Mechanical properties and cytoskeletal protein changes after
ANLN deficiency

doi:10.3969/j.issn.2095-4344.1875

Abstract

Abstract:

BACKGROUND: To date, ANLN has definite roles in altering cell shape, regulating cell-cell junction integrity in interphase and stabilizing actomyosin contractile rings in cytokinesis, but its effects on cell mechanical properties and on cytoskeletal proteins have rarely been reported.

OBJECTIVE: To investigate the effect of ANLN deletion on the mechanical properties and cytoskeleton of interphase Hela cells.

METHODS: Surface elastic modulus and membrane rupture force of normal untreated Hela cells and ANLN RNA stably knocked down Hela cells were measured by atomic force microscopy. We screened for the cells that stably expressed mCherry-Myosin II A, and observed the distribution characteristics of cytoskeletal proteins by laser scanning confocal microscopy.

RESULTS AND CONCLUSION: (1) The elastic modulus of Hela cells with ANLN stably knocked down was significantly higher than that of normal Hela cells, and the elastic modulus of normal cells were more prone to polar distribution (gradually decreasing between the two poles) than that of ANLN knockdown Hela cells. However, there was no significant difference in the membrane rupture force at the long-axis edge region between the two groups. (2) Myosin IIA lowly expressed in the marginal region of ANLN knockdown cells. (3) The actin fibers tended to be scattered in the near-bottom cell-cell junction region of the ANLN knockdown group, and there were no obvious intracellular fibers bundles arranging along the long axis. The cell gap tended to enlarge in the middle layer. To conclude, ANLN knockdown cells have the greatest impact in the marginal region, the deficiency of ANLN leads to a more frequent remodeling in the cell marginal region, and the cells need to accumulate more cytoskeletal proteins and binding proteins to stabilize the cell state, resulting in higher modulus of elastics.

Key words: ANLN protein, cell spreading, atomic force microscopy, force curve, surface elastic modulus, membrane rupture force, cytoskeleton, actin fiber, National Natural Science Foundation of China

CLC Number:

Xu Limeng, Luo Wenyu, Zhang Shuwei, Wang Li. Mechanical properties and cytoskeletal protein changes after ANLN deficiency[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(1): 87-92.

