Cofilin’s activity to nucleate actin filament assembly, is regulated by phosphorylation at a single site on the amino terminus, Serine 3. Phosphorylation at this site abolishes the ability of ADF/cofilin to bind to F-actin and inhibits its severing function. This work characterizes the ability of dephosphorylated cofilin (mutation at Serine 3 site) to navigate prostate cancer actin cytoskeleton and metastatic properties in response to TGF-β. TGF-β increased Lim Domain Kinase 2 (LIMK-2) activity leading to cofilin phosphorylation and decrease actin filament severing in wild type cofilin (WTCFL) PC-3 cells. Constitutively active cofilin in Serine 3 cofilin mutants (S3ACFL) promoted prostate cancer cell filopodia formation, actin severing and directed TGF-β mediated migration and invasion. Co-culture of prostate cancer cells with prostate cancer associated fibroblasts induced cell invasion in WTCFL and S3ACFL cells. Active cofilin further enhanced the invasive response, even in the presence of a TGF-β-neutralizing antibody, implicating the contribution of the microenvironment. Active cofilin led to a significant increase in prostate cancer cell metastatic potential in vivo and cofilin correlated with metastasis in a mouse model of prostate tumor progression. In human prostate cancer, cofilin expression was significantly higher in metastasis compared to the primary tumors. Cofilin thus emerges as a regulator of the actin cytoskeleton remodeling capable of coordinating the cellular responses to TGF- β towards prostate cancer metastasis. Understanding how cancer cells interprete TGF-β signals from the microenvironment, is critical for defining the mechanism via which TGF- β function is switched from a growth suppressor to a metastasis promoter. Here we show that in prostate cancer, TGF-β action is directed by active cofilin enabling actin cytoskeleton changes and metastatic behavior. The significant association of cofilin with prostate cancer metastatic progression supports its predictive and targeting value in metastasis.

A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.

A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.

Prions are infectious protein entities capable of self-replication. Prions are the causal agents behind the transmissible spongiform encephalopathies causing neurodegeneration and death in affected organisms. Prions have been identified in yeast with the best-characterized prions being [PSI+] and [PIN+], whose respective native proteins are the Sup35 translation termination factor and Rnq1 (function unknown). Autophagy is a cellular housekeeping mechanism mediating the degradation of damaged proteins and superfluous organelles. It is a highly sequential process regulated by autophagy related genes (ATGs). Autophagy has also been implicated in the clearance of amyloidogenic proteins including prions. However, the mechanistic basis underlying this activity is poorly understood, and a key objective of this project was to characterize how autophagy prevents spontaneous prion formation. Our study found that the deletion of core ATGs correlated with an increase in de novo [PSI+] and [PIN+] formation as well as Sup35 aggregation. Enhancement of autophagic flux through spermidine treatment attenuated the increased levels of de novo [PSI+] formation in mutants that normally show elevated levels of [PSI+] formation. Defective autophagy correlated with increased oxidatively damaged Sup35 in an atg1 mutant whereas anaerobic growth abrogated the increased [PSI+] formation in the atg1 mutant to wild-type levels. Our data suggest that autophagy serves a protective role in the clearance of oxidatively damaged Sup35 proteins that otherwise has a higher propensity towards [PSI+] prion formation. We also investigated the role of prion formation and autophagy during yeast chronological ageing which is the time that non-dividing cells remain viable. Prion diseases are associated with advanced age which correlates with a decline in cellular protective mechanisms including autophagy. Our study found an age dependent increase in the frequency of de novo [PSI+] formation with chronological age of yeast cells, more so in an atg1 mutant relative to the wild-type. Autophagy competent cells carrying the [PSI+] and [PIN+] prions also had improved chronological lifespan relative to prion free cells and atg1 cells. Cells carrying the [PSI+] prion elicited elevated autophagic flux that may promote improved lifespan thus suggesting a beneficial role of the [PSI+] prion during chronological ageing. The actin cytoskeleton provides the structural framework essential for a multitude of cellular processes to occur. We investigated the role of the Arp2/3 complex responsible for branching of actin filaments towards prion formation. Knockout mutants of the nucleation promoting factors of the Arp2/3 complex, in particular the abp1 mutant, showed reduced de novo [PSI+] formation and Sup35 aggregation under basal and oxidative stress conditions. Similarly, treatment with latrunclin A, an actin monomer-sequestering drug also abrogated de novo [PSI+] formation. Colocalization studies revealed that Sup35 often does not colocalize with Rnq1, a marker for the insoluble protein deposit (IPOD) in an abp1 mutant. This suggests a role for the Abp1 protein in the efficient transport of Sup35 molecules to the IPOD that may facilitate de novo [PSI+] prion formation under vegetative states and oxidant challenges.

Cell migration is required for a number of physiological processes and is implicated in pathologies such as tumor metastasis. Cell motility is dependent upon dynamic actin reorganization, and is regulated by the Rho family of small GTPases. Rho GTPases are molecular switches that cycle between their active and inactive conformations. The best-studied members of this family are Rac1, RhoA, and Cdc42. Each is responsible for the formation of specific actin structures. Diacylglycerol kinases (DGKs) act at the membrane to convert diacylglycerol (DAG) and phosphatidic acid (PA), maintaining the balance of these two lipid second messengers. Previous studies from our lab have demonstrated that the ζ isoform of DGK facilitates the release of Rac1 and RhoA from their inhibitor, RhoGDI. Here we studied a closely related isoform, DGKι, using mouse embryonic fibroblasts (MEFS) in which the gene for DGKι had been deleted. Aberrations in cell morphology, spreading, and migration were identified in DGKι-null MEFs. We showed that the activity of Rac1 and RhoC, but not RhoA, was impaired in the absence of DGKι, yet only RhoC protein levels were affected. Reduced activation of these Rho GTPases was accompanied by defects in Rac1- and RhoC- related actin structures. These data demonstrate that DGKι, in addition to DGKζ, contributes to the regulation of GTPase activation and remodeling of the actin cytoskeleton.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23551号 / 生博第462号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 中野 雄司, 教授 見学 美根子, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

Yes / Cells migrating over 2D substrates are required to polymerise actin at the leading edge to form lamellipodia protrusions and nascent adhesions to anchor the protrusion to the substrate. The major actin nucleator in lamellipodia formation is the Arp2/3 complex, which is activated by the WAVE regulatory complex (WRC). Using inducible Nckap1 floxed mouse embryonic fibroblasts (MEFs), we confirm that the WRC is required for lamellipodia formation, and importantly, for generating the retrograde flow of actin from the leading cell edge. The loss of NCKAP1 also affects cell spreading and focal adhesion dynamics. In the absence of lamellipodium, cells can become elongated and move with a single thin pseudopod, which appears devoid of N-WASP. This phenotype was more prevalent on collagen than fibronectin, where we observed an increase in migratory speed. Thus, 2D cell migration on collagen is less dependent on branched actin.

This research examines the role of actin cytoskeleton filaments in chondroinduction by cyclic hydrostatic pressurization. A chondroinductive hydrostatic pressurization system was developed and characterized. A pressure of 5 MPa at 1 Hz frequency, applied for 7200 cycles (4 hours intermittent) per day, induced chondrogenic differentiation in C3H10T1/2 cells while 1800 cycles (1 hour intermittent) did not induce chondrogenesis. Quantitative analysis of chondrogenesis was determined as sulfated glycosaminoglycan synthesis and rate of collagen synthesis while qualitative analysis was obtained as Alcian Blue staining and collagen type II immunostaining. Actin disruption using 2 uM Cytochalasin D inhibited the enhanced sGAG synthesis in the chondroinductive hydrostatic pressurization environment and significantly inhibited rate of collagen synthesis to the mean level lower than that of the non-pressurized group. These results suggest an involvement of actin cytoskeleton filaments in mechanotransduction of cyclic hydrostatic pressure.

No description available.