ObjectiveTo explore the effects of cytoskeleton depolymerizing agent and stabilizer on the clathrin/caveolae-mediated endocytosis, the expression of membrane vascular endothelial cadherin (VE-cad), and the vascular permeability by the transformation of cytoskeleton structure after lipopolysaccharide (LPS) treatment. MethodsCRL-2922 cells were used in the experiments. Indexes were tested at corresponding time point according to name of group, but in blank control group indexes could be tested at any time point. CRL-2922 cells were divided into blank control group, LPS-1 h group, and LPS-4 h group to observe cytoskeleton structure; CRL-2922 cells were divided into LPS-1 h group, Cyt D+LPS-1 h group, LPS-4 h group, and Jasp+LPS-4 h group to determine the expression of membrane VE-cad, and to determine the expression of its co-immunoprecipitation with clathrin and caveolin-1 (Cav1); besides, CRL-2922 cells were divided into blank control group, LPS-1 h group, Cyt D+LPS-1 h group, LPS-4 h group, and Jasp+LPS-4 h group to detect the cumulative infiltration rate. Results①The cytoskeleton showed a dynamic change after LPS treat-ment, the F-actin polymerized and stress fibers formed at 1 hour after LPS treatment, but depolymerized at 4 hours after LPS treatment. ②Compared with LPS-1 h group, the level of co-immunoprecipitation of VE-cad with clathrin in Cyt D+ LPS-1 h group decreased (P<0.05), the level of co-immunoprecipitation of VE-cad with Cav1 increased (P<0.05), and expression level of VE-cad in plasma membrane decreased (P<0.05); compared with LPS-4 h group, there was no significant difference in the level of co-immunoprecipitation of VE-cad with clathrin of Jasp+LPS-4 h group (P>0.05), but the level of co-immunoprecipitation of VE-cad with Cav1 decreased in Jasp+LPS-4 h group (P<0.05), and expression level of VE-cad in plasma membrane increased (P<0.05). ③Compared with blank control group, the cumulative infiltration rates of LPS-1 h group and LPS-4 h group were both higher (P<0.05); compared with LPS-1 h group, the cumulative infiltration rate of Cyt D+LPS-1 h group was higher (P<0.05); compared with LPS-4 h group, the cumulative infiltration rate of Jasp+LPS-4 h group was lower (P<0.05). ConclusionActin cytoskeleton shifts from polymerization to depoly-merization after LPS treatment, the structural change of actin cytoskeleton is an important reason for the transformation of VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated after LPS treatment.
Cell autophagy plays a key role in maintaining intracellular nutritional homeostasis during starvation through elimination of aberrant or obsolete cellular structures. The cellular cytoskeleton has a crucial role in multiple processes involving membrane rearrangements and vesicle-mediated events. Autophagy is mediated by both microtubules and actin networks: microtubules promote the synthesis of autophagosome and are related to the movement of autophagosome; actin networks have been implicated in structurally supporting the expanding of phagophore, moving autophagosomes and enabling their efficient fusion with the lysosome; non-muscle myosinⅡoperates in the early stages of autophagy during the initiation and expansion of the phagophore, whereas myosinⅥ and myosin 1C are involved in the late stages of autophagosome maturation and fusion with the lysosome, respectively. This review summarizes the multiple regulation of cytoskeleton on autophagy and focuses on the regulation of autophagy by actin and myosin, providing a new approach for the study of pathogenesis and innovative therapies of autophagy related diseases.
Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.
Mechanical stress modulates almost all functions of cells. The key to exploring its biological effects lies in studying the perception of mechanical stress and its mechanism of mechanotransduction. This article details the perception and mechanotransduction mechanism of mechanical stress by extracellular matrix, cell membrane, cytoskeleton and nucleus. There are two main pathways for the perception and mechanotransduction of mechanical stress by cells, one is the direct transmission of force, and the other is the conversion of mechanical signal into chemical signal. The purpose of this study is to provide some reference for the exploration of precise treatment of mechanical stress-related diseases and the optimization of construction of tissue engineered organs by mechanical stress.