Understanding how cells invade through basement membrane in vivo

在发育和免疫细胞运输过程中，特化细胞获得了突破基底膜（BM）基质屏障的能力，从而迁移到感染和损伤部位。在许多疾病中，细胞侵袭也被不恰当地启动，是哮喘、关节炎、多发性硬化症和转移性癌症组织破坏的基础[2-6]。因此，了解细胞如何穿越脑屏障对人类健康具有重要意义。隐杆线虫子宫锚定细胞（AC）侵入外阴上皮是一种高度刻板的细胞入侵体内模型，它结合了许多强大的实验方法，包括活体成像、细胞- bm相互作用的亚细胞视觉分析、快速基因组编辑以及强大的正向遗传和功能基因组方法（图1）[7,8]。利用这些优势，本研究旨在：(1)阐明入侵细胞如何获取和使用能量来促进基底膜的入侵；(2)确定脂质生物合成如何构建一个大的突出物，通过基底膜屏障打开路径；(3)确定入侵细胞如何适应基质金属蛋白酶（MMPs）缺乏的侵袭；(4)揭示在基底膜突破过程中导致、预防和愈合质膜损伤的机制。这些研究与NIH的使命相关，因为它们将导致对细胞侵袭行为的基本生物学过程有更深入的了解，从而允许更好的治疗策略来调节人类疾病的侵袭。所需补充设备（NOT-GM-22-017）的科学依据：母奖R35GM118049-07的所有目的都要求对内源性荧光团标记的蛋白质进行定量实时图像分析。我的小组每周花50个小时在共聚焦显微镜上，以满足奖项的目标，需要一台专用的共聚焦显微镜。我们目前的共聚焦使用的是横河CSU10旋转盘共聚焦头，已经超过15年了。该系统缺乏空间和时间分辨率，无法进行有效完成拟议工作目标所需的分析。我们需要一台横河CSU-W1-T2旋转盘共聚焦头和滨松qCOMS Quest相机，以显着提高光学分辨率并改进我们的实时成像定量分析。CSU-W1-T2的针孔间距更宽，并使用微透镜来收集更多的光。此外，相机能够快速成像帧速率（120 fps），其4.6微米像素尺寸与我们的100X 1.4 NA蔡司物镜的衍射限制分辨率相匹配。这种新系统在收集荧光信号和抑制失焦荧光方面更有效，从而增加了信噪比，同时最大限度地减少了光漂白。它还大大提高了横向、轴向和时间分辨率。我们有机会在我们的共聚焦显微镜上演示CSU-W1-T2旋转盘共聚焦头（图2-4），并发现在检测和成像分辨率方面有了显着改善，这将是完成工作目标的关键。Aim 1的一个关键目标是描述和定义在AC中构建和极化特化高容量线粒体的机制。我们发现线粒体极化到入侵位点，并产生高水平的ATP，以促进侵入性和侵入性突起的形成（图1）[9,10]。我们利用基因组编辑技术，用mNeonGreen （mNG）标记了20多个线粒体电子传递链（ETC）复合物I、II、III、IV和V的组分。ETC通过氧化磷酸化产生ATP。这是ETC的首次内源性标记，我们已经确认了所有菌株的生存能力和健康状况。尽管AC线粒体的体积与邻近的非侵袭性子宫细胞相同，但我们发现AC线粒体内许多ETC成分的荧光增加，特别是在侵袭前（图2）。引人注目的是，我们的初步研究表明，ETC成分不是简单地从非侵入性线粒体中扩增出来的，而是一部分成分在不同水平上富集。这表明侵入性高容量线粒体具有独特的ETC。评估特定线粒体的精确组成需要更好的信噪比成像，而横河CSU-W1-T2提供了这一功能（图2）。此外，该目标的一个关键目标是了解高容量线粒体如何极化到侵入性前沿，以及它们如何与非侵入性线粒体区分开来。这需要线粒体动力学（裂变和融合）的实时成像和线粒体网络分析，这需要Yokogawa CSU W1-T2的高分辨率（图3）。Aim 2的一个关键目标是确定脂质生物合成如何构建破坏BM的侵袭性突起。我们已经确定了超过10种脂质生物合成酶，这些酶在AC侵袭过程中对侵袭性突起的形成和功能很重要，包括对转移至关重要的基因（例如，SREBP[11]）。我们对这些脂质合成酶进行了内源性标记，并发现许多酶定位于内质网（ER） （ELO-1，拉长酶，图2）。我们还发现，在入侵之前，急诊室会急剧扩张。为了表征内质网的扩展，内质网网络中蛋白质的亚细胞定位和内质网的组成，需要横河CSU-W1-T2的高信噪比和更高的分辨率（图2和3）。Aim 3的一个关键目标是了解侵袭性细胞如何适应缺乏mmmp的侵袭程序，这可能解释了为什么MMP疗法在临床癌症试验中失败。我们的初步结果表明，mRNA翻译在MMP丢失后上调和重塑。我们成功地内源性标记了两个核糖体蛋白[12]。随着CSU-W1-T2分辨率的提高，我们现在可以观察到核糖体在侵入前特异性地定位于内质网，这在邻近的非侵入性子宫细胞中没有观察到（图2）。我们还发现核糖体在AC中含量较高（图2）。为了确定在没有MMPs的情况下交流如何适应转换，需要横河CSU-W1-T2的高分辨率和高信噪比。Aim 4的一个关键目标是了解侵袭细胞在侵入过程中冲破基底膜是如何损伤质膜的。为此，我们正在改变BM成分的水平，以确定损害AC的成分。我们已经内源性标记了60多种BM成分，并可以操纵它们在BM中的水平[13,14]。横河CSU-W1-T2旋转盘共聚焦头和Hamamatsu qCOMS Quest相机显着提高了BM的分辨率，并且它使我们能够可视化BM内的纤维型IV胶原结构（图4），我们假设这些结构正在破坏质膜。我们目前的共聚焦系统无法可视化这些结构，因此需要横河CSU-W1- T2来完成母公司的目标。预期的未来成本和所需设备的培训：横河CSU-W1-T2旋转盘共焦相机和滨松qCOMS Quest相机将取代我们现有的CSU-10和ImageEM BT-EM CCD相机。这两种升级都将由micromanager软件控制，该实验室有使用经验，因此不需要额外的培训。Biovision将继续免费为我们的共聚焦提供服务（就像他们16年来所做的那样），因此没有维护成本。

RESEARCH STRATEGY Summary of Parent Award R35GM118049-07: Understanding How Cells Invade Through Basement Membrane In Vivo During development and immune cell trafficking, specialized cells acquire the ability to breach basement membrane (BM) matrix barriers to migrate to sites of infection and injury [1]. Cell invasion is also inappropriately initiated during numerous diseases and underlies tissue destruction in asthma, arthritis, multiple sclerosis, and metastatic cancer [2-6]. Understanding how cells traverse BM barriers is thus of fundamental importance in human health. Uterine anchor cell (AC) invasion into the vulval epithelium in C. elegans is a highly stereotyped in vivo model of cell invasion that combines many powerful experimental approaches, including live imaging, subcellular visual analysis of cell-BM interactions, rapid genome editing, and powerful