiotemporal scales within the cell10,11. Altogether, such a scale-crossing approach based on live imaging provided the first rationale linking cytoskeletal agitation of the nucleus to the developmental success of oocytes.
The protocol provides the imaging and image analysis pipeline used to study the transmission of cytoplasmic forces (generated primarily by F-actin and partly by microtubules) to the nucleus and its internal components in mouse oocytes. The outcome of these experiments is to capture the continuum of forces across spatial scales, from the cytoskeleton in the cytoplasm to the nuclear interior via high temporal resolution movies as shown in two recent studies10,11, that established the link between cytoplasmic active movements, fluctuations of the nuclear outline, as well as movement and surface fluctuations of a single type of nuclear biomolecular condensates: nuclear speckles. The same approach may be applied to other model systems where cytoplasmic forces are expected to change, such as in the context of malignant cancer cells17.

저자 스포트라이트: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

공간 규모에 걸쳐 마우스 난모세포핵의 세포골격 기반 교반 캡처

The lab aims to better understand the mechanisms involved in the acquisition of female gamut quantity, which is essential for the reproduction of sexual species using the mouse oocytes model system. Recently, we identified a novel mechanical transduction process that promotes agitation of the nucleus and its content, leading to the fine regulation of maternal RNAs in mouse oocytes. The imaging and image analysis pipeline presented in this protocol allows us to highlight the transmission of cytoskeletal forces as a continuum across scales from the cytoplasm to the nucleus and its components, including nuclear RNA processing bodies in mouse oocytes.This protocol provides simple tools that allow us to address first transmission across multiple cellular scales in a single pipeline for the first time. Complemented with biophysical modeling, this protocol constitutes a non-invasive technique to address changes in nuclear mechanics and the dissipation of energy across cellular compartments.

도 3의 이미지 패널은 전형적인 완전히 성장한 난모세포(그림 3A), YFP-Rango를 발현하는 완전히 성장한 난모세포의 핵질(그림 3B), 올바르게 발현하는 완전히 성장한 난모세포의 핵질(왼쪽 패널; 그림 3C) 또는 과도한(오른쪽 패널; 그림 3C) SRSF2-GFP cRNA의 투여량과 SC35 항체를 사용하여 완전?...

Watch this Scientific Journal Video about Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes at JoVE.com

이 프로토콜은 생쥐 난모세포 시스템의 핵 모양과 내부 막이 없는 세포 소기관에 대한 세포골격의 물리적 영향을 문서화하기 위한 실험 프레임워크를 제공합니다. 이 프레임워크는 다른 세포 유형 및 컨텍스트에서 사용하도록 조정할 수 있습니다.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...

이 연구실은 마우스 난모세포 모델 시스템을 사용하여 유성 종의 번식에 필수적인 암컷 영역의 양 획득과 관련된 메커니즘을 더 잘 이해하는 것을 목표로 합니다. 최근에 우리는 핵과 그 내용물의 교반을 촉진하여 마우스 난모세포에서 모계 RNA의 미세 조절로 이어지는 새로운 기계적 형질 전달 과정을 확인했습니다. 이 프로토콜에 제시된 이미징 및 이미지 분석 파이프라인을 통해 세포질에서 핵 및 그 구성 요소(마우스 난모세포의 핵 RNA 처리 본체 포함)에 이르는 규모에 걸쳐 연속체로서 세포골격력의 전달을 강조할 수 있습니다.이 프로토콜은 단일 파이프라인에서 여러 세포 규모에 걸친 첫 번째 전송을 처음으로 처리할 수 있는 간단한 도구를 제공합니다. 생물물리학적 모델링으로 보완된 이 프로토콜은 핵 역학의 변화와 세포 구획에 걸친 에너지 소실을 해결하기 위한 비침습적 기술을 구성합니다.

capturing-cytoskeleton-based-agitation-mouse-oocyte-nucleus-across

Research

JoVE Journal

Biology

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

499 조회수.  Collège de France, CNRS, INSERM, Université PSL. 이 연구실은 마우스 난모세포 모델 시스템을 사용하여 유성 종의 번식에 필수적인 암컷 영역의 양 획득과 관련된 메커니즘을 더 잘 이해하는 것을 목표로 합니다. 최근에 우리는 핵과 그 내용물의 교반을 촉진하여 마우스 난모세포에서 모계 RNA의 미세 조절로 이어지는 새로운 기계적 형질 전달 과정을 확인했습니다. 이 프로토콜에 제시된 이미징 및 이미지 분석 파이프라인?...

비디오: 저자 스포트라이트: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.