«THE MICRORHEOLOGY OF LIPID BILAYERS by TRISTAN HORMEL A DISSERTATION Presented to the Department of Physics and the Graduate School of the University ...»
THE MICRORHEOLOGY OF LIPID BILAYERS
Presented to the Department of Physics
and the Graduate School of the University of Oregon
in partial fulﬁllment of the requirements
for the degree of
Doctor of Philosophy
DISSERTATION APPROVAL PAGE
Student: Tristan Hormel Title: The Microrheology of Lipid Bilayers This dissertation has been accepted and approved in partial fulﬁllment of the requirements for the
Doctor of Philosophy degree in the Department of Physics by:
John Toner Chair Raghuveer Parthasarathy Advisor Eric Corwin Core Member Kelly Sutherland Institutional Representative and Scott L. Pratt Dean of the Graduate School Original approval signatures are on ﬁle with the University of Oregon Graduate School.
Degree awarded June 2015 ii c 2015 Tristan Hormel This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs (United States) License.
This dissertation includes previously published and unpublished coauthored material.
CURRICULUM VITAENAME OF AUTHOR: Tristan Hormel
GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED:
University of Oregon, Eugene, OR Reed College, Portland, OR
Doctor of Philosophy, Physics, 2015, University of Oregon Bachelor of Arts, Physics, 2009, Reed College
AREAS OF SPECIAL INTEREST:
Rheology of lipid bilayers
Research Assisstant, Parthasarathy Group
GRANTS, AWARDS AND HONORS:
NSF GK-12 Fellow, Scientist in Residence, University of Oregon, 2015
T. Hormel, S. Kurihara, M. Brennan, M. Wozniak and R. Parthasarathy. Measuring lipid membrane viscosity using rotational and translational probe diﬀusion. Physical Review Letters, 112:188101, 2014.
Several undergraduates helped with experiments and deserve thanks: Morgan Hynson, Katie Brennan, Matthew Wozniak, and especially Sarah Kurihara and Matthew Reyer. Labmates also helped with experiments, even if they didn’t conduct them: Ryan Baker, Matthew Jemelieta, Andrew Loftus and Mike Taormina all provided valuable discussions and insight. For nonacademic support, friends and family and in particular Marial Torres my cats Cheepie Caloo and Barnaby Jones. The National Science Foundation funded my research. My advisor Raghu Parthasarathy, from whom I have learned so much about being a scientist, deserves special thanks.
Finally, support from NSF Awards 1006171 and 0742540 funded this work.
If you can read this, you are doing so with biological machinery selected through a four billion year history of evolution. Your eyes, that can see the words, and your brain, than can understand them, are products of this rich history, as is every organ used to construct a creature capable of reading. As is a human being in its entirety, and every other living thing on Earth.
This history is a story as much of accident as much innovation. Its result are the millions of species alive today.
To understand this staggering diversity in all of its complexity is the unenviable task of the biologist. As a biophysicist, I’m tempted to seek unifying principles analogous to Newton’s gravity, that explains both falling apples and orbiting planets. But from a superﬁcial inspection, this seems nothing short of hopeless. That there are unifying principles that can describe a whale as well as a ﬂea seems unlikely. But nonetheless, that such unifying principles do in fact exist is obvious inasmuch as we can learn about ourselves by studying Eschericia coli. Some such unifying principles are conceptual- there is a logic to evolution, and biology is as subject to physics as is chemistry. But others are concrete and material- all living things make use of DNA, of the same amino acids.
My dissertation research is an attempt to develop tools that will help us understand another such universal feature of cellular life: the lipid bilayer. This structure is the foundational, essential component of all biological membranes, and by any assessment one of the most important structures in biology.
While the lipid bilayer is the essential component of a cell membrane, you might not know it by looking at one. In fact even in the middle of the twentieth century there was debate a to whether lipids or proteins constituted the essential component of a living membrane. This isn’t really an odd confusion. Cell membranes often contain an appreciable amount of proteins by weight, sometimes even exceeding the lipid content. The variety of these proteins is a testament to the importance of the membrane structure to life: in E. Coli, which has the most understood genome, almost a quarter of genes code for membrane proteins. There are many reasons for this variety, a corollary to the many roles that cell membranes play in cellular function. As the barrier that separates a cell’s interior from its exterior, any interaction with a cell’s environment must be mediated through the membrane. It also serves as a semi-permeable barrier, keeping biological molecules and cellular contents localized to diﬀerent regions where they can be useful to the cell.
Flexibility, or deformability, is another key requirement, in part because cells will sometimes wish to ingest large amounts of material, for instance through phagocytosis, but especially because major requirement for all life on a cellular scale is division. All of these functions require families of proteins and other macromolecules. The physical mechanisms that govern the organization of these proteins on the cell’s surface is an area of research rife with controversy, particularly with respect to lipid rafts- the point being that now is a good time to study membranes.
The ﬂuid mosaic model of Singer and Nicholson established a view of membranes according to which the bilayer serves as a matrix on which proteins are able to move and spatially organize. Singer and Nicholson emphasized the ﬂuidity of the bilayer, a feature responsible for the membrane’s ability to dynamically and chemically reorganize in response to a cell’s needs.
Additionally, the magnitude of ﬂuid properties of the bilayer, such as bilayer viscosity, are in their own right biological parameters of important scope. The timescale for diﬀusion in a membrane is set by bilayer viscosity, and therefore viscosity sets a physical limit on the speed with which cells can perform certain functions. At a diﬀerent lengthscale, viscosity also determines how much a membrane will deform in response to an in-plane force, and so will also be a determining feature for how quickly cells can stretch and broadly change their shape.
