Journal article
Jade M. Noble, Netta Vidavsky, LaDiedra Monet Roberts, Aaron E. Chiou, M. Paszek, C. Fischbach, L. Estroff, L. Kourkoutis
Microscopy and Microanalysis, 2018
Microscopy and Microanalysis, 2018
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APA
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Noble, J. M., Vidavsky, N., Roberts, L. D. M., Chiou, A. E., Paszek, M., Fischbach, C., … Kourkoutis, L. (2018). Revealing Mechanisms of Microvesicle Biogenesis in Breast Cancer Cells via in situ Microscopy. Microscopy and Microanalysis.
Chicago/Turabian
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Noble, Jade M., Netta Vidavsky, LaDiedra Monet Roberts, Aaron E. Chiou, M. Paszek, C. Fischbach, L. Estroff, and L. Kourkoutis. “Revealing Mechanisms of Microvesicle Biogenesis in Breast Cancer Cells via in Situ Microscopy.” Microscopy and Microanalysis (2018).
MLA
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Noble, Jade M., et al. “Revealing Mechanisms of Microvesicle Biogenesis in Breast Cancer Cells via in Situ Microscopy.” Microscopy and Microanalysis, 2018.
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@article{jade2018a,
title = {Revealing Mechanisms of Microvesicle Biogenesis in Breast Cancer Cells via in situ Microscopy},
year = {2018},
journal = {Microscopy and Microanalysis},
author = {Noble, Jade M. and Vidavsky, Netta and Roberts, LaDiedra Monet and Chiou, Aaron E. and Paszek, M. and Fischbach, C. and Estroff, L. and Kourkoutis, L.}
}
Abstract
Tumors secrete microvesicles (MVs) through direct shedding from the plasma membrane. When introduced into the extracellular matrix, MVs are believed to aid in tumor invasiveness [1]. Despite their importance, the mechanisms of MV biogenesis, as well as their ultrastructure and chemical cargos, are poorly understood. MV shedding is believed to occur through membrane bending of the cell [2]. Researchers have proposed that asymmetric molecular crowding on the lipid bilayer drives membrane bending [3]. One key structure that possesses such asymmetric crowding is the glycocalyx, a dense network of glycosylated proteins surrounding the exterior of most cells. Here we used both room temperature and cryo-SEM and cryo-TEM to better understand the biogenesis of MVs from MCF10A-HAS3 (hyaluronan synthase 3) cells. Compared to more traditional approaches in which vesicles are isolated from the growth medium through centrifugation, here, cells are cultured directly on TEM grids, thereby reducing the risk of structural damage due to sample preparation. The adherence and densities of cells cultured on TEM grids were similar to those grown using standard methods. We determined that HAS3 cells contained tubular structures that extended from the cell surface. These tubules contained smaller vesicles, suggesting a mechanism of vesicle trafficking through pearling and potential actin dynamics. We find that vesicle expression is increased in HAS3 cells as compared to the non-malignant control. However, we observe a discrepancy in the particle diameter distributions of HAS3 vesicles obtained by nanoparticle tracking analysis (NTA) and those from direct inspection with cryo-TEM. A large peak at ~100 nm dominates the NTA data while the signal is distributed across other vesicle diameters in the cryo-TEM data. This may suggest that NTA not only captures vesicles but also biological debris present in the analyzed media. Cryo-TEM further shows that the majority of secreted HAS3 vesicles contain surface structure suggesting the presence of the glycocalyx. For HAS3 cells these surface features are not very pronounced. This is in stark contrast to the vesicles secreted by MCF10A-Muc1 cells. This difference may be due to the weak covalent linking of the glycocalyx in HAS3 vesicles. The presence of a glycocalyx confirms the MVs’ origin from the plasma membrane.
