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Direct observation of ?-actinin tension and recruitment at focal adhesions during contact growth.

ABSTRACT: Adherent cells interact with extracellular matrix via cell-substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive ?-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in ?-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in ?-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of ?-actinin and paxillin to the adhesion sites. Changes in ?-actinin tension and correlated relocation of ?-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, ?-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in ?-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in ?-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that ?-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion?s growth.

SUBMITTER: Ye N

PROVIDER: S-EPMC4153383 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth.

Ye Nannan N Verma Deepika D Meng Fanjie F Davidson Michael W MW Suffoletto Kevin K Hua Susan Z SZ

Experimental cell research 20140801 1

Adherent cells interact with extracellular matrix via cell-substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we ...[more]

PMID: 25088253

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Project description:Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F2α (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes contain miRNA that are targeting the focal adhesion (FA) pathway. The hypothesis of this study is embryo contact with endometrium causes a change in abundance of focal adhesion molecules (FA) in the endometrium leading to decrease in PGF secretion. Mares (n = 3/day) were utilized in a cross-over design with each mare serving as a pregnant and non-pregnant (non-mated) control on days 9 and 11 PO. Mares were randomly assigned to collection day and endometrial samples and embryos were collected on the specified day. Biopsy samples were divided into five pieces, four for culture for 24 hours and one immediately snap frozen. Endometrial biopsies for culture were placed in an incubator with one of four treatments: [1] an embryo in contact on the luminal side of the endometrium, [2] beads in contact on the luminal side of the endometrium, [3] peanut oil in contact on the luminal side of the endometrium or [4] the endometrium by itself. Biopsies and culture medium were frozen for further analysis. RNA and protein were isolated from biopsies for PCR and Western blot analysis for FA. PGF assays were performed on culture medium to determine concentration of PGF. Statistics were performed using SAS (P ≤ 0.05 indicated significance). The presence of beads on day 9 impacted samples from pregnant mares more than non-pregnant mares and had very little impact on day 11. Presence of oil decreased FA in samples from pregnant mares on day 9. On day 11, oil decreased FA abundance in samples from non-pregnant mares. Embryo contact caused multiple changes in RNA and protein abundance in endometrium from both pregnant and non-pregnant mares. The PGF secretion after 24 hours with each treatment was also determined. On day 9, there was no change in PGF secretion compared to any treatments. On day 11, presence of peanut oil increased PGF secretion in samples from non-pregnant mares. In samples from non-pregnant mares, presence of an embryo decreased PGF secretion compared to control samples from non-pregnant mares. Results revealed that while beads and peanut oil may impact abundance of FA RNA and protein in endometrial samples, it does not appear to impact PGF secretion. Conversely, embryo contact for 24 hours with endometrium from a non-pregnant mare causes a decrease in PGF secretion. These results suggest that it is not just contact of any substance/object causing attenuation of PGF secretion, but the embryo itself is necessary to decrease PGF secretion.

Dataset Information

Direct observation of ?-actinin tension and recruitment at focal adhesions during contact growth.

Publications

Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth.

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