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| Author(s): |
Daniela Dover de Araujo
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2003-08-27 |
| Examining board members: |
Enilza Maria Espreafico;
Roger Chammas;
Auro Nomizo
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| Advisor: | Enilza Maria Espreafico |
| Abstract | |
Class V myosins are actin-based molecular motors involved in severa! celular processes such as organelle trafficking, secretion, calcium-dependent signaling, expression of cell surface molecules; transport of mRNA and protein complexes, and probably centrosome and mitotic spindle dynamics. In order to better understand the function ofthe three known members of class V myosins (MV), we selected an approximately 50 amino acid segment displaying low degree of conservation among them, reasoning that when expressed in mammalian cells, they would interfere with each member\'s specific functions. MVa1274-1328 and MVc1179-1232 segments were successfully amplified by RT-PCR and cloned either into bacterial (pGEX) or mammalian (pEGFP and pDsRed) expression vectors. Phenotypic studies on B16-F10 murine melanoma cells revealed that MVa1274-1328-EGFP expressjon induces about 37% of cell loss. In contrast, the number ofcontrol cells expressing EGFP alone increases nearly 35% during the same time course. Cells overexpressing MVal274-1328EGFP exhibited unusually small size, round shape, and the majority of them detached from the substrate during the time course of the experiment. Analysis of DNA and F-actin in cells transfected with MVa1274-1328EGFP, 20 hours after transfection, showed that about 70% ofthe transfected cells exhibited condensed or fragmented chromatin, as well as the presence of a cortical ring of filamentous actin and membrane blebbing. It has been shown that MV plays a role in the sequestering ofthe pro-apoptotic protein, Bmf, to the actin cytoskeleton through dynein light chain-2 (DLC2) and damage signals, such as loss of cell attachment, lead to the release of Bmf and its subsequent binding to the pro-survival Bcl2 protein, triggering apoptosis. We demonstrated here that bacterially expressed MVa, but not MVc1179-1232GST, was capable ofinteracting with DLC2, in vitro, suggesting that the loss of cell viability caused by expression of MVa1274-1328EGFP is due to its role in competing for DLC2/Bmf, releasing this pro-apoptotic factor from its anchorage sites. Regarding MVc1179-1232EGFP, we have no evidence for underlying mechanisms of its effect in cell death or proliferation. Also, we demonstrated that MVa is differentially expressed and its subcellular localization varies among melanoma cell lines from radial, vertical or metastatic growth phases. Our data reinforce the involvement of MV in the apoptotic response and in tumor progression, since evading apoptosis is an important step in the development of malignancy. Additionally, this work provides a new molecular tool for the induction of apoptosis in melanoma cells, that can be used both to clarify MVa function in the apoptotic response and as a potential reagent to arrest tumor growth. (AU) | |