Cell biology meets engineering in collaborative XSeed grant for Professor Sergey Plotnikov to study wound closure in fruit flies

CSB Professor Sergey Plotnikov has been awarded XSeed funding to identify protein targets that can accelerate wound healing.  The XSeed program catalyzes cross-disciplinary partnerships between investigators from the Faculty of Applied Science and Engineering and other faculties.  

The grant was awarded in collaboration with Professor Fernandez-Gonzalez from the Institute of Biomaterials and Biomedical Engineering. Plotnikov and Fernandez-Gonzalez will leverage their expertise in wound repair and cell mechanics to probe the mechanisms of cell movement to the site of wounding.

Animal cells are embedded in an extracellular matrix (ECM) and protein interactions through the ECM allow cells to maintain their shape and structure. Cells adhere and migrate through the ECM using multi-protein structures called focal adhesions. Plotnikov and Fernandez-Gonzalez will examine the dynamics of cell-ECM adhesions after wounding.

They will investigate how talin, a core protein in focal adhesions, allows cells to move to the site of an injury, thereby closing the wound. Wound closure in talin mutants with either weaker or stronger ECM binding will be compared to normal talin. Wounds will be induced through laser nanosurgery, and the accumulation of talin and the assembly of focal adhesions at the injured site will be recorded using cutting-edge spinning disc confocal microscopy. The results of this study will establish whether talin-based cell-ECM adhesions that allow cells to move are required for wound healing.

Cell movement in response to wounding is regulated using transient receptor protein (TRP) ion channels. These channels respond to mechanical stresses exerted on cells by altering cellular calcium ion levels. TRP-mediated calcium signaling controls cell-ECM adhesions to regulate cell movement. Plotnikov and Fernandez-Gonzalez will suppress different components of the calcium signaling pathway to investigate how calcium signaling controls the assembly of focal adhesions and the movement of cells during wound closure.

Adult wounds heal much slower than embryonic wounds and often result in scarring and infection. By studying cell-ECM adhesions and wound closure in fruit fly embryos, Plotnikov and Fernandez-Gonzalez will be able to identify components of the protein pathway that can be modified with the goal of accelerating wound healing in adults.

Parasitic plant biology study by Professor Shelley Lumba reveals unexpected pathway to germination of witchweed seeds

The crop fields of sub-Saharan Africa have fallen under a spell cast by the witchweed Striga hermonthica. Every year, this parasitic plant targets and destroys over eight billion dollars worth of staple crops, leading the UN to declare Striga infestations as a major impediment to poverty alleviation in Africa. A new discovery from researchers at U of T could provide tools to prevent Striga seeds from germinating in farmers’ fields.

Striga’s success comes from its ability to lie dormant in the soil for years, lying in wait for a crop host to infect. When a host plant begins to grow, it emits a small molecule called strigolactone into the soil.  This chemical acts as a cue for Striga to germinate, telling the parasite that a host is nearby.

Due to its parasitic nature, studying Striga in the laboratory is challenging. Plant scientists use the model plant Arabidopsis thaliana to study germination but unlike Striga, Arabidopsis does not germinate in response to strigolactone. Professor Shelley Lumba’s lab in the Department of Cell & Systems Biology (CSB) decided to learn more about the action of strigolactone on germination by expressing Striga’s strigolactone receptor protein in Arabidopsis

As the results came in, excited discussions arose in the hallways of CSB and at the coffee stand, provoked by the surprising revelation that strigolactone could trigger germination independent of Arabidopsis’ dominant germination pathway.

Normally Arabidopsis seeds (and crop seeds) don’t germinate in storage because of the activity of a group of proteins called DELLA domain repressors. When these seeds sense enough water and warmth, their cells produce a hormone known as gibberellin that enables breakdown of DELLA proteins, kickstarting the process of germination. Lumba found that activated Striga receptors in Arabidopsis circumvented gibberellin to cause germination.

The signal that bypasses gibberellin takes an unexpected path from the strigolactone receptor through the cellular KAI2 protein, which had no previously known receptor partner. The KAI2 signaling pathway inactivates the SMAX1 protein to allow germination even in the presence of DELLA repressors. These details, revealing how Striga strigolactone receptors act as the dominant signaling component in parasitic plant germination, will refine the methods used to prevent the growth of these devastating weeds.

The approach used by the Lumba lab shows that receptors for known hormones can be adopted to analyze orphan receptors, which may have broader utility in understanding hormone signaling in both plants and animals. These results were published in a recent Nature Plants article: “SMAX1-dependent seed germination bypasses GA signalling in Arabidopsis and Striga“.

Sensing light synthetically yields insight into retinal disease

“Can we make yeast see?” seems an odd question for biologists to ask, but students in the Department of Cell & Systems Biology (CSB) have used synthetic biology