Understanding how cells invade through basement membrane in vivo

Duke University, Durham NC

Investigators

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Abstract

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.