New Method Improves Quantification of Endocytosis in Living Cells

Living cells and tissues show a high capacity to absorb various chemical compounds from the surrounding environment. Although the absorption of small molecules is quite simple for cells and tissues, it becomes more problematic to absorb larger molecules. In order to do this, cells have to use certain methods of transport, and one of them is endocytosis.

The larger the molecule, the more difficult it is not only to absorb but also to transport it within the cell. This makes the delivery of large molecules even of biological importance like drugs a research challenge. In addition to discovering new and effective methods to deliver specific compounds into the cell, researchers also look for new methods to quantify this process, i.e., to provide parameters such as the number of molecules entering the cell and their time of entry. Recently, researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences led by prof. Robert Hołyst showed a novel approach in the quantitative method to describe the endocytosis process using the example of transferrin uptake. Let's take a closer look at their findings.

Each cell is separated from its environment by a barrier called a cell membrane that protects the cell from the outside environment. However, this barrier can be overcome by certain chemicals that sustain the basic functions of the cell. The movement of substances across the membrane into or out of the cell is called cellular transport, although highly efficient, it sometimes requires an additional factor such as a protein that is a receptor for a specific chemical serving as a "named ticket to entry" into the cell. One can call its endocytosis that is a process of absorption of molecules, or chemical compounds, by a cell. In other words, in this process, macromolecules can enter cells only by being captured and enclosed within membrane-bound carriers. Cells use many different mechanisms for endocytosis. In clathrin-mediated endocytosis, cell surface receptors selectively bind macromolecules that concentrate in coated pits on the surface. Next, they pinch off to form clathrin-coated vesicles that carry the cargo into the cell interior. That way, the nutrients uptake as well as cell signaling takes place. How does it work?

In human cells, a single protein can exist in numbers ranging from several to even tens of millions of copies per cell. This makes observing such an intricate and challenging process, and in order to observe mechanisms involving several proteins, one needs measurement methods sensitive enough to be able to detect single molecules. The standard method for observing molecules inside a cell is imaging by fluorescence microscopy. This method is widely used and extremely helpful, but it only allows observations like a binary system - whether something is inside a cell or not, and only after a certain limit of molecules has been exceeded, without determining how many of these objects there are.

The method developed by researchers at the Institute of Physical Chemistry Polish Academy of Sciences study led by prof. Robert Hołyst addresses these limitations. The researchers use detection based on counting single photons - the basic unit that light is made of - we can see individual particles and count them. Scientists calibrated their devices to prepare measurements of molecular brightness and to eliminate the light from the background. From this measurement, they understand how many photons emit from one molecule.

They used transferrin in their research. Why was this particular molecule used in the study? Transferrin is a glycoprotein that reversibly binds iron and is responsible for its transport in the human body. For such a cargo role in iron delivery, transferrin has been extensively studied by different approaches, and thus results obtained with the presented methodology could be compared to existing literature data. Endocytosis is a multistep process engaging dozens of different proteins to work together in a highly coordinated manner to drive endocytic vesicle formation. Given the complexity of this process, how long does it take? Does it take seconds or minutes? Additionally, why does counting time matter? Endocytosis, apart from providing a mechanism for cells to take up nutrients, is the principal route of entry for many drugs. Therefore, understanding of endocytosis by determining, among others, time resolution, can benefit the development of different therapeutic strategies.

Dr. Marta Pilz, the first author of the work says "Our study demonstrated that the quantitative analysis of fluorescence images with single-molecule sensitivity is a simple and efficient tool ideally suited for studying the endocytosis of fluorescent molecules. We therefore expect that quantitative imaging will play an important role in assessing the efficiency of drug delivery."

Researchers, as a method, prepared unique software to analyze fluorescence images with the possibility of counting photons emitted from individual molecules. Applying that system, let's measure the lower transferrin concentration inside the cell. Thanks to an innovative combination of software and detection system, it was possible to measure 100 times lower concentration outside the cell than previously used methods. Thus, they can easily say how transferrin accumulates in the cell and whether it depends on outside concentration.

The transferrin cycle time – the time from transferrin entry to cell exit – was determined by measuring changes in cell transferrin concentration over time. Researchers also observed that the cycle time was not dependent on the concentration of transferrin outside the cell. However, the transferrin concentration inside cells was dependent on the transferrin concentration in the medium. Once a certain external concentration is exceeded, the value inside does not increase. And this is because transferrin needs the previously mentioned "ticket", which is a unique receptor for input, the number of which is limited on the cell surface. The research of scientists from the IPC PAS shows that the amount of transferrin receptors can be easily determined. Moreover, from the dependency of transferrin concentration inside and outside cells, researchers developed a mathematical formula that resolved times of different steps of the endocytosis process. The study showed that the amount of time transferrin spends outside the cell is determined by the search time of the free receptor on the surface, internalization time, plus the external transferrin concentration. At a sufficiently high transferrin concentration, most of the surface receptors are occupied, so transferrin has to wait for a free one that returns back to the cell surface only after the cycle is completed.

Why is the method proposed by researchers from ICP PAS important? The developed method, along with its demonstration on a specific example - transport of transferrin shows the possibility of its potential application to other biological problems, for example, to study the transport of molecules with therapeutic properties, including the drugs. The methodology opens up the possibility of quantitative evaluation of the efficiency of delivery of any fluorescent molecules into living cells. That is, we can say not only whether something is present in the cells, but also how fast it enters and leaves them and in what amount.

"The results presented here show that an integrated approach addressing the challenges of uptake measurements enables the quantification of endocytosis in living cells. With quantitative fluorescence imaging and the resulting matrix of photon numbers, concentrations in any ROI (region of interest) can be determined. Consequently, the approach enables parallel concentration measurements inside and outside the cell as well as tracking their changes over time, as illustrated here by the study of transferrin uptake kinetics to extract transferrin cycle time" – remarks dr. Marta Pilz.

The proposed method has a lower limit of detection to compare with the existing methods. In addition, the procedure is safe for cells, background elimination is achievable, and measurements can be taken in real time (during the experiment).

This research published in the Talanta under support of Polish Science Fund, Poland within the framework of the Virtual Research Institute; grant WIB-1/2020-O11 - WIB_HERO.