Biomembranes are traditionally viewed as flat lipid-bilayer sheets, defining the cell boundaries or dividing the cell into multiple subcellular organelles with specialized functions. However, biomembranes may also fold up into 3-dimensional periodic arrangements, termed cubic membranes, which can adopt extraordinary complexity with up to 12 parallel layers of membranes. The same geometry is mathematically well described and extensively studied in synthetic liquid crystals and block copolymers systems, with a wide range of technological applications. Inner mitochondrial membranes of amoeba Chaos adopt cubic morphology upon starvation (Figure 1), which thus represents a unique experimental system to address the molecular mechanisms involved in controlling membrane morphology and associated functions. Cubic membrane formation has been studied in detail in our laboratory by computer simulation of 2-dimensional transmission electron microscopic projections and tomographic 3D reconstruction (Figure 2). Transformation of membrane morphology is accompanied by significant changes in phospholipid and fatty acid profiles of Chaos cells, and liposomes prepared from the lipids of starved Chaos cells display a high propensity to form hexagonal and cubic arrangements in vitro. These data demonstrate the importance of lipids in forming highly order membrane structures. We have further shown that isolated amoeba mitochondria containing cubic membranes efficiently incorporate and retain DNA oligonucleotides, suggesting a role of cubic membranes in intracellular macromolecule transport, which opens potential application in gene delivery and therapy approaches. Currently, we are pursuing two major directions of cubic membrane research in our laboratory: |
1. Studies on cubic membrane biogenesis and function(s) |
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By electron tomography (Figure 2B), we have proven that the inner mitochondrial membrane organization of amoeba Chaos is taking up "cubic" morphology upon starvation. The physiological significance of this transition is unclear, and may be related to a cellular stress response. A lipid profiling study of "fed" and "starved" mitochondria (in collaboration with Dr. Wenk at Dept. of Biochemistry, NUS) has shown that cubic membrane transformation is accompanied by a major increase of the very long-chain polyunsaturated fatty acid, n-6 DPA (C22:5), which appears predominantly in plasmalogen PC, plasmalogen PE and diacyl PI. Liposomes produced in vitro from lipids of starved Chaos cells show a high propensity to form hexagonal and cubic morphologies. Notably, addition of n-6 DPA, but not of n-3 DPA, to the cell culture induces inner mitochondrial membrane transformation into cubic morphology, also in fed cells. These findings demonstrate for the first time an important structural role of n-6 DPA-containing lipids in cell membrane organization. A Chaos mitochondrial proteomics study is currently in progress, in collaboration with Dr. Qingsong at Dept. of Biological Science, NUS to address potential changes in protein profiles associated with cubic membrane transformation (Figure 3). |
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The crystalloid ER of UT-1 cells is a specialized region of the smooth ER that is characterized by an extensive 2D or 3D periodicity of the ER membrane. The structural determinants of this morphology are unclear. Crystalloid ER houses HMG-CoA reductase, a membrane protein and key enzyme of endogenous cholesterol synthesis, and the biogenesis of this crystalloid ER is known to be associated with overexpression of this enzyme. We aim at understanding the general mechanism underlying crystalloid ER biogenesis in UT-1 cells (statin-resistant CHO cells), which may also shed light on the mechanisms underlying (1) biogenesis and function of virus-induced host membrane complexes and (2) so-called tubular aggregates or tubuloreticular inclusions in various myopathies and human diseases with immunological basis. Lipidomic and proteomic studies are planning to be undertaken in collaboration with Dr. Sepp D. Kohlwein at University of Graz (Austria) and Dr. Lin Qingsong at Dept. of Biological Sciences (NUS, Singapore), respectively, to reveal the role of lipids and proteins involved in the formation of highly ordered crystalloid ER membranes. |
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Due to the absence of a clear view of the 3D nature of cytomembraneous inclusions, numerous nicknames have been given to the observed TEM micrographs in various viral-induced host membrane complexes, such as "tubulocrystalline inclusion" in HCV infected liver, "convoluted membraneous mass" in viral St. Louis Encephalitis and "tubuloreticular structure (TRS)" in AIDS and recently in SARS virus infected Vero cells. TRS is even considered as a specific ultrastructural marker of AIDS in various organs. Using the direct template matching method, we have characterized TEM micrographs of host membranes that were induced by viral infection as cubic membranes. The mechanisms behind the host cubic membrane formation post viral infection remain unknown, however, the virus-induced formation of convoluted membrane complexes provides an excellent model to study the mechanism of crystalloid membrane biogenesis and its potential function(s). This project will be in collaboration with Dr. Ng Mah Lee at Dept. of Microbiology and Dr. Lin Qingsong at Dept. of Biological Sciences (NUS, Singapore). |
2. Cubic membranes: a novel carrier for gene therapy |
Cellular DNA and gene delivery systems are dependent on carrier materials with high efficacy and low cytotoxicity, limiting the currently established DNA transfection agents based on virus capsids or cationic lipids and which are characterized by substantial cytotoxicity. Published data (Almsherqi et al. 2008) from our basic research in amoeba Chaos cells demonstrated that special lipid arrangements that resemble cubic morphologies display a high capacity for DNA uptake and cellular delivery (Figure 4 ). This project aims at exploiting the biomedical potential of cubic membrane-forming lipids, derived from natural sources or chemical synthesis, as a novel carrier for DNA delivery into mammalian cells, with potential applications in gene therapy. |
