Studying the influence of membrane curvature on positioning lipids and proteins in bacterial membranes
Many proteins in bacteria are preferentially located at specific sites within the cell. These proteins can control anything from cell shape, growth, division, movement of intracellular components, or assembly of motility systems to cell polarization. An untested hypothesis is that microdomains of a uniquely-curved phospholipid, cardiolipin, function as 'landmarks' to localize proteins in bacteria at regions of high negative curvature via a mechanism similar to lipid rafts. The aim of this proposal is to determine the relationship between the localization of CL microdomains, protein localization, and membrane curvature using spheroplasts of Escherichia coli, giant unilamellar vesicles (GUVs), and supported lipid bilayers (SLBs). By compressing spheroplasts and GUVs in microfluidic channels, or by conforming SLBs to either micropatterned curvy substrates or subject to deformation over a micropore, the temporal and spatial organization of CL and its putative binding proteins can be measured. An understanding of the reorganization of lipid microdomains in response to membrane curvature will be critical in developing a complete picture of the physical forces that influence biomolecule localization in bacteria. It is envisioned that the strain induced reorganization of membranes may be a widely spread phenomenon in biology that extends to eukaryotes.
Training objectives include developing an expertise in bacterial lipid metabolism and its quantification by in vivo labeling or through in vitro analysis. Broader impacts of this work include educational outreach through the Research Experience for Teaching Program. The PI will mentor local teachers by developing educational kits that will be incorporated into after-school programs in the Madison Metropolitan School District through the MicroExplorers Program.
This National Science Foundation (NSF) Postdoctoral Fellowship, based at UW-Madison Biochemistry, was awarded to perform studies of organization in bacteria. Despite their small size and low amount of genetic material (in comparison to higher order life), bacteria are highly complex organisms that regulate and localize, often with astounding precision, the machinery that maintains their viability and reproduction. The localization of proteins at the poles in rod-shaped bacteria is a particularly prevalent observation. A greater understanding of how and why these proteins localize to the poles may lead to more effective antimicrobial agents, which is a growing need with the advent of multi-drug resistant pathogenic bacteria, such as MRSA. I contributed to this understanding of bacterial organization by investigating the distribution of molecules within the natural fluid barrier enclosing the cell, the cell membrane. It has been shown that the poles of bacteria are negatively charge due to the concentration of a highly-curved lipid, cardiolipin, at the poles. This lipid, which comprises approximately 5-10% of the total lipid content in the Escherichia coli membrane, is believed to shuttle to the poles due to the preference of small aggregates of the lipid to seek areas of higher-curvature (i.e. the cell poles). The negative character of the poles appears to be functionally important for the cell and may serve as a landmark to recruit certain proteins to the cell poles. I discovered that other negatively-charged lipids, such as phosphatidylglycerol (~20% of the membrane), can also be shuttled to the poles of the cell in E. coli, even in the absence of cardiolipin. The results of this study on lipid organization in bacteria were recently published in the peer reviewed Journal of Bacteriology (2014). We are not sure how phosphatidylglycerol moves to the poles and maintains its negative charge, which is the subject of ongoing work. My in vivo studies with E. coli membranes will likely impact research into the fields of bacteriophage infection and mechanisms of antimicrobial peptide action, both of which have significant ties to membrane organization. My results may also affect the interpretations of researchers working with eukaryotic cardiolipin—an area of particular interest because a number of diseases have an association with misregulated cardiolipin metabolism, such as Barth's syndrome and Alzheimer's disease. Outreach: In addition to research into lipid organization in bacteria, I mentored an undergraduate student to develop hands-on 15-minute presentations about microfluidics intended for a middle-school audience. The presentation was expanded to include an educational "macro"-fluidic device, which was introduced at the 2013 UW-Madison Engineering Expo and is seeing ongoing use with school groups at the Wisconsin Institutes of Discovery. Cross-funding was provided by the Materials Research Science and Engineering Center (MRSEC). In the summer of 2012, I collaborated with two members of the Weibel lab at UW-Madison and a chemistry teacher from Beaver Dam High School, Beaver Dam, WI through the Research Experience for Teaching (RET) Program. We developed a new acid/base equilibrium lab using an inquiry-based pedagogical approach with microfluidics. The lab is currently used in a number of other settings throughout southern Wisconsin and is the subject of a manuscript published in the Journal of Chemical Education (2014).
