Yangzhe Wu

BRMS1 expression alters the ultrastructural, biomechanical and biochemical properties of MDA-MB-435 human breast carcinoma cells: An AFM and Raman microspectroscopy study

Restoring BReast cancer Metastasis Suppressor 1 (BRMS1) expression suppresses metastasis in MDA-MB-435 human breast carcinoma cells at ectopic sites without affecting tumor formation at orthotopic site in the body. BRMS1 expression induces many phenotypic alterations in 435 cells such as cell adhesion, cytoskeleton rearrangement, and the down regulation of epidermal growth factor receptor (EGFR) expression. In order to better understand the role of cellular biomechanics in breast cancer metastasis, the qualitative and quantitative detection of cellular biomechanics and biochemical composition is urgently needed. In the present work, using atomic force microscopy (AFM) and fluorescent microscopy we revealed that BRMS1 expression in 435 cells induced reorganization of F-actin and caused alteration in cytoarchitectures (cell topography and ultrastructure). Results from AFM observed increase in biomechanical properties which include cell adhesion, cellular spring constant, and Youngs modulus in 435/BRMS1 cells. Raman microspectroscopy showed weaker vibrational spectroscopic bands in 435/BRMS1 cells, implying decrease in concentration of cellular biochemical components in these cells. This was despite the similar spectral patterns observed between 435 and 435/BRMS1 cells. This work demonstrated the feasibility of applying AFM and Raman techniques for in situ measurements of the cellular biomechanics and biochemical components of breast carcinoma cells. It provides vital clues in understanding of the role of cellular biomechanics in cancer metastasis, and further the development of new techniques for early diagnosis of breast cancer.

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy

AbstractThe molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures. FigureAFM deflection images, Raman spectra, SEM images, and FTIR of Mycobacterium sp. KMS

Biophysical assessment of single cell cytotoxicity: Diesel exhaust particle-treated human aortic endothelial cells

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs.

Probing nanostructures of bacterial extracellular polymeric substances versus culture time by Raman microspectroscopy and atomic force microscopy.

The structure of a bacterial cell wall may alter during bacterial reproduction. Moreover, these cell wall variations, on a nanoscale resolution, have not yet fully been elucidated. In this work, Raman spectroscopy and atomic force microscopy (AFM) technique are applied to evaluate the culture time-dependent cell wall structure variations of Pseudomonas putida KT2440 at a quorum and single cell level. The Raman spectra indicate that the appearance of DNA/RNA, protein, lipid, and carbohydrates occurs till 6 h of cultivation time under our experimental conditions. AFM characterization reveals the changes of the cellular surface ultrastructures over the culture time period, which is a gradual increase in surface roughness during the time between the first two and eight hours cultivation time. This work demonstrates the feasibility of utilizing a combined Raman spectroscopy and AFM technique to investigate the cultivation time dependence of bacterial cellular surface biopolymers at single cell level.

Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells.

Epidermal growth factor receptor (EGFR) plays an important role in signaling pathway of the development of breast cancer cells. Since EGFR overexpresses in most breast cancer cells, it is regarded as a biomarker molecule of breast cancer cells. Here we demonstrated a new AFM technique-topography and recognition (TREC) imaging-to simultaneously obtain highly sensitive and specific molecular recognition images and high-resolution topographic images of EGFR on single breast cancer cells.

Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

Fluctuations in adhesion behavior of dividing/budding mycobacterium sp. strains JLS, KMS, MCS: An AFM evaluation

AFM was used to evaluate topography and adhesion properties of dividing Gram-positive mycobacterium sp. strains JLS, KMS, and MCS. This research revealed that these three mycobacterium strains divided by budding, and the adhesion force of mother cells differed from their daughter (or budding) cells. At the early stage of division, the adhesion force of the mother cell was larger than that of newly formed daughter cell (10-30%); however, at the anaphase of division, the mother cell possessed smaller adhesion force compared with daughter cell (4-15%). According to the range of the adhesion force measured on the dividing (budding) mycobacterium JLS (M.JLS) cells, the schematic division (or budding) models were proposed, and these mycobacterium strains were found to follow well these division models. Altogether, AFM-based topography and adhesion force measurements at the nanoscale resolution may offer new insightful views that would further facilitate the understanding and elucidation of the biophysical behaviors during division (or budding) processes of microorganisms.

Topography, nanomechanics, and cell surface components of cancer cells examined by combined atomic force microscopy and Raman microspectroscopy

The investigation of the nanostructures and hydrophobic properties of cancer cell membranes is of importance for elucidating the plasma membrane roles in protein folding, membrane fusion, and cell adhesion that are directly related to cancer cell biophysical properties, such as aggressive growth and migration. On the other hand, the chemical component analysis of the cancer cell membrane could be potentially applied in the clinical diagnosis of cancer by the identification of specific biomarker receptors expressed on cancer cell surfaces. In the present work, a combined atomic force microscopy (AFM) and Raman microspectroscopy technique was applied to detect the difference in nanomechanics and membrane chemical components between two cancer cell lines, human lung adenocarcinoma epithelial cells (A549) and human breast cancer cells (MDA-MB-435 with and without expression of BRMS1 metastasis suppressor). The membrane surface adhesion forces for these cancer cells acquired in culture medium were measured using AFM at 0.478±0.091 nN for A549 cells, 0.253±0.070 nN for 435 cells, and 1.114±0.281 nN for 435/BRMS1 cells, and the cell spring constant was measured at 2.62±0.682 mN/m for A549 cells, 2.105±0.691 mN/m for 435 cells, and 5.448±1.081 mN/m for 435/BRMS1 cells. Raman spectral analysis indicated similar peaks between the A549 cells and the breast cancer cell lines 435 and 435/BRMS1 including ~720 cm-1 (guanine band of DNA), 940 cm-1 (skeletal mode polysaccharide), 1006 cm-1 (symmetric ring breathing phenylalanine), and 1451 cm-1 (CH deformation). Slight variations were observed between ~780 - 985 cm-1 (DNA/RNA and proteins) and 1035 - 1210 cm-1 (lipid and proteins).