University of Minnesota
School of Physics & Astronomy

Spotlight

Understanding Cell Membranes

Jonathan Garamella
Jonathan Garamella
Annie Bartels
                                                       

Biological physicists at the University of Minnesota are breaking apart the components of a cell, taking out the molecular machinery responsible for protein synthesis from DNA to reconstruct specific cellular functions such as cytoskeleton-based cell division. The goal of this research is ultimately to create a synthetic cell in a test tube that can divide and evolve on its own.

Jonathan Garamella is a graduate student in Professor Vincent Noireaux’s research lab. Garamella is studying cytoskeleton proteins that are responsible for the cylindrical shape and the division of E. coli. First, Garamella prepares a cell-free expression system by taking the raw components for protein synthesis, squeezed out of E.coli bacteria with a 12,000 psi hydraulic press, to produce proteins from synthetic DNA programs cloned into the laboratory. He encapsulates the reaction into cell-sized spherical liposomes. To image the liposomes, he uses molecular cloning techniques to attach a reporter protein to the cytoskeletal proteins, so that the cell can be seen under a microscope by fluorescence. "We are trying to see if we can deform the liposomes by recapitulating gene expression." Garamella says that the protein responsible for division in E. coli forms little rings at the inner membrane, a constriction ring, which cinches off and splits the membrane into two. While other groups have observed the mechanism itself, Garamella is taking it a step further, starting with the DNA sequence. "Other groups have done these experiments with pure proteins that have been encapsulated. They have induced their synthetic membrane to form different shapes. But it hasn’t been done starting with the DNA."

The group uses E. coli genes, which are readily available from the genome of the bacterium. "E. coli is one of the best understood and documented organisms. Its genome has been entirely sequenced," Garamella says. The group also uses E. coli because of its large repertoire of transcriptional regulatory parts that allows them to form complex biological circuits. "We can use DNA as logic circuits. One DNA sequence will encode one protein and in turn this protein activates another DNA sequence to encode another protein, and from that we can do a genetic cascade." The cascade can end in a functional protein that will be used for division and cell shape, or end in reporter protein that can be used for florescence.

Garamella says that there are a lot of variables to cope with, leading to a fair amount of trial and error. "You look at what works in E.coli, what people have done with just the protein, but introducing DNA in the cell-free expression system changes the outcome." The group is looking at how specific proteins interact with the membrane, because the proteins were evolved by over millions of years and optimized for specific membrane compositions. The challenge is to mimic these outcomes in the laboratory. "We spent quite a bit of time cloning plasmid vectors (circular sequence of DNA) used in cell division. If there is linear DNA introduced to bacteria, they will destroy it as a defense mechanism to protect against viruses."

The long-term goal of the lab is to recapitulate complex biological functions into synthetic liposomes with the minimal cell as the ultimate goal —a cell created in a test tube able to divide and show some kind of selective evolution. "We utilize a bottom up approach. In one experiment I’m trying to break this down just for membrane proteins. In another experiment I’m trying to look at just these proteins that enable division. I’m trying to break it down systematically, to see if they can work separately and, later, in concert." These systems have such an expansive parameter space that a lot of the effort is spent trying to understand how these mechanisms behave in vitro.