Lipid Bilayers on Planes and in Micropipettes - Two model systems to study binding of DivIVA to flat and negatively curved membranes

Abstract

The aim of this thesis work was to form and characterize model systems of cell membranes
on planar supports and in micropipettes. Firstly, supported lipid bilayers (SLBs) were
formed on glass slides after an existing experimental procedure. It was shown possible
to obtain fluid SLBs from different kinds of lipids as well as on glass slides that were
cleaned with different techniques. Custom-written Matlab scripts were used to assess
the mobilities of the lipids. It was found that all SLBs showed diffusivities within the same
order of magnitude independent of the lipid used, while the fraction of lipids that were
immobile within the SLB were higher for Escherichia coli (E. coli) compared to POPC
lipids. Secondly, a new protocol was established for the lipid-coating of pipettes, which
was successfully demonstrated as well. Using again a custom-written Matlab program
to analyze the mobility of the lipids, it was found that they diffused significantly slower
than on the planar SLBs. It is suggested that this was due to deficient cleaning or that it
could be inherent to the geometry of the lipid bilayer. Additionally, both model systems
were used to investigate the membrane binding behavior of the protein DivIVA which is
known to localize to regions of high negative curvature in the cell. The results indicated
that besides curvature, lipid charge and composition are also features that affect the
membrane binding behavior of the protein. Overall, this work presents the groundwork
for a cheap model system of the curved cell membrane, which also allows to investigate
many different curvature radii at the same time.

Popular Abstract

Whether bacteria or blue whale- every living organism is made up of the common, fascinating building block of life: the cell. It is, as its origin from the Latin word cella suggests, a "small room" crammed with all kinds of things. There are for example the cell nucleus containing the DNA with the genetic instructions, the mitochondria as power plants of the cell or the endoplasmic reticulum as an intracellular highway to quickly transport molecules from one place to another. Naturally, the cell is also surrounded by a kind of wall which separates it from its surroundings. This wall, called the cell membrane, was the subject of this thesis work. In principle, it is just a greasy coating, since the membrane is built up of fat molecules - lipids. The lipid is an amphiphilic (Greek amphis = both and philia = love) molecule which means that one end of it wants to be in contact with water (the head) while another does not (the tail region). Due to this, lipids in water spontaneously assemble into structures in which the lipid heads are exposed to water molecules, while the lipid tails are not. We have all seen this when trying to mix water and fat, like for example in a soup. One of these structures is the lipid bilayer. As the name suggests, it is composed of two chains of lipids facing each other. The cell membrane is therefore simply a lipid bilayer. Simply? Far wrong! While its main component are the lipids, it is also embedding countless different kinds of proteins. These we do not only need to grow muscles, but they also fulfill complex and intertwined functions regarding e.g. intercellular communication or molecule transport into and out of the cell among many others. We were particularly interested in one of the proteins binding to the cell membrane which is called DivIVA. It is found in bacteria that have a rigid cell wall which shapes the fluid cell membrane. DivIVA seems to be involved in regulatory processes for cell division and growth. An interesting feature of it is that it tends to bind to the regions of the membrane that are more curved than others, for example the two ends in a rod-shaped bacterium. Understanding why this is the case will certainly shed light on how the bacterial
cell manages to divide exactly in the middle or avoids excessive growth. However, it turns out that it is rather difficult to study DivIVA or other proteins within the living cell, not only because of all the efforts associated with culturing cells but also because of all the intertwined interactions of proteins
on the membrane which make it very hard to isolate a particular facet. Therefore, researchers have been utilizing model systems of the cell membrane. The advantage of these is that the conditions, such as the kinds of lipids the membrane is composed of or the concentrations of the proteins present in the systems, are highly controllable and therefore particular issues can be investigated. One of these model systems is the supported lipid bilayer (SLB), which is a lipid bilayer formed on a solid support. The goal of this thesis work was to obtain an SLB from different lipids on a plane glass slide, characterize it regarding its quality (i.e. investigate the movement of the lipids within the SLB) and use it to study the membrane binding behavior of DivIVA. Moreover, it was tried to obtain and characterize a model system of the curved membrane, since DivIVA is known to bind to these. For this, the walls of a glass pipette were coated with a lipid bilayer. Due to the conical shape of the pipette, many different curvatures, higher ones close to the pipette opening and lower ones further away from it, could be utilized to investigate the curved membrane binding preference of proteins such as DivIVA. In this work, I showed it possible to obtain SLBs on glass slides as well as in micropipettes. I investigated DivIVA binding in both systems and found that the charge and type of lipid in the bilayer are important factors affecting DivIVA binding. To put this into a broader context, I showed that lipid-coated pipettes can be used as a model system of the curved membrane. This could be useful to conduct studies under highly controlled experimental conditions not only with DivIVA but also other proteins that are known to preferably bind to curved membranes. An example is the protein alpha-synuclein which is associated with aggregation in the brain in patients with Parkinson’s disease.

• author: Baumann, Elisabeth
• supervisor: Peter Jönsson
• organization: Physical Chemistry; Department of Physics
• course: FYSM60 20161
• year: 2016
• type: H2 - Master's Degree (Two Years)
• subject: Physics and Astronomy
• keywords: supported lipid bilayer, DivIVA, curvature, TIRF, FRAP, epifluorescence microscopy, micropipette, model system
• language: English
• id: 8875599
• date added to LUP: 2016-06-01 17:38:04
• date last changed: 2016-06-01 17:38:04