Cell membrane thickness measured inside living cells for first time; Scripps method reveals hidden variations

A Scripps team has adapted advanced imaging and computation to measure cell membrane thickness inside intact animal and yeast cells, uncovering organelle-specific differences and local patterns around energy-making proteins that could sharpen understanding of cell function and support drug discovery

Scientists have measured the thickness of cellular membranes inside intact cells for the first time, after researchers at Scripps Research, La Jolla, California, USA, developed a methodology that reveals variations that standard models have previously missed.

The study has built on a computational analysis framework called Surface Morphometrics, which Dr. Danielle Grotjahn’s laboratory at Scripps Research has developed to quantify the shapes and properties of biological membranes within near-native cellular environments.

Cell membranes form the thin lipid barriers that define the boundaries of cells and the internal structures known as organelles. These organelles include the energy producing mitochondria and the endoplasmic reticulum, which processes proteins and lipids. Although membranes appear as simple lines in many microscopy images, their thickness varies by composition and context, and those differences can influence how proteins fit within membranes, how molecules cross them, and how organelles perform specialised tasks.

Until now, researchers have largely relied on simplified systems to study thickness, such as synthetic lipid mixtures in vitro. Such models help to reveal basic physical principles, but they cannot fully reproduce the crowded, protein-rich environment of real cells, where membranes constantly bend, touch, fuse and can remodel themselves in response to cellular demands.

“Membranes don’t exist in isolation – they’re shaped by the proteins and structures around them,” said Grotjahn, an associate professor at Scripps and senior author of the study.

“By measuring thickness inside intact cells, we can start to understand how all of these components work together,” she said.

Grotjahn’s team combined high-resolution imaging with computational reconstruction and quantitative analysis to estimate membrane thickness at specific locations within cells. By measuring membranes in their native context rather than in stripped-down laboratory substitutes, the researchers have aimed to capture the physical and molecular constraints that shape membrane architecture in vivo.

The scientists applied the method to mammalian cells and yeast cells and both reported marked differences between organelles and between membrane regions within the same organelle. In mitochondria, the team found that the outer membrane was significantly thinner than the inner membrane in both cell types. The result has supported the idea that each mitochondrial membrane has a distinct molecular composition, shaped by different lipid and protein content and that these differences help to maintain mitochondrial function.

In mammalian cells, the researchers reported further variation within the inner mitochondrial membrane itself. The inner membrane forms folded structures known as ‘cristae’ which increase the surface area available for energy-producing machinery. The study found that cristae membranes were thicker than regions of the inner membrane that sat close to the outer membrane, a pattern that could reflect differences in local protein packing, lipid distribution, or mechanical constraints.

“The Surface Morphometrics pipeline allows for an unprecedented look at cellular organisation,” said Dr. Michaela Medina, a researcher within the Grotjahn laboratory and a co-first author of the study.

“Even the smallest changes in membrane thickness can have an outsized impact on biological function,” Medina said.

Beyond thickness differences across organelles, the study has linked thickness to membrane curvature. In other words, where membranes curve sharply, they also tend to show a different thickness profile. The authors suggested that proteins that prefer curved membranes, or proteins that impose curvature as they assemble, may also influence local membrane thickness. This matters because curvature acts as a biological signal in its own right, helping cells to sort proteins, organise reactions and regulate processes such as trafficking and division.

A key feature of the work was an extension called patch-based analysis, which the team used to connect local membrane properties to specific proteins. This component, developed by Ya-Ting ‘Atty’ Chang, a graduate student at the Skaggs Graduate School of Chemical and Biological Sciences – part of Scripps – and co-first author, isolates small circular regions of membrane around defined protein positions. By comparing these ‘patches’ with surrounding membrane regions that lack the protein, researchers can test whether a protein’s presence correlates with distinctive membrane characteristics.

“There are likely many proteins on membrane surfaces that we haven’t discovered yet. Patch-based analysis gives us a way to find them – by scanning for patterns that hint at a protein’s presence before we even know to look for it,” Chang said.

To demonstrate the potential of patch-based analysis, the researchers examined adenosine triphosphate (ATP) synthase, the enzyme complex that helps cells to generate ATP, the energy currency that powers many biological processes. ATP synthase sits within the inner mitochondrial membrane and forms higher-order structures that relate to cristae shape and function. Using their approach, the Scripps team reported that ATP synthase tended to cluster in regions where membranes were both strongly curved and unusually thick, a combination that would have remained difficult to detect with conventional methods that average measurements across large areas.

The ability to measure thickness and curvature together in intact cells could help researchers to probe long-standing questions in membrane biology. One such question concerns cause and effect: whether proteins assemble because a membrane has the right shape and thickness, or whether protein assembly forces the membrane to adopt those properties. In reality, both processes may occur, but tools that quantify physical features at precise sites could help scientists to disentangle the sequence of events.

The authors also suggested that the approach could improve understanding of disease, since many conditions disrupt membrane composition, organelle shape, or protein distribution. Mitochondrial disorders, neurodegenerative disease, viral infection, and metabolic syndromes can all alter membrane behaviour and subtle shifts in thickness may influence how protein complexes function or misfunction. A clearer picture of membrane architecture could therefore help to identify mechanisms that underpin pathology, as well as potential targets for therapeutic intervention.

With Surface Morphometrics and patch-based analysis in place, the team has positioned itself to tackle problems that previously lay beyond reach, including the ability to quantify how membrane properties change over time and how proteins relocate or reorganise across dynamic cellular landscapes. If future work can extend the method across more cell types, disease models and physiological conditions, then scientists may add more items to the list of how physical rules govern cell organisation.

For cell biology, the central significance lies in what the method makes visible. Thickness has long appeared as an abstract parameter in textbooks and model membranes, but this study has brought it into real cells, where molecular crowding, curvature, and protein assemblies make membrane behaviour more biologically meaningful.

For further reading please visit: 10.1101/2025.04.30.651574v1