Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy
Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy
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DOI：
10.1002/jlb.3hi0120-589r
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发表时间：
2020-04-23
影响因子：
5.5
通讯作者：
Lieschke, Graham J.
中科院分区：
文献类型：
--
作者：
Manley, Harriet R.;Potter, David L.;Lieschke, Graham J.
中性粒细胞和巨噬细胞（M phi）迁移是炎症反应的基础。然而，吞噬细胞的快速运动、多向瞬时运动和可塑性、不断变化的形状混淆了高分辨率活体成像。点阵光片显微镜（LLSM）捕捉高度动态的细胞形态在特殊的时空分辨率。我们证明了LLSM在体内白细胞的第一次广泛应用，利用光学透明的斑马鱼，白细胞特异性报告细胞系，突出亚细胞结构，和白细胞迁移的创伤试验。LLSM揭示了迁移的白细胞形态的细节，并允许复杂的，体积的询问高度动态的活动在其原生的生理环境。非常薄，经常性的尾足延伸现在必须被认为是中性粒细胞迁移的特征。LLSM解决尾随尾足延伸，证明其令人惊讶的长度，并允许定量评估细胞骨架的贡献，其渐逝的形式。在LLSM的时空分辨率下对血管微环境中的白细胞进行成像，显示了血流诱导的中性粒细胞动力学，并显示了意想不到的白细胞-内皮相互作用，如白细胞诱导的内皮变形对抗血管内压力。吞噬作用和细胞死亡的LLSM提供了亚细胞的见解，并揭示了新的行为。总的来说，我们提供了高分辨率LLSM白细胞结构（丝状伪足板状伪足，尾足延伸，囊泡）和活动（间质和血管内迁移，白细胞滚动，吞噬作用，细胞死亡和细胞质气球样变）的例子。LLSM应用于活体白细胞成像为吞噬细胞生物学的细胞和亚细胞复杂性的变革性研究奠定了基础。
Neutrophil and macrophage (M phi) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.