Shedding light on a decades-old protein sorting mystery

Christian de Caestecker, Ph.D. student in the laboratory of Ian Macara, the Louise B. McGavock Professor and chair of the Department of Cellular and Developmental Biology, proposed and validated a mechanism that addresses a decades-old mystery surrounding epithelial cells. Caestecker’s research, published in Natural cellular biologyhighlights the process by which epithelial cells, polarized cells that face the outside world, sort and deliver the specialized proteins they need to the top (outermost) surface of each cell.

Epithelial cells are organized like boxes, with tops and sides called apical and lateral surfaces. To properly perform their functions, epithelial cells must precisely sort proteins on each surface, and defects in the delivery of these proteins may play a role in several human diseases such as cancers.

“Most human cancers are of epithelial origin and have defects in the distribution of polarized membrane proteins,” Macara said. “How childbirth occurs all the way to the upper, or apical, surface has remained a mystery for decades, but Christian’s genius and hard work have led to an important breakthrough.”

Freshly synthesized proteins are sent to the Golgi for further processing and to be sorted to their final destination via a process analogous to the postal service. The distribution along the sides of the cell is well understood, due to the presence of “zip codes” in proteins that tell the cell which path the protein should take to the cell surface.

Apical proteins do not have equivalent zip codes, so the apical sorting process has remained more elusive. Whereas many apical membrane proteins have very short, if any, cytoplasmic domains, meaning that most of their mass resides inside or outside the plasma membrane and not inside the cell, de Caestecker hypothesized that proteins destined to reach the apical membrane are sorted. to the Golgi by the physical size of the cytoplasmic domains.

To test this idea, he looked at three representative apical proteins with short cytoplasmic tails and used synthetic biology approaches to change the size of their tails and observe how this affected sorting. de Caestecker found that when their cytoplasmic tails were elongated, proteins experienced a significant delay in their processing and exit from the Golgi and were misrouted to the sides of the cell rather than the top.

Additionally, using super-resolution microscopy from the Cellular Imaging Shared Resource, they observed the segregation of small and large cargos to distinct regions of the Golgi during trafficking, suggesting that a large cytoplasmic tail blocks the access to specialized areas of the Golgi that could be involved. in deliveries to the apical membrane.

de Caestecker also tested whether apical proteins that have binding partners are sorted individually or bound and found that although the proteins and their binding partners co-traffic to the Golgi, they dissociate before being trafficked to the apical membrane. These results support the hypothesis that the Golgi uses a size filter to determine whether a protein should be trafficked to the apical membrane or not.

“How cells sort and deliver polarized proteins to their appropriate domains is a fundamental question in cell biology,” de Caestecker said. “We first need to understand this process in normal cells so we can identify how it is disrupted in diseases such as cancers.”