Cell Blebbing in Confined Microfluidic Environments

Migrating cells can extend their leading edge by forming myosin-driven blebs and F-actin-driven pseudopods. When coerced to migrate in resistive environments, Dictyostelium cells switch from using predominately pseudopods to blebs. Bleb formation has been shown to be chemotactic and can be influenced by the direction of the chemotactic gradient. In this study, we determine the blebbing responses of developed cells of Dictyostelium discoideum to cAMP gradients of varying steepness produced in microfluidic channels with different confining heights, ranging between 1.7 μm and 3.8 μm. We show that microfluidic confinement height, gradient steepness, buffer osmolarity and Myosin II activity are important factors in determining whether cells migrate with blebs or with pseudopods. Dictyostelium cells were observed migrating within the confines of microfluidic gradient channels. When the cAMP gradient steepness is increased from 0.7 nM/μm to 20 nM/μm, cells switch from moving with a mixture of blebs and pseudopods to moving only using blebs when chemotaxing in channels with confinement heights less than 2.4 μm. Furthermore, the size of the blebs increases with gradient steepness and correlates with increases in myosin-II localization at the cell cortex. Reduction of intracellular pressure by high osmolarity buffer or inhibition of myosin-II by blebbistatin leads to a decrease in bleb formation and bleb size. Together, our data reveal that the protrusion type formed by migrating cells can be influenced by the channel height and the steepness of the cAMP gradient, and suggests that a combination of confinement-induced myosin-II localization and cAMP-regulated cortical contraction leads to increased intracellular fluid pressure and bleb formation.

迁移中的细胞可通过形成肌球蛋白（myosin）驱动的膜泡（bleb）与纤维状肌动蛋白（F-actin）驱动的伪足（pseudopod）来延伸其迁移前沿。当被迫在阻力环境中迁移时，盘基网柄菌（Dictyostelium discoideum）细胞会从主要使用伪足转向使用膜泡。已有研究表明，膜泡形成具有趋化性，且可受趋化梯度方向的影响。
本研究针对盘基网柄菌（Dictyostelium discoideum）的发育态细胞，探究其在受限高度介于1.7 μm至3.8 μm的微流控通道中产生的不同陡度的环磷酸腺苷（cAMP）梯度下的膜泡响应。研究发现，微流控通道受限高度、梯度陡度、缓冲液渗透压以及肌球蛋白II（Myosin II）活性，是决定细胞以膜泡还是伪足进行迁移的关键影响因素。
实验中观察到盘基网柄菌细胞在微流控梯度通道的受限空间内迁移。当环磷酸腺苷梯度陡度从0.7 nM/μm提升至20 nM/μm时，在受限高度低于2.4 μm的通道中进行趋化迁移的细胞，会从混合使用膜泡与伪足的移动模式，转变为仅使用膜泡的移动模式。此外，膜泡尺寸随梯度陡度增加而增大，且与细胞皮层处的肌球蛋白II定位增强呈正相关。通过高渗透压缓冲液降低细胞内压，或通过blebbistatin抑制肌球蛋白II活性，均可减少膜泡形成并缩小膜泡尺寸。
综上，本研究数据表明，迁移细胞形成的突起类型可受通道高度与环磷酸腺苷梯度陡度的调控，且提示：受限诱导的肌球蛋白II定位与环磷酸腺苷调控的皮层收缩共同作用，会提升细胞内流体压力并促进膜泡形成。