A friend of mine posts on his blog “lay summaries” of papers he writes, which I’ve always thought is a good use for a blog, though I’ve never gotten around to doing it. One of my graduate students will defend his Ph.D. in a few days, and so this seems like a good opportunity to describe the paper on his last major project, which we published a few months ago (Andrew F. Loftus, Sigrid Noreng, Vivian L. Hsieh, and Raghuveer Parthasarathy, “Robust Measurement of Membrane Bending Moduli Using Light Sheet Fluorescence Imaging of Vesicle Fluctuations,” Langmuir 29: 14588–14594 (2013)).
The topic is the stiffness of lipid membranes – the two-dimensional materials that make up the boundaries of all cells, and of all organelles within cells. As I’ve noted before, cells sculpt membranes into highly curved shapes as they perform various tasks. For example, the trafficking of cargo from place to place involves proteins binding to membranes, bending them into gently and then highly curved buds, and then pinching them to form little membrane-bound capsules (see illustration, above). The rigidity of the membrane is the key material property that governs this process. Membrane rigidity, especially as modified by proteins, remains poorly measured, however, limiting our understanding of normal cellular function as well as diseases associated with trafficking.
We’ve been measuring membrane rigidity for a few years, relying on the conceptually simple but experimentally challenging method of pulling on membranes with optical traps (focused laser light that “grabs” objects) — see e.g. this video:
With this approach, we discovered that a key trafficking protein, called Sar1, dramatically lowers the rigidity of the membranes to which it binds. We’ve been interested, however, in developing other experimental methods.
Since the 1980s, a handful of researchers have been watching the shape fluctuations of giant lipid vesicles. “Giant” means hundredths of a millimeter in diameter (!), and vesicles are spherical shells of membrane:
We realized that a neat way to look at shape fluctuations, that hadn’t been done before, is to use “light sheet microscopy” — illuminating the vesicle with a thin sheet of laser light, and thereby looking just at the form of the equatorial plane with high speed and sensitivity. Here’s what light sheet imaging of vesicles looks like:
The cross-sections of the vesicle aren’t circles, due to ever-present thermal energy driving undulations…
Analysis of the amplitudes of the fluctuation modes allows us to determine the rigidity of the vesicle membrane.
There are advantages to using light sheet fluorescence imaging to look at vesicle fluctuations, compared to other approaches. Usually, people examine vesicles sitting at the bottom of some imaging chamber, leading to vesicles that are gravitationally distorted, which affects their fluctuation modes. With light sheet imaging, we can image free-falling (or rising, or stationary) vesicles. Moreover, using membrane fluorescence rather than other imaging modes allows straightforward evaluation of the accuracy of mode analysis, for reasons I won’t go into here.
We describe these things in the paper, and also show that our fluctuation-based measurements of membrane rigidity, with or without trafficking proteins, agree with the results of our “pulling” measurements. Remarkably, no one before us had ever applied vesicle-based and non-vesicle-based membrane rigidity measurement methods to the same system!
Despite its importance to cellular processes, membrane rigidity remains largely mysterious. We hope that our development of better methods to quantify it, and our investigation of the behaviors of membrane-active proteins, helps illuminate the generation of shape and form in cells.
2 thoughts on “Dancing Membranes”
a great layperson’s summary and beautiful water colors.