The cover image of the April 21 issue of Biophysical Journal, which appears as a constellation of bright puncta scattered across the surface of living cells, captures a highly dynamic and essential process known as endocytosis. Endocytosis is the mechanism by which cells internalize nutrients, receptors, and signaling molecules from their environment.
This image was acquired by using confocal fluorescence microscopy, which allows us to selectively visualize specific molecular components within living cells. Multiple fluorescent labels were used to distinguish different features at the plasma membrane. Clathrin-coated pits, which are small, transient membrane invaginations responsible for endocytosis, appear as diffraction-limited fluorescent puncta. Overlaid on these structures is the signal from fluorescently tagged sialic acids, which are negatively charged sugars, on glycoproteins localized at the cell surface.
By combining these fluorescent signals into a single image, we can visualize and quantitatively measure how proteins with and without sialic acid partition into clathrin-coated pits. This image and the research in this article represent our efforts to understand how the biophysical and biochemical properties of proteins influence their behavior at the cell surface. Specifically, we are interested in how glycoproteins, which are proteins decorated with carbohydrate chains, interact with endocytic structures like clathrin-coated pits. Each fluorescent punctum corresponds to an active site of endocytosis, where the cell is constantly sampling its environment and turning over lipids and proteins to maintain membrane homeostasis. Quantifying the overlap between glycoproteins and these sites provides insight into how molecular features such as charge and glycosylation influence clathrin-mediated endocytosis.
Glycosylation plays a central role in many biological processes, and disruptions in this pathway are linked to numerous diseases. For example, many cancer cells, including breast cancer cells, have altered glycosylation and sialylation patterns compared to healthy cells. Understanding how these molecular changes influence endocytosis provides important insight into the mechanisms behind cancer signaling and metastasis. Therapeutically, many biologics are glycosylated, and glycosylation is important in their structure and stability. Understanding how the physical properties of glycan chains affect how molecules enter cells can inform the design of targeted therapeutics that more efficiently reach their intracellular targets. Our work provides a mechanistic foundation toward understanding how glycosylation influences cellular uptake, contributing to these broader biophysical and therapeutic goals. You can learn more about our lab’s recent work at https://www.stachowiaklab.org/.
—Advika Kamatar, Jose A. Villalobos, Jr., Carl C. Hayden, Fabiola G. Rodriguez Flores, Stephanie Archer-Hartmann, Parastoo Azadi, Brian Belardi, Sapun H. Parekh, and Jeanne C. Stachowiak