How was the double membrane structure of cells discovered?

Author: Chinese Biophysics Society

Reviewer: Ruan Ke, Professor at the University of Science and Technology of China.

Produced by: Science Popularization Department, China Association for Science and Technology

Producer: China Science and Technology Publishing House Co., Ltd., Beijing Zhongke Xinghe Cultural Media Co., Ltd.

Source: Science Popularization China Creation and Cultivation Program

This is a very interesting question. In fact, it took a long time to arrive at the correct answer. The composition of the entire biological membrane involves understanding the types of biomolecules present, how these biomolecules are organized to construct the membrane, and how this organization allows for functional diversity.

In the 19th century, although microscopes already existed, it was still unclear whether cells had a membrane on their surface. This uncertainty was a matter of debate for a long time. Many scientists believed that even when cells were stained, it remained uncertain whether there was a barrier distinguishing the internal and external environments of the cell.

Indeed, we can refer to a well-known experiment in botanical research called plasmolysis. During the plasmolysis experiment, plant cells, which possess a rigid cell wall made of polysaccharides, act as a tough barrier. Inside this cell wall, however, plant cells have a more flexible cell membrane. When we alter the osmotic pressure around these plant cells, they dehydrate rapidly, causing a significant shrinkage of the cell. One direct effect of this shrinkage is that the plasma membrane collapses away from the cell wall, a phenomenon we refer to as plasmolysis. This experiment provides intuitive evidence for the existence of a layer on the cell surface, visible through simple light microscopy. When plasmolysis occurs, the plasma membrane can be clearly observed, marking it as one of the earliest pieces of evidence supporting the concept of a cellular membrane.

The phenomenon of plasmolysis was formally reported by scientists in the 1860s. The first scientist we will discuss is Overton, who utilized the plasmolysis phenomenon in 1895, discovering that the plasma membrane is comprised of lipid molecules.

Next, we have Langmuir, who developed a technique called membrane balance. This method allows for the extraction of lipid molecules using organic solvents, enabling these lipid molecules to spread in a monolayer on the water surface. By adjusting the baffle of the water tank, the area covered by the lipid molecules can be calculated. At that time, the chemical structures of lipid molecules were already known, meaning that the projected area of lipid molecules had specific parameters. When all lipid molecules spread on the water surface, they form a measurable film. By dividing the area of this film by the projected area of each lipid molecule, one can roughly estimate the total amount of lipid molecules within that film.

Why do we emphasize the two-dimensional projection of lipid molecules? This is because lipid molecules are amphiphilic, possessing a hydrophilic head and a hydrophobic tail. When these amphiphilic molecules are dispersed on the water's surface, they orient themselves according to the principle of like dissolves like, with the hydrophilic head in contact with water while the hydrophobic tail is somewhat repelled, standing upright.

In this orientation, the projected area on the water surface corresponds to the area occupied by each of its molecules. This method does not directly answer whether biological membranes are monolayers or bilayers, but it builds the groundwork for later scientists to address this question. This underscores the crucial role new technologies, methods, or equipment play in advancing scientific research.

The third key scientists, Gert and Grendel, were two Dutch researchers who used red blood cells for their studies. The advantage of using red blood cells is that mature red blood cells lack internal organelles, having lost them during maturation and leaving only a cell membrane. Thus, the visible biological membrane area reflects the total area of the red blood cell's membrane. By estimating the surface area of red blood cells and employing organic solvents to extract the lipid molecules, they utilized membrane balance techniques to determine the area occupied by these lipid molecules when they spread as a monolayer on water. They arrived at a surprising ratio of two.

What does this "two" signify? It indicates that if all lipid molecules were arranged into a single layer, they would cover an area twice that of a red blood cell's surface area. Therefore, we can deduce that the lipid molecules forming the red blood cell membrane are organized in a bilayer arrangement. This clever method allowed scientists to infer that lipid membranes are bilayer-structured, even before lipid molecules were visible through microscopy.

Furthermore, an indirect method was also developed to assess whether the biomembrane consists of a single layer or two layers of lipid molecules using an electrophysiological technique. As early as 1925, scientists devised an electrical method similar to the patch-clamp principle to measure the capacitance of the red blood cell membrane. They estimated the thickness of the membrane to be approximately 3.5 nanometers.

For those familiar with lipid molecular structures, when two lipid molecules are stacked back-to-back, their thickness measures precisely 3.5 nanometers. This indicates that earlier electrophysiological measurements were accurate. However, there was still speculation that there might be three layers or more, and as a result, the thickness of a single lipid molecule was not factored into the calculations confirming the bilayer structure. While Gorter and Grendel obtained relatively accurate results in 1925, this issue remained a topic of debate among scientists until later advancements in electron microscopy allowed for direct observation of the true bilayer of lipid molecules, gradually settling the related disputes.

This article is supported under the Science Popularization China Creative Cultivation Program.