Figures/Tables 4

References

[1] LIU J, FAIRN GD, CECCARELLI DF, et al. Cleavage furrow organization requires PIP(2)-mediated recruitment of anillin. Curr Biol. 2012;22(1):64-69.
[2] FIELD CM, ALBERTS BM. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J Cell Biol. 1995;131(1):165-178.
[3] STRAIGHT AF, FIELD CM, MITCHISON TJ. Anillin binds nonmuscle myosin II and regulates the contractile ring. Mol Biol Cell. 2005;16(1):193-201.
[4] OEGEMA K, SAVOIAN MS, MITCHISON TJ,et al. Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis. J Cell Biol. 2000;150(3):539-552.
[5] D'AVINO PP, TAKEDA T, CAPALBO L, et al. Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site. J Cell Sci. 2008;121(Pt 8):1151-1158.
[6] GREGORY SL, EBRAHIMI S, MILVERTON J, et al. Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring. Curr Biol. 2008; 18(1):25-29.
[7] PIEKNY AJ, GLOTZER M. Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis. Curr Biol. 2008;18(1):30-36.
[8] TRACHE A, MEININGER GA. Atomic force microscopy (AFM). Curr Protoc Microbiol. 2008;Chapter 2:Unit 2C.2.
[9] REYES CC, JIN M, BREZNAU EB, et al. Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin. Curr Biol. 2014; 24(11):1263-1270.
[10] WANG D, CHADHA GK, FEYGIN A, et al. F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells. Cell Mol Life Sci. 2015;72(16): 3185-3200.
[11] FRENETTE P, HAINES E, LOLOYAN M, et al. An anillin-Ect2 complex stabilizes central spindle microtubules at the cortex during cytokinesis. PLoS One. 2012;7(4):e34888.
[12] MCKENZIE AJ, HICKS SR, SVEC KV, et al. The mechanical microenvironment regulates ovarian cancer cell morphology, migration, and spheroid disaggregation. Sci Rep. 2018;8(1): 7228.
[13] SCHIFFHAUER ES, ROBINSON DN. Mechanochemical Signaling Directs Cell-Shape Change. Biophys J. 2017;112(2): 207-214.
[14] PILLET F, CHOPINET L, FORMOSA C, et al. Atomic Force Microscopy and pharmacology: from microbiology to cancerology. Biochim Biophys Acta. 2014;1840(3): 1028-1050.
[15] JINSCHEK JR, HELVEG S. Image resolution and sensitivity in an environmental transmission electron microscope. Micron. 2012;43(11):1156-1168.
[16] GUO Q, XIA Y, SANDIG M, et al. Characterization of cell elasticity correlated with cell morphology by atomic force microscope. J Biomech. 2012;45(2):304-309.
[17] LEKKA M, GIL D, POGODA K, et al. Cancer cell detection in tissue sections using AFM. Arch Biochem Biophys. 2012; 518(2):151-156.
[18] ZEMŁA J, DANILKIEWICZ J, ORZECHOWSKA B, et al. Atomic force microscopy as a tool for assessing the cellular elasticity and adhesiveness to identify cancer cells and tissues. Semin Cell Dev Biol. 2018;73:115-124.
[19] NI Y, QIN Y, FANG Z, et al. ROCK Inhibitor Y-27632 Promotes Human Retinal Pigment Epithelium Survival by Altering Cellular Biomechanical Properties. Curr Mol Med. 2017;17(9): 637-646.
[20] CASCIONE M, DE MATTEIS V, RINALDI R, et al. Atomic force microscopy combined with optical microscopy for cells investigation. Microsc Res Tech. 2017;80(1):109-123.
[21] SMITH JR, TSIBOUKLIS J, NEVELL TG, et al. AFM friction and adhesion mapping of the substructures of human hair cuticles. Applied Surface Science. 2013; 285(Part B): 638-644.
[22] MILLER KG, ALBERTS BM. F-actin affinity chromatography: technique for isolating previously unidentified actin-binding proteins. Proc Natl Acad Sci U S A.1989;86(13):4808-4812.
[23] CAPPELLA B , DIETLER G. Force-distance curves by atomic force microscopy. Surf Sci Rep.1999;34(1-3):1-3,5-104.
[24] SIRGHI L, BRETAGNOL F, MORNET S, et al. Atomic force microscopy characterization of the chemical contrast of nanoscale patterns fabricated by electron beam lithography on polyethylene glycol oxide thin films. Ultramicroscopy. 2009;109(3):222-229.
[25] LI QS, LEE GY, ONG CN, et al. AFM indentation study of breast cancer cells. Biochem Biophys Res Commun.2008; 374(4):609-613.
[26] CROSS SE, JIN YS, RAO J, et al. Nanomechanical analysis of cells from cancer patients. Nat Nanotechnol.2007;2(12): 780-783.
[27] ZHOU W, WANG Z, SHEN N, et al. Knockdown of ANLN by lentivirus inhibits cell growth and migration in human breast cancer. Mol Cell Biochem.2015;398(1-2):11-19.
[28] FIELD CM, COUGHLIN M, DOBERSTEIN S, et al. Characterization of anillin mutants reveals essential roles in septin localization and plasma membrane integrity. Development. 2005;132(12):2849-2860.
[29] ATILLA-GOKCUMEN GE, MURO E, RELAT-GOBERNA J, et al. Dividing cells regulate their lipid composition and localization. Cell. 2014;156(3):428-439.
[30] YOKOKAWA M, TAKEYASU K, YOSHIMURA SH. Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope. J Microsc. 2008; 232(1):82-90.
[31] 李海涛,梁婷,邵毅杰,等.针刺与异常机械应力对大鼠椎间盘纤维环纳米级生物力学特性影响的对比[J].中国组织工程研究,2018, 22(34):5510-5517.
[32] TOMAR S, PLOTNIK JP, HALEY J, et al. ETS1 induction by the microenvironment promotes ovarian cancer metastasis through focal adhesion kinase. Cancer Lett. 2018;414: 190-204.