If, then, we wish to model any such cellular process, we will require good measurements of membrane viscosity. My dissertation research is an attempt to make such measurements, but also to develop improved methodologies for making them. The hope is that, in the future, we will have the means to make relatively easy and precise measures of the viscosity of two-dimensional ﬂuids.
This document is divided in to ﬁve chapters. In the second, I discuss some of the chemistry and ﬂuid mechanics that inform our understanding of lipid bilayers. This includes the chemical nature of the lipids that form bilayers, and how that chemistry informs bilayer viscosity. I also discuss continuum models of diﬀusion in membranes; these models have been broadly, but not universally, successful in describing the motion of inclusions within membranes.
In the third chapter, I discuss the analytical and computational tools needed to perform a microrheology experiment like those I performed. These are speciﬁc cases of problems, such as feature localization, within the larger ﬁeld of image analysis. The information in this chapter is summary and general.
The fourth chapter contains the experimental details and results of my experiments. These are the main conclusions of my dissertation research. The speciﬁcs of analytic and experimental approaches we used for each project will be found here. This chapter includes material coathored with Sarah Kurihara, Kathleen Brennan, Matthew Wozniak, Matthew Reyer and Raghuveer Parthasarathy.
The ﬁnal chapter oﬀers concluding remarks and some ideas for extensions of my work that could be performed by another researcher.
Even though the cell membrane is a small structure, there are a lot of ways to characterize it. In this chapter, I’ll present details of two such characterizations, one which relies on the physical chemistry of the bilayer and another which considers the ﬂuid mechanics of twodimensional membranous structures. Throughout, the membranes I’ll discuss will be simpliﬁed, cell-free lipid bilayers, devoid of the proteins and other cellular machinery that complicate the real things in real life.
The experiments I performed are amenable to continuum descriptions of lipid bilayers.
That is, given the size of relevant experimental features, we were able to ignore the molecular nature of the bilayer in our calculations and measurements. Nonetheless a few words on the constituents of these membranes, lipids and otherwise, are in order. In particular, lipid structural properties can illuminate some of the characteristics of the bilayers that they form, including bilayer viscosity.
Lipids are an enormous class of molecules, and approximately 5% of genes are used in synthesizing many diﬀerent types. Taken in the broadest sense, they comprise not just the bilayer forming lipids present in cell membranes, but also a host of other compounds with varying degrees of complexity and size. The latter include detergents (such as sodium dodecylsulfate, familiar to shampoo users) and some vitamins, while an important example of the former are the phospholipids.
All lipids are amphipathic molecules, composed of a hydrophilic (water-loving) region, and a hydrophobic (water-hating) region. In phospholipids the hydrophobic region is termed a “headgroup”, while the hydrophobic region is composed of hydrocarbon (acyl) chains of varying length (Fig. 1 and 2). The core, or conserved, portion of phospholipids is a glycerol molecule, or backbone, attached to a phosphomoiety containing headgroup (Fig. 1 and 2).1. Many diﬀerent headgroups are used by cells, and the headgroup is one feature that distinguishes diﬀerent categories of phospholipids. Phosphatidylcholines, or PC lipids, comprise one of the most studied and most common groups, and are the subject of my research, but phosphatidylethanolamines (PE) and phosphatidylserines (PS) are also abundant and well-studied. The glycerol core is also bound to two acyl chains. The length and saturation of these chains is another distinguishing feature of phospholipids2. While phospholipids diﬀerentiated by acyl chain structure always carry diﬀerent chemical names, due to the large number of phospholipid varieties these names can be clunky; and it is therefore often easier and more illuminating, especially when considering lipids with a common headgroup, to refer to lipids by (chain length):(number of double bonds). For instance, 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, and 18:1 PC all refer to the same molecule (Fig. 2(b)), as do 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC and 16:0 PC, respectively (Fig 2(c)), but the 18:1 PC and 16:0 PC nomenclature make the diﬀerences between the two most obvious, at least for a physicist. When one is interested in discussing the location of a particular bond in the acyl chain, this is done by referring to a ∆ position, which is the number of carbon bonds from the headgroup. So, for example, DOPC has a double bond at the ∆ 9 position. Furthermore, the conformation of the tail chains is important in lipid phase behavior.
Carbon bonds in lipid acyl chains have two conformations, either cis or trans3 (Fig 2). The cis conformation places a bend in the acyl chain that increases the cross-sectional area of the lipid molecule. Unsaturated double bonds are almost always found in the cis conformation, while single bonds can be found in either, dependent upon bilayer phase (see section 2.1.2).
Phospholipids are not the only lipids found in biological membranes. Table 1 gives the lipid composition of several membranes from several lifeforms. Like phospholipids, sphingolipids are complex lipids with a polar headgroup and acyl chains. Some biological membranes also contain cholesterol (Fig 3). Cholesterol is a sterol lipid that, on its own, does not form bilayers. It is, however, readily incorporated into phospholipid and especially sphingolipid bilayers. It is present in large quantities in the plasma membrane of eukaryotes, where it can be present at 1 Phospholipids are also termed glycerophospholipids, but in practice they are almost always referred to using the briefer designation.
2 In saturated chains, every carbon atom in the chain is bound to other carbons with just single bonds, while the other bonds are ﬁlled with hydrogen; unsaturated chains contain one or more double bonds between carbon atoms.
3 These terms come from the latin cis = this side, trans = the other side.