Seeing into the cell membrane using light

Scientists from the McKelvey School of Engineering at Washington University in St. Louis have developed a new technique that allows us to see inside a cell membrane.

Therefore, the cell membrane has two functions: first, to be a barrier keeping the constituents of the cell in and unwanted substances out and, second, to be a gate allowing transport into the cell of essential nutrients and movement from the cell of waste products.

Picturing a cell likely brings to mind several discrete, blob-shaped objects; maybe the nucleus, mitochondria, ribosomes, and the like. But, one part remains overlooked: the cell’s membrane.

Now, the technique developed in the lab of Matthew Lew, assistant professor in the Preston M. Green Department of Electrical and Systems Engineering, enabled scientists to differentiate collections of lipid molecules of the same phase and to determine the chemical composition within those domains.

The technique called single-molecule orientation localization microscopy, or SMOLM, directly measures the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes.

Lew said, “Using traditional imaging technologies, it’s difficult to tell what’s “inside” versus “outside” a squishy, transparent object like a cell membrane, particularly without destroying it.”

“We wanted a way to see into the membrane without traditional methods” — such as inserting a fluorescent tracer and watching it move through the membrane or using mass spectrometry — which would destroy it.”

Scientists used a fluorescent probe to probe the membrane without destroying it. This fluorescent probe emits light to directly “see” where the probe is and where it is “pointed” in the membrane. The probe’s orientation reveals information about both the phase of the membrane and its chemical composition.

Scientists found numerous lipid molecules in cell membranes—some form liquid, some form a more solid or gel phase. Molecules in a solid phase are rigid, and their movement is constrained. They are, in other words, ordered. However, when they are in a liquid phase, they have more freedom to rotate; they are in a disordered phase.

Using a model lipid bilayer to mimic a cell membrane, Lu added a solution of fluorescent probes, such as Nile red, and used a microscope to watch the probes briefly attach to the membrane.

When light is shined on the system, the probe releases photons. An imaging method previously developed in the Lew lab then analyzes that light to determine the molecule’s orientation and whether it’s fixed or rotating.

Jin Lu, a postdoctoral researcher in Lew’s lab, said, “Our imaging system captures the emitted light from single fluorescent molecules and bends the light to produce special patterns on the camera.”

“Based on the image, we know the probe’s orientation, and we know whether it’s rotating or fixed, and therefore, whether it’s embedded in an ordered nanodomain or not.”

Several times, repeated the process could provide enough information to create a detailed map, showing the ordered nanodomains surrounded by the ocean of the disordered liquid regions of the membrane.

The probe was able to distinguish between lipid derivatives within the same nanodomains. In this manner, the probe can reveal whether or not the lipid molecules are hydrolyzed when a certain enzyme was present.

Lew said, “This lipid, named sphingomyelin, is one of the critical components involved in nanodomain formation in cell membranes. An enzyme can convert a sphingomyelin molecule to ceramide. We believe this conversion alters the way the probe molecule rotates in the membrane. Our imaging method can discriminate between the two, even if they stay in the same nanodomain.”

“This resolution, a single molecule in the model lipid bilayer, cannot be accomplished with conventional imaging techniques.”

Lew said, “At this scale, where molecules are constantly moving, everything is self-organized. It’s not like solid-state electronics where each component is connected in a specific and importantly static way.”

“It’s not like solid-state electronics where each component is connected in a specific and importantly static way.”

Individual molecules can organize into these nanodomains that can collectively inhibit or encourage certain things — like allowing something to enter a cell or keeping it outside.

“These are processes that are notoriously difficult to observe directly. Now, all you need is a fluorescent molecule. Because it’s embedded, its movements tell us something about what’s around it.”

Journal Reference:
  1. Dr. Jin Lu et al. Single‐Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes. DOI: 10.1002/anie.202006207

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