Understanding how cells invade through basement membrane in vivo

了解体内细胞如何侵入基底膜

• 批准号: 10795365
• 负责人: David R Sherwood
• 金额: $ 23.29万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10795365
• 关键词: 15 year old; Address; Arthritis; Asthma; Award; Basement membrane; Behavior; Biological Process; Caenorhabditis elegans; Cell membrane; Cell model; Cell physiology; Cells; Charge; Collagen Type IV; Complex; Computer software; Confocal Microscopy; Dedications; Detection; Development; Disease; Disseminated Malignant Neoplasm; Electron Transport; Endoplasmic Reticulum; Enzymes; Epithelium; Equipment; Fluorescence; Future; Genes; Genetic; Genomic approach; Goals; Head; Health; Hour; Human; Image; Image Analysis; Immune; Immune System Diseases; Infection; Injury; Invaded; Lateral; Light; Lipids; Malignant Neoplasms; Matrix Metalloproteinases; Microscope; Mission; Mitochondria; Multiple Sclerosis; Neoplasm Metastasis; Noise; Optics; Oxidative Phosphorylation; Parents; Pathway Analysis; Photobleaching; Proteins; Research; Resolution; Ribosomal Proteins; Ribosomes; Services; Signal Transduction; Site; Stereotyping; Stress; Structure; System; Therapeutic; Tissues; Training; Translations; United States National Institutes of Health; Uterus; Visual; Visualization; Work; cancer clinical trial; cell injury; charge coupled device camera; cost; developmental disease; enzyme biosynthesis; experience; fluorophore; functional genomics; genome editing; healing; human disease; improved; in vivo; in vivo Model; lens; lipid biosynthesis; mRNA Translation; migration; novel therapeutic intervention; prevent; programs; temporal measurement; trafficking

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.

父母奖R35GM118049-07的研究策略摘要：了解细胞在发育过程中如何通过体内的基底膜侵袭和免疫细胞运输，专门细胞获得了违反地基膜（BM）基质壁垒的能力，以迁移到感染和受伤的部位[1]。在许多疾病中，在许多疾病，关节炎，关节炎，多发性硬化症和转移性癌症中的组织破坏是不适当的[2-6]。因此，了解细胞如何穿越BM屏障在人类健康中至关重要。秀丽隐杆线虫中的子宫锚细胞（AC）侵袭进入外阴上皮是一种高度定型的细胞浸润模型，结合了许多强大的实验方法，包括实时成像，细胞-BM相互作用的亚细胞视觉分析，快速基因组编辑，强大的前瞻性遗传和功能性基因组方法（图1）[7，8]。使用这些优势，这项研究的目的是：（1）阐明入侵细胞如何获取和利用能量来燃烧BM的入侵，（2）确定脂质生物合成如何构建一种大型蛋白质，该蛋白质通过BM屏障打开路径，（3）确定侵入性细胞如何使其对侵袭性的缺失，从而揭示了Matrix Metalloprix sypers（MMMMMMMMMMESS），并且（4 MMMMMMMES）（4MMMMMMMES）（4）和4（4）（4），并且（4）（4）和4.4mmmmmmmms）（4），并且（4）及其对侵袭性的侵袭（4），并揭示了（4）的侵入性。在BM破裂过程中治愈质膜损伤。这些研究与NIH的使命相关，因为它们将使对细胞侵入性行为的基本生物学过程有更深入的了解，从而使更好的治疗策略可以调节人类疾病的入侵。请求补充设备的科学依据（INT-GM-22-017）：父级奖R35GM118049-07的所有目的都需要对内生荧光团标记的蛋白质进行定量实时图像分析。我的小组每周在共聚焦显微镜上花费约50个小时，以解决该奖项的目标，即必要的共聚焦显微镜。我们目前的共聚焦使用了15年以上的横川CSU10旋转磁盘共聚焦头。该系统缺乏空间和临时解决方案，用于进行有效完成拟议工作目标所需的分析。我们要求横视CSU-W1-T2旋转磁盘共聚焦头和Hamamatsu Qcoms Quest摄像头，以显着提高光学分辨率并改善对我们的实时成像的定量分析。 CSU-W1-T2中的针孔间距更宽，并使用微镜来收集更多的光线。此外，摄像头能够具有快速成像帧速率（120 fps），其4.6微米像素尺寸与我们的100x 1.4 Na Zeiss物镜的衍射限制分辨率相匹配。这个新系统在收集荧光信号和抑制异常荧光的情况下更有效，从而增加信号到噪声，同时最大程度地减少光漂白。它还大大增加了横向，轴向和临时分辨率。我们有机会在共聚焦显微镜上演示CSU-W1-T2旋转磁盘共聚焦头（图2-4），并发现对成像的检测和分辨率有了显着改善，这对于完成工作的目标至关重要。目标1的一个关键目标是表征和定义AC中构建和极化高容量线粒体的机制。我们发现线粒体偏振到入侵部位，并产生高水平的ATP来燃烧侵袭性和浸润性蛋白质的形成（图1）[9，10]。我们已经使用基因组编辑来将复合物I，II，III，IV和V与Mneongreen（MNG）的复合物I，II，III，IV和V的20个线粒体电子传输链（ETC）进行标记。 ETC通过氧化磷酸化产生ATP。这是ETC的第一个内源标记，我们已经确认了所有菌株的生存能力和健康。尽管AC线粒体体积与邻近的非侵入性子宫细胞相同，但我们发现AC线粒体内许多ETC成分的荧光增加了，特别是在侵入性方面（图2）。令人惊讶的是，我们的初步研究表明，ETC成分不仅是从非浸润性线粒体中放大的，而且要在各个层面上富集了一部分组件。这表明侵入性的高容量线粒体具有独特的建筑。评估专业线粒体的精确构成需要更好的信噪成像，而横川CSU-W1-T2提供了这一点（图2）。此外，此目标的一个关键目标是了解高容量的线粒体如何偏振与侵入性阵线以及如何保持与非侵入性线粒体不同。这需要对线粒体动力学（裂变和融合）和线粒体网络分析进行实时成像，这需要横川CSU W1-T2的高分辨率（图3）。目标2的一个关键目标是确定脂质生物合成如何产生破坏BM的侵入性突出。我们已经确定了10多种脂质生物合成酶对AC侵袭过程中侵入性蛋白质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]）。我们已经对这些脂质合成酶进行了内源性标记，并且发现许多局部位于内质网（ER）（ELO-1，延伸酶，图2）。我们还发现，急诊室在入侵之前大大扩展。为了表征ER的膨胀，蛋白质在ER网络中的亚细胞定位以及ER组成需要高的信噪比，并增加了横川CSU-W1-T2的分辨率（图2和3）。目标3的一个关键目标是了解侵入性细胞如何使其入侵计划适应MMP的缺乏，这可以解释为什么MMP疗法在临床癌症试验中没有成功。我们的初步结果表明，MMP损失后更新和重塑mRNA翻译。我们已经成功地标记了两个核糖体蛋白[12]。随着CSU-W1-T2的分辨率增加，我们现在可以观察到核糖体在侵袭之前特有在ER上定位，这在邻近的非侵入性子宫细胞中未见（图2）。我们还发现核糖体在AC中存在较高的水平（图2）。为了确定在不存在MMP的情况下AC如何适应翻译，需要高分辨率和高信号对横川CSU-W1-T2。目标4的关键目标是了解在入侵期间破坏BM的侵入性细胞的质膜如何受损。为此，我们正在改变BM组件的水平，以确定损坏AC的组件。我们的内源性标签超过60 bm，可以在BM中操纵其水平[13，14]。横川CS​​U-W1-T2旋转磁盘共聚焦头和Hamamatsu Qcoms Quest摄像机大大提高了BM的分辨率，它使我们能够可视化BM中的IV型IV胶原蛋白型结构（图4），我们假设这是损害了质膜。我们无法用当前的共焦点可视化这些结构，因此要求横正在横视CSU-W1-T2完成父母奖的目标。预期的未来成本和培训使用请求的设备：横视CSU-W1-T2旋转磁盘共焦和Hamamatsu Qcoms Quest摄像头将取代我们现有的CSU-10和Imageem BT-EM CCD摄像头。这两个升级都将由Micromanager软件控制，该软件具有经验，因此不需要额外的培训。 Biovision将继续免费为我们的公共焦点提供服务（就像16年的时间一样），因此没有维护成本。

项目成果

Tissue linkage through adjoining basement membranes: The long and the short term of it.

• DOI: 10.1016/j.matbio.2018.05.009
• 发表时间: 2019-01
• 期刊: Matrix biology : journal of the International Society for Matrix Biology
• 作者: Keeley DP;Sherwood DR
• 通讯作者: Sherwood DR

Endogenous expression of UNC-59/Septin in C.elegans.

• DOI: 10.17912/micropub.biology.000200
• 发表时间: 2019-12-20
• 期刊: microPublication biology
• 作者: Chen, David;Hastie, Eric;Sherwood, David
• 通讯作者: Sherwood, David

Basement membrane remodeling guides cell migration and cell morphogenesis during development.

• DOI: 10.1016/j.ceb.2021.04.003
• 发表时间: 2021-10
• 期刊: Current opinion in cell biology
• 影响因子: 7.5
• 作者: Sherwood DR

Fueling Cell Invasion through Extracellular Matrix.

• DOI: 10.1016/j.tcb.2021.01.006
• 发表时间: 2021-06
• 期刊: Trends in cell biology
• 影响因子: 19
• 作者: Garde A;Sherwood DR
• 通讯作者: Sherwood DR

Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling.

边界细胞限制肌营养不良聚糖的运输，以控制组织重塑过程中基底膜的滑动。

• DOI: 10.7554/elife.17218
• 发表时间: 2016
• 期刊: eLife
• 影响因子: 7.7
• 作者: McClatchey,ShellyTh;Wang,Zheng;Linden,LaraM;Hastie,EricL;Wang,Lin;Shen,Wanqing;Chen,Alan;Chi,Qiuyi;Sherwood,DavidR
• 通讯作者: Sherwood,DavidR