Lee Hyunsu, Bonin Keith, Guthold Martin
Department of Physics, Wake Forest University, Winston-Salem, NC 27109, USA.
Department of Physics, Wake Forest University, Winston-Salem, NC 27109, USA.
Biochim Biophys Acta Gen Subj. 2021 Jun;1865(6):129891. doi: 10.1016/j.bbagen.2021.129891. Epub 2021 Mar 6.
The epithelium forms a protective barrier against external biological, chemical and physical insults. So far, AFM-based, micro-mechanical measurements have only been performed on single cells and confluent cells, but not yet on cells in mature layers.
Using a combination of atomic force, fluorescence and confocal microscopy, we determined the changes in stiffness, morphology and actin distribution of human mammary epithelial cells (HMECs) as they transition from single cells to confluency to a mature layer.
Single HMECs have a tall, round (planoconvex) morphology, have actin stress fibers at the base, have diffuse cortical actin, and have a stiffness of 1 kPa. Confluent HMECs start to become flatter, basal actin stress fibers start to disappear, and actin accumulates laterally where cells abut. Overall stiffness is still 1 kPa with two-fold higher stiffness in the abutting regions. As HMECs mature and form multilayered structures, cells on apical surfaces become flatter (apically more level), wider, and seven times stiffer (mean, 7 kPa) than single and confluent cells. The main drivers of these changes are actin filaments, as cells show strong actin accumulation in the regions where cells adjoin, and in the apical regions.
HMECs stiffen, flatten and redistribute actin upon transiting from single cells to mature, confluent layers.
Our findings advance the understanding of breast ductal morphogenesis and mechanical homeostasis.
上皮细胞形成了一道抵御外部生物、化学和物理损伤的保护屏障。到目前为止，基于原子力显微镜的微机械测量仅在单细胞和汇合细胞上进行过，尚未在成熟层的细胞上进行。
我们结合原子力显微镜、荧光显微镜和共聚焦显微镜，测定了人乳腺上皮细胞（HMECs）从单细胞转变为汇合细胞再到成熟层过程中其硬度、形态和肌动蛋白分布的变化。
单个HMECs呈高而圆（平凸透镜状）的形态，基部有肌动蛋白应力纤维，皮质肌动蛋白呈弥漫状，硬度为1kPa。汇合的HMECs开始变得更扁平，基部的肌动蛋白应力纤维开始消失，肌动蛋白在细胞邻接处侧向积累。整体硬度仍为1kPa，但邻接区域的硬度高出两倍。随着HMECs成熟并形成多层结构，顶端表面的细胞变得更扁平（顶端更平整）、更宽，且比单细胞和汇合细胞硬七倍（平均为7kPa）。这些变化的主要驱动因素是肌动蛋白丝，因为细胞在细胞邻接区域和顶端区域显示出强烈的肌动蛋白积累。
HMECs从单细胞转变为成熟的汇合层时会变硬、变扁平并重新分布肌动蛋白。
我们的研究结果推进了对乳腺导管形态发生和机械稳态的理解。
