Research in our group is focused on understanding intermolecular interactions in biological membranes and the processing of molecules and nanoparticles as they enter the cell. We study the dynamics and distribution of molecules within the membrane as a means of understanding their function in events such as cell-cell communication, signal transduction, endocytosis and cell differentiation. To this end, we use spectroscopic and microscopic techniques that are suited to study single molecules or molecules at low concentration. There are four major themes:
1. Studies of the distribution and extent of aggregation and interaction of membrane receptors in living cells.
This work is based on analysis of laser scanning confocal microscopy images (Fig 1A) to estimate the density of protein clusters and their sizes. We have developed specific tools - Image Correlation Spectroscopy (Fig 1B) and Image Cross-correlation Spectroscopy for this purpose.
Figure 1: Receptor Distribution (A) and the corresponding correlation function (B)
2. Studies of the dynamics of movement of molecules, clusters, and nanoparticles in living cells.
This work applies fluorescence photobleaching, fluorescence correlation spectroscopy and dynamic image correlation spectroscopy (Fig 2) to study the diffusion and flow of individual molecules or complexes on the surface or within the cell. This provides detailed information of which molecules interact and how the dynamics relates to their function
Figure 2: Decay of correlation function amplitude reveals rate of movement
3. Studies of interactions of nanoparticles with cell membranes
This work aims to understand the mechanism(s) whereby nanoparticles attach to cell surfaces and the pathways whereby they are internalized (endocytosed) in living cells. We also wish to understand the fate of these nanoparticles as well as the effect they may have on cellular functions, such as growth, division, and differentiation. The major tools are electron microscopy and fluorescence microscopy, but in the future, we wish to explore other tools, such as secondary ion mass spectrometry. Figure 3 shows an example of the distribution and colocalization (yellow) of gold nanoparticles (green) and lysosomes (red).
Figure 3: 20 nm lipid coated gold nanoparticles (left) are seen as green fluorescence in cells. They colocalize with red fluorescent markers for lysosomes as seen by the yellow in the superimposed images
4. Studies of lung surfactants from healthy and dysfunctional animals.
The purpose is to understand how the particular lipids and proteins present in lung surfactants affect the function and malfunction of the surfactant films in lungs. The objective is to establish the phase behaviour of the systems (Fig 4A), to identify the composition and physical characteristics of different phases (Fig 4B) and to establish the mechanism whereby these components create a low surface tension environment dynamically. The program combines imaging by fluorescence, atomic force microscopy (Fig 4A) and secondary ion mass spectrometry (Fig 4B).
Figure 4: Distribution of solid phases of DPPC in a liquid phase (A) and the distribution of deuterated palmitic acid from DOPC in a monolayer detected by TOF-SIMS in a mixture (B) confirm that DOPC is in the liquid phase only
M. Anikovski, Z.D. Wiltshire, K. Weisshart, and N.O. Petersen “Photon Counting Histogram Analysis for Two-Dimensional Systems” ChemPhysChem (in press – published electronically August 2011) Cover Article
Y. Jiang, A.J. Nohe, B. Gragdon, C. Tian, N. Rudarakanchana, N.W. Morrell, and N.O. Petersen, “Trapping of BMP receptors in distinct membrane domains inhibits their function in pulmonary arterial hypertension” American Journal of Physiology – Lung Cellular and Molecular Physiology 301:L218-L227 (2011)
E. Keating, A.J. Waring, F.J. Walther, F. Possmayer, R. A. Veldhuizen, and N.O. Petersen “ A TOF-SIMS study of the lateral organization of lipids and proteins in pulmonary surfactant systems” BBA-Biomembranes 1808, 614-621 (2011) (CIHR)
B. Bragdon, S. Thinakaran, O. Moseychuk, D. King, K. Young, D.W. Litchfield, N.O. Petersen, and A. Nohe “Casein Kinase 2 beta-Subunit is a regulator of Bone Morphogenetic Protein 2 Signaling.” Biphysical Journal 99, 897-904 (2010)
F. Possmayer, S.B. Hall, T. Haller, N.O. Petersen, Y.Y. Zuo, J. Bernardino de la Serna, A.D. Postle, R. A.W. Veldhuizen, S. Orgeig “Recent Advances in Alveolar Biology: Some new looks at the alveolar interface” Respiratory Physiology and Neurobiology 173S, S55-S64 (2010)
M.S. Bakshi, V.S. Jaswal, F. Possmayer, and N.O. Petersen
“Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State” J. Nanoscience and Nanotechnology 10
, 1747-1756 (2010)