forward genetic and functional genomic approaches (Fig 1) [7, 8]. Using these strengths, this study is aimed at: (1) elucidating how invading cells acquire and use energy to fuel BM invasion, (2) determining how lipid biosynthesis builds a large protrusion that opens paths through BM barriers, (3) establishing how invasive cells adapt invasion to the absence of matrix metalloproteinases (MMPs), and (4) revealing the mechanisms that cause, prevent, and heal plasma membrane damage during BM breaching. These studies are relevant to NIH’s mission as they will lead to a deeper understanding of the fundamental biological process of cell invasive behavior, allowing for better therapeutic strategies to modulate invasion in human disease. Scientific Justification for Requested Supplement Equipment (NOT-GM-22-017): All aims of the parent award R35GM118049-07 require quantitative live image analysis of endogenously fluorophore-tagged proteins. My group spends ~50 hours/week on confocal microscopy addressing the aims of the award, necessitating a dedicated confocal microscope. Our current confocal uses a Yokogawa CSU10 Spinning Disk Confocal Head that is over 15 years old. This system lacks the spatial and temporal resolution to conduct the analysis needed to effectively complete the aims of the proposed work. We are requesting a Yokogawa CSU-W1-T2 Spinning Disk Confocal Head and Hamamatsu qCOMS Quest camera to significantly increase optical resolution and improve quantitative analysis of our live imaging. The pinhole spacing in the CSU-W1-T2 is wider and uses microlenses for collecting more light. Further, the camera is capable of fast imaging frame rates (120 fps) and its 4.6-micron pixel size matches the diffraction-limited resolution of our 100X 1.4 NA Zeiss objective. This new system is more efficient in collecting fluorescent signals and in suppressing out-of-focus fluorescence and thus increases signal-to-noise while minimizing photobleaching. It also substantially increases lateral, axial, and temporal resolution. We had an opportunity to demo the CSU-W1-T2 Spinning Disk Confocal Head on our confocal microscope (Fig 2-4) and found a dramatic improvement in the detection and resolution of our imaging that will be crucial in completing the aims of the work. A key goal of Aim 1 is to characterize and define the mechanisms that build and polarize specialized high-capacity mitochondria in the AC. We discovered that mitochondria polarize to the site of invasion and generate high levels of ATP to fuel invadopodia and invasive protrusion formation (Fig 1) [9, 10]. We have used genome editing to tag over 20 mitochondrial electron transport chain (ETC) components of complexes I, II, III, IV, and V with mNeonGreen (mNG). The ETC generates ATP through oxidative phosphorylation. This is the first endogenous tagging of the ETC and we have confirmed the viability and health of all strains. Although AC mitochondria volume is the same as neighboring non-invasive uterine cells, we found increased fluorescence for many ETC components within the AC mitochondria specifically at the invasive front (Fig 2). Strikingly, our preliminary studies indicate that ETC components are not simply amplified from non invasive mitochondria, but rather a subset of components are enriched at various levels. This suggests that invasive high-capacity mitochondria have a uniquely built ETC. Assessing the precise makeup of specialized mitochondria requires better signal-to-noise imaging, which the Yokogawa CSU-W1-T2 provides (Fig 2). In addition, a key goal of this aim is to understand how high-capacity mitochondria polarize to the invasive front and how they are kept distinct from non-invasive mitochondria. This requires live imaging of mitochondria dynamics (fission and fusion) and mitochondrial network analysis, which requires the high resolution of the Yokogawa CSU W1-T2 (Fig 3). A key goal of Aim 2 is to determine how lipid biosynthesis builds an invasive protrusion that breaches BM. We have identified over 10 lipid biosynthesis enzymes important for invasive protrusion formation and function during AC invasion, including genes critical to metastasis (e.g., SREBP [11]). We have endogenously tagged these lipid synthesis enzymes and are finding many localized to the endoplasmic reticulum (ER) (ELO-1, elongase, Fig 2). We have also discovered that the ER expands dramatically prior to invasion. To characterize the expansion of the ER, subcellular localization of proteins in the ER network, and ER composition, requires the high signal-to-noise and increased resolution of the Yokogawa CSU-W1-T2 (Fig 2 and 3). A key goal of Aim 3 is to understand how invasive cells adapt their invasion program to the absence of MMPs, which may explain why MMP therapies have been unsuccessful in clinical cancer trials. Our preliminary results suggest that mRNA translation is upregulated and remodeled after MMP loss. We have successfully endogenously tagged two ribosomal proteins [12]. With the increased resolution of the CSU-W1-T2, we can now observe that ribosomes localize to the ER specifically prior to invasion, which is not seen in neighboring non-invasive uterine cells (Fig 2). We also discovered that ribosomes are present at higher levels in the AC (Fig 2). To determine how the AC adapts translation in the absence of MMPs requires the high resolution and high signal to-noise of the Yokogawa CSU-W1-T2. A key goal of Aim 4 is to understand how plasma membrane of invasive cells are damaged from breaching BM during invasion. To do so, we are altering the levels of BM components to determine components that damage the AC. We have endogenously tagged over 60 BM components and can manipulate their levels in BM [13, 14]. The Yokogawa CSU-W1-T2 Spinning Disk Confocal Head and Hamamatsu qCOMS Quest camera dramatically increases resolution of BM, and it has allowed us to visualize fibril type IV collagen structures within the BM (Fig 4), which we hypothesize are damaging plasma membrane. We cannot visualize these structures with our current confocal and thus require the Yokogawa CSU-W1- T2 to complete the aim of the parent award. Anticipated future costs and training with the requested equipment: The Yokogawa CSU-W1-T2 Spinning Disk Confocal and Hamamatsu qCOMS Quest camera will replace our existing CSU-10 and ImageEM BT-EM CCD Camera. Both upgrades will be controlled by micromanager software, which the lab has experience using and so no additional training is required. Biovision will continue to service our confocal at no charge (as they have done for 16 years) so there are no upkeep costs.