Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO) cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology.
In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.The signal transduction of the epidermal growth factor receptor (EGFR) 2 is important for normal cell physiology (1, 2). During the search for novel EGFR substrates, Eps8 (EGFR pathway substrate number 8) as suggested by its designation was originally identified as a putative EGFR target devoid of phosphotyrosine-binding SH2 domain (3). Among its 97-and 68-kDa isoforms, only the former is well characterized and thus referred to as Eps8. Later, Eps8 also is the substrate for Src tyrosine kinase (4). In addition to tyrosyl phosphorylation, expression of Eps8 is also affected by Src activity (4, 5). Remarkably, its aberrant overexpression in murine fibroblasts can lead to cellular transformation (6), and of note, Eps8 overexpression contributes to Src-mediated transformation (7).As an adaptor protein, Eps8 contains several structural features such as a split pleckstrin homology, a putative nuclear targeting sequence, a central SH3 domain, and several prolinerich regions. Although the split pleckstrin homology confers the ability of Eps8 to associate with plasma membrane in response to serum stimulation and conveys signals to ERK activation (6), Eps8 can also complex with Abi-1/E3b1 and RN-tre separately through its SH3 domain (8, 9). By interacting with Abi-1 or RN-tre, Eps8 integrates signals leading to actin cytoskeleton via Rac and receptor endocytosis via Rab5, respectively (10, 11). Recently, IRSp53 has been demonstrated as an Eps8-binding protein whose complex with Eps8 reinforces Rac activation and cell migration in fibrosarcoma cells (12). Besides, Eps8 is also identified as an actin capper, which is capable of regulating actin-based motility (13).Focal adhesion kinase (FAK) is an intracellular tyrosine kinase localized prominently within focal adhesion (14, 15) and participates in a variety of integrin-elicited biological ac...
The oncoprotein Eps8 facilitates proliferation in fibroblasts and colon cancer cells. However, its role in human cervical cancer is unclear. By immunohistochemical staining and Western blotting, aberrant Eps8 expression was observed in cervical carcinoma compared with normal cervical epithelial cells. Clinicopathologic analysis of 45 patients indicated that Eps8 expression was associated with parametrium invasion and lymph node metastasis, two major poor prognostic factors for early-stage cervical cancer. Kaplan-Meier analysis of cervical cancer specimens also indicated an inverse relationship between the level of Eps8 and the patients' survival rate. Using small interfering RNA of eps8, we observed reduced proliferation and tumorigenesis in Eps8-attenuated HeLa and SiHa cells cultured in dishes or inoculated in mice. Furthermore, diminished Eps8 impeded G 1 -phase progression in HeLa and SiHa cells that might be attributable to reduced expression of cyclins D1, D3, and E, elevated accumulation of p53 and its downstream target p21 Waf1/Cip1 , and suppressed hyperphosphorylation of retinoblastoma. Alteration of these cell cycle -related proteins could be reversed by ectopic Eps8, implicating that the effect of Eps8 on the mentioned cell cycle modulators was specific. Notably, the augmented expression of p53 by diminished Eps8 was at least due to its decreased turnover rate. Concurrent with p53 up-regulation and the decrement of Src and AKT activity, Eps8-attenuated HeLa and SiHa cells exhibited increased chemosensitivity to cisplatin and paclitaxel. Together, our findings implicate the involvement of Eps8 in chemoresistance and show its importance in prognosis of cervical cancer patients. [Mol Cancer Ther 2008;7(6):1376 -85]
The Toll-like receptors (TLRs) play a pivotal role in innate immunity for the detection of highly conserved, pathogen-expressed molecules. Previously, we demonstrated that lipopolysaccharide (LPS, TLR4 ligand)-increased macrophage motility required the participation of Src and FAK, which was inducible nitric oxide synthase (iNOS)-dependent. To investigate whether this iNOS/Src/FAK pathway is a general mechanism for macrophages to mobilize in response to engagement of TLRs other than TLR4, peptidoglycan (PGN, TLR2 ligand), polyinosinic-polycytidylic acid (polyI:C, TLR3 ligand) and CpG-oligodeoxynucleotides (CpG, TLR9 ligand) were used to treat macrophages in this study. Like LPS stimulation, simultaneous increase of cell motility and Src (but not Fgr, Hck, and Lyn) was detected in RAW264.7, peritoneal macrophages, and bone marrow-derived macrophages exposed to PGN, polyI:C and CpG. Attenuation of Src suppressed PGN-, polyI:C-, and CpG-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by the reintroduction of siRNA-resistant Src. Besides, knockdown of FAK reduced the mobility of macrophages stimulated with anyone of these TLR ligands. Remarkably, PGN-, polyI:C-, and CpG-induced Src expression, FAK Pi-Tyr861, and cell mobility were inhibited in macrophages devoid of iNOS, indicating the importance of iNOS. These findings corroborate that iNOS/Src/FAK axis occupies a central role in macrophage locomotion in response to engagement of TLRs.
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