Investigators from Baylor College of Medicine and Rice University have made an important discovery that can potentially help in understanding the pathogenesis of different diseases and cancers. Structural biologists have now been able to capture the first 3-dimensional crystalline snapshot of actin formation in the cell.
Actin filaments are vital for the cellular functioning within the human body, such as for the maintenance of cell shape, cell division, muscle contraction and other vital activities. The actin formation takes place hundreds of times a second as a result of assembling, tearing and reassembling of actin monomers, or G-actin.
F-Actin was first discovered in 1887, and since then over 18,000 research studies have been published to explain various functional and structural aspects of filamentous assembly of actin; however, little was known about the monomeric assembly of G- actin (monomeric building blocks) to form F- actin (nucleation product). This assembly known as nucleation is the basis of all cellular activities and vital functions.
In the words of BCM biochemist and study co-author Qinghua Wang:
“Nucleation is critical for this continual building and rebuilding. For healthy cells, nucleation is the starting place for robust shape. For unhealthy cells, like cancer, nucleation processes may play a crucial role in unregulated growth. That’s one reason we want to better understand nucleation.”
The study appeared in the peer-reviewed scientific journal Cell Reports. The co authors of the study, Professor Jianpeng Ma of Rice University’s bioengineering department and Dr. Lodwick T. Bolin, Professor of Biochemistry at BCM, commented:
“One of the major distinctions between cancerous cells and healthy cells is their shape. There is a correlation between healthy shape and well-regulated cell growth, and cancer cells are often ugly and ill-shaped compared to healthy cells.”
Double Mutant Strategy:
The researchers from Rice University and Baylor College of Medicine — Ma, Wang and Xiaorui Chen — used x-ray crystallography to study the structure of the actin nucleus. After a few failed initial attempts, the team created two mutant versions of G-actin that was incapable of polymerizing but can nevertheless nucleate.
Researchers learned that actin monomers bind with neighboring monomers in end-end and top-bottom fashion to create polymers. In order to stop the polymerization process without interrupting nucleation, the researchers created a mutant version of G–actin that allows the monomers to bind either on top or on bottom with neighboring monomers and not both.
“This dual-mutant strategy was the key. After that, we had to overcome problems related to forming and growing the crystal samples needed for crystallography.”
Chen then prepared the crystals by using super saturation in order to spur initial crystal formation, and with the help of a seeding processes, transferred the crystals in another medium for better examination. The crystals were then analyzed by x-ray diffraction to reveal “nucleated, dual-mutant pair.”
This process is the basis of actin nucleation and researchers were able to capture for the first time how actin nucleation begins.