A new method for looking through the (cyto)skeletons in the closet
Rachel Dvorak was the lead author on this study. She is interested in studying the biochemical mechanisms by which mutations in gamma smooth muscle actin cause visceral myopathy. Patients with visceral myopathy present with severe abdominal distension, intractable constipation, feeding intolerance, and growth delays. Because there are no targeted therapies for this disease, many patients die in adolescence. She hopes that by understanding how disease-causing mutations alter actin biochemistry we can develop treatments for patients with visceral myopathy and other rare conditions caused by actin mutations.
or technically,
A solution to the long-standing problem of actin expression and purification
[See Original Abstract on Pubmed]
Authors of the study: Rachel H. Ceron, Peter J. Carman, Grzegorz Rebowski, Malgorzata Boczkowska, Robert O. Heuckeroth, and Roberto Dominguez
Take a moment to picture the last evening you spent with friends. Now, think about something that happened over 10 years ago.
Throughout both of those events- in fact, throughout every day of your life- you’ve had the same exact cells living in your brain, helping you navigate each situation. These cells are called neurons, and your body can’t replace them; when they’re gone, they’re gone for good. This means it’s especially important for neurons to be well-built to withstand the everyday challenges that cells face, and well-equipped to respond to emergencies that could threaten their existence.
One contributor to neurons’ ability to last a lifetime lies in how they’re built; much like buildings that are constantly exposed to the elements, neurons need structural integrity to last a lifetime. To do this, there are several types of miniature “skeletons” that exist within the cell. Making up these miniature cellular skeletons, collectively called the cytoskeleton, are proteins. Proteins are large molecules that perform specific jobs intended to keep cells alive. One type of protein is called actin. Similar to links in a chain, actin proteins can attach to each other to form a long structure called a filament. As part of the cytoskeleton, actin filaments are important for helping neurons and many other types of cells keep their shape. Correspondingly, neurons are only as structurally sound as their components; issues within actin filament structures cause severe defects in overall brain structure. This includes lissencephaly, a condition where the brain lacks the grooves that normally sprawl across its surface, leaving it completely smooth. By understanding how actin behaves, both on its own and in the presence of other proteins, scientists hope to develop better treatments for diseases where actin isn’t acting the way it normally would.
Rachel Dvorak, a PhD candidate in Dr. Roberto Dominguez’s lab at the University of Pennsylvania, wanted to develop a robust way to study how actin works. Specifically, she was interested in studying how actin interacts with itself and other proteins- much like people, proteins can interact with each other in ways that influence their behavior in the cell. To do this, she aimed to isolate actin from cells. Isolating a single type of protein is a common method to study the way it functions. The inside of a cell is a bustling place, and it can be hard to tease out the specific interactions that proteins have with one another when they exist within that cellular environment.
This is far from a simple undertaking. Inside of an actual cell, there are many actin proteins, and they are not all identical. Instead, there are several possible varieties of actin. One way to understand this is by comparing it to different flavors of ice cream. Although chocolate and vanilla are made of slightly different ingredients and paired with different foods, they are both still ice cream. Actin also comes in different “flavors” called isoforms. While each of their structures are slightly different, and each isoform might be best suited for use in different scenarios, they are all still considered actin due to their overall similarities and jobs in the cell. Moreover, even two actin proteins that are the same isoform can be slightly different, because the cell can modify actin by attaching other molecules to it. These attachments are called post-translational modifications, and they also influence actin’s behavior. However, despite their important effect on actin behavior, many of these post-translational modifications are usually lost during the process of isolating actin to study its behavior.
Another challenge of isolating specific isoforms of actin lies in getting cells to produce large quantities of the actin isoform of interest. Making actin is an involved, multi-step process for the cell that requires a lot of molecular machinery. Because of this, most scientists use only one type of actin isolated from muscle cells to conduct experiments outside of the cellular environment. This severely limits our ability to study whether different isoforms of actin behave in slightly different ways. It also means that oftentimes, our out-of-the-cell reconstructions of cellular events are created using actin filaments that lack much of the nuance they would have in cells. This limits the accuracy of this model and makes it impossible to study how actin that is incorrectly produced causes human disease.
To tackle this, Rachel decided to use modified human kidney cells to produce actin. This meant that the cells would already contain the machinery unique to humans that is necessary to carry out production of actin. This is in contrast with existing methods that use cells like bacteria or insect cells; these cell types can also be used to churn out proteins for isolation, but lack the machinery to make some proteins native to human cells or add post-translational modifications the way that human cells would. Rachel was able to introduce genetic instructions that caused the human kidney cells she worked with to essentially become actin-making factories, synthesizing large quantities of whichever actin isoform she was interested in.
However, the actin the kidney cells made based on the genetic instructions that Rachel provided was a bit different than any other actin in the cells; each of these actin proteins was also attached to two other man-made proteins called tags. Tags are helpful because scientists have materials that can grab onto them, allowing for the separation of the tags (and anything attached to them) from everything else in the cell. Most tags don’t occur naturally, so only the protein (or, in this case, the actin isoform) that you’ve instructed the cell to produce will have this unique feature. To further ensure she was isolating only the type of actin she wanted, Rachel also used a different protein her lab had engineered to grab and release actin based on how much calcium is in the environment. By using a combination of materials that grab the tags attached to actin in tandem with the protein that grabs onto actin itself, Rachel was able to isolate specific isoforms of actin with post-translational modifications from the rest of the contents of the kidney cells.
Rachel and her colleagues ultimately invented a new method for isolating actin exactly as it would exist in the cell. This is important because it means scientists now have a new way to study the exact things that are going awry in diseases that involve issues with interactions between actin and other proteins, such as the one that causes lissencephaly. The more we understand about how actin functions differently in diseases like this, the better our ability to develop effective treatments.
About the brief writer: Julia Riley
Julia is a PhD candidate in Dr. Erika Holzbaur’s lab studying the consequences that damaged mitochondria (the powerhouses of the cell) have on the function of astrocytes, a cell type found in the brain. This is important for understanding diseases like Parkinson’s, where we know mitochondrial damage occurs but don’t fully understand how it impacts the health of brain cells.