Complicated CAP Does It All

Cells in the human body are made to move. They assemble during embryo development, migrate to repair tissue, hunt pathogens, and perform a host of other tasks requiring travel. As part of the cytoskeleton, actin filaments help maintain a cell’s shape, but they also help the cell move and divide. They do much of this by treadmilling, a process in which actin monomers are added to one end of the filament and removed from the other end. This pushes on the inner cell membrane and causes the cell to roll in one direction. According to the dogma, these monomers are typically added to just one end of the actin filament, called the barbed end, and removed from the opposite end, called the pointed end. Cyclase-associated protein (CAP), which helps disassemble the filament’s pointed ends, is one of the most important proteins involved in this treadmilling process.

In a new pre-print posted on bioRxiv, researchers at Emory University showed that CAP helps disassemble, or depolymerize, the barbed ends as well.¹ These findings support and extend results from another paper, recently published by a different group in 2023, which demonstrated that the rate of barbed end depolymerization in vitro changed according to the concentration of CAP.² However, the researchers responsible for the previous paper did not directly observe CAP-actin interactions. In the present study, the Emory team observed CAP in action, directly visualizing the protein landing on the filaments’ barbed ends, which then rapidly depolymerized.

“I think together the two papers change what we think is going on at the barbed end,” said Jeffrey Michael Field, a molecular biologist at the University of Pennsylvania who discovered CAP in 1990 and was not involved in this study.³ “What this shows is there’s actually machinery to depolymerize at the barbed end … as if there’s a reverse gear.”

“In a cell, you … break down the old networks so you can form new networks,” said study co-author Shashank Shekhar, a biophysicist at Emory University. “Our question really has been, how does the cell break apart old actin networks?” To investigate the disassembly of these old networks, graduate student Ekram Towsif attached actin filaments to a coverslip with the barbed ends floating free. He ran buffer containing CAP through the system across some actin filaments and pure buffer across the others. He found that the barbed ends of filaments exposed to CAP depolymerized faster than control filaments.

Next, the researchers explored the mechanisms of CAP-mediated depolymerization in greater detail. CAP has two halves that function autonomously, N-terminal (N-CAP) and C-terminal (C-CAP). Typically, N-CAP controls depolymerization at the pointed end, but the team wanted to know whether it also depolymerized the barbed end. They put free barbed ends in solution with either N-CAP, C-CAP, or the full protein and found that C-CAP and full-length CAP increased depolymerization by more than four-fold, while N-CAP did nothing. In addition, because there are two actin-binding domains in the C-terminal end, WASP-Homology 2 (WH2) and CAP and X-linked retinitis pigmentosa 2 protein (CARP), the team expressed peptides of each and found that WH2 was necessary for barbed-end depolymerization while CARP was not, painting a clearer picture of how C-CAP depolymerizes the barbed end.

The team then used single molecule microscopy to watch individual CAPs in action. They visualized labelled CAP and saw that it directly associated with the barbed ends of actin filaments; the filaments with fluorescent CAP depolymerized quickly, whereas those without CAP only slowly depolymerized. “You see that a filament that was depolymerizing slowly immediately starts depolymerizing fast as soon as a CAP molecule is visible at the end,” said Shekhar. This is a step forward from the 2023 paper, where the researchers inferred that CAP caused the depolymerization but did not directly visualize the process.

Finally, Shekhar’s team wanted to know how CAP affected the activity of other actin regulatory proteins such as formin, which helps polymerize the barbed end, and capping protein, which halts filament elongation. The researchers found that CAP reduced the capping protein’s lifetime on the barbed end. In contrast, when CAP colocalized with formin, it increased the length of time that formin remained on the barbed end. However, formin caused filaments to grow at the same rate, whether or not CAP was present.

The formin result differs from the findings of the 2023 paper, which found that CAP promotes formin dissociation from barbed ends. Guillaume Romet-Lemonne, a molecular biologist at Institut Jacques Monod, whose actin dynamics laboratory uses the same techniques as those in this paper but who was not involved in the research, said that understanding why the two research groups had different results is important. “I would first look for tiny details in the buffer, the preparation, and the temperature. There could be minor things like this that could end up having a big effect … It could be that they are both right and there is a difference hidden in the details that turns out to be super interesting. At this stage we just don’t know,” said Romet-Lemonne.

Field emphasized that the preprint needs to be subjected to peer review, and studying this process in vivo is also necessary. “We need to go back in the cell and validate our findings. That’s my next big goal,” said Shekhar.

References

1. Towsif E, Shekhar S. Cyclase-associated protein is a pro-formic anti-capping processive depolymerase of actin barbed and pointed ends. bioRxiv. 2023;2023.11.30.569482.
2. Alimov N, et al. Cyclase-associated protein interacts with actin figment barbed ends to promote depolymerization and forming displacement. J Biol Chem. 2023;299(12):105367.
3. Field J, et al. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990;61(2):319-327.