Elucidating spatio-temporal nuclear dynamics in 4D using state-of-the-art imaging in beating hearts

使用最先进的跳动心脏成像以 4D 方式阐明时空核动力学

• 批准号: 2888380
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2888380
• 关键词: Elucidating; spatio; temporal; nuclear; dynamics

The nuclear envelope and associated Linker between Nucleoskeleton and Cytoskeleton (LINC) complex proteins are essential for proper cardiac and skeletal muscle development (Ross and Stroud, 2021; Stroud, 2018; Stroud et al., 2017). This is highlighted by the numerous mutations that lead to cardiac and skeletal myopathies in humans and mice that are collectively known as laminopathies. Interestingly, laminopathies predominantly affect striated muscle tissue which are under constant mechanical load. Pathological mechanisms underlying the laminopathies are poorly understood, but seem to involve multiple, often overlapping factors: altered or weakened structural integrity at the nucleus, which appears to be particularly important in contractile cells, leading to aberrant morphologies, ruptures and DNA damage; altered genome organisation, adversely affecting gene expression; and altered chemical and biomechanical signalling, affecting a host of cellular functions (Jaalouk and Lammerding, 2009; Stephens et al., 2018; Strom et al., 2021).Currently, the majority of these studies are performed in vitro and require physical interventions to drive nuclear deformation and rupture in 2D cell culture. Therefore, new imaging and system modalities will be required to drive our understanding of laminopathies further in environments that better recapitulate those observed in vivo. In regards to this, live light-sheet imaging provides unprecedented spatio-temporal resolution in thick tissues with minimal phototoxicity (https://www.m2lasers.com/microscopy-aurora.html). Here, we propose live light-sheet imaging of beating intact hearts that are under endogenous physical load as the next forefront required to extend our knowledge of changes to nuclear morphology and dynamics that are frequently observed in laminopathies. In this project, we propose to develop an imaging platform allowing live imaging of cardiomyocyte nuclei in beating hearts. This will enable interrogation of how forces extrinsic to nuclei (driven by muscle contraction) and intrinsic to nuclei (chromatin organization) drive nuclear deformation in control and LINC complex mutant mouse hearts. The findings will be of broad relevance to understanding nuclear dynamics in live, intact hearts in healthy hearts, as well as in disease states.In this project, we propose to develop an imaging platform allowing live imaging of cardiomyocyte nuclei in beating hearts. This will enable interrogation of how forces extrinsic to nuclei (driven by muscle contraction) and intrinsic to nuclei (chromatin organization) drive nuclear deformation in control and LINC complex mutant mouse hearts. The findings will be of broad relevance to understanding nuclear dynamics in live, intact hearts in healthy hearts, as well as in disease states.To achieve this objective, we propose the following aims:Aim 1: Establishing a physical platform for imaging hearts on the light-sheet microscope.Aim 2: Optimization of heart extraction and culture conditions for imaging hearts ex vivo.Aim 3: Establishment of mouse colony with nuclear indicator mice.Aim 4: Imaging fluorescently labelled nuclei in control and nuclear envelope mutant mouse models.

核骨骼和细胞骨架（LINC）复合蛋白之间的核包膜和相关连接器对于适当的心脏和骨骼肌发育至关重要（Ross and Stroud，2021; Stroud，2018; Stroud et et al。，2017）。众多突变导致人类和小鼠的心脏和骨骼肌病的众多突变强调了这一点，这些突变统称为椎板病。有趣的是，椎板病主要影响在恒定的机械负荷下的肌肉组织。椎板病的病理机制知之甚少，但似乎涉及多个经常重叠的因素：细胞核的结构完整性改变或弱化，这在收缩细胞中似乎尤其重要，导致异常的形态，破裂和DNA损伤；基因组组织改变，对基因表达产生不利影响；化学和生物力学信号传导改变，影响了许多细胞功能（Jaalouk和Lammerding，2009; Stephens等，2018; Strom等，2021）。当然，这些研究中的大多数是在体外进行的，并且需要进行物理干预以驱动2D细胞培养的核畸形和破裂。因此，将需要新的成像和系统方式来推动我们在体内观察到的环境中进一步推动我们对椎板病的理解。关于这一点，实时的灯页成像在厚的组织中提供了前所未有的时空分辨率，具有最小的光毒性（https://www.m2lasers.com/microscopy-aurora.html）。在这里，我们提出了在内源性物理负载下跳动完整心脏的实时灯页成像，这是扩展我们对椎板病经常观察到的核形态和动力学变化的下一个前沿。在这个项目中，我们建议开发一个成像平台，允许在跳动心脏中对心肌细胞核进行现场成像。这将质疑如何强迫外部向核（由肌肉收缩驱动）和对核（染色质组织）固有的驱动驱动对照和林型复合物突变小鼠心脏的核变形。这些发现将与理解健康心脏和疾病状态中的活力，完整心脏中的核动态具有广泛相关性。在该项目中，我们建议开发一个成像平台，以允许跳动心脏中心肌细胞核的实时成像。这将质疑如何强迫外部向核（由肌肉收缩驱动）和对核（染色质组织）固有的驱动驱动对照和林型复合物突变小鼠心脏的核变形。这些发现将与理解健康心脏以及疾病状态中的核动力学相关。为了实现这一目标，我们提出以下目的：目标1：建立一个物理平台，以在光片表上进行成像成像心脏，以光 - 片段的显微镜进行成像。MAIM2：对心脏提取和培养条件的优化和培养条件，以实验室3：用核的核定核定的核定核定的核定材料。对照和核包膜突变小鼠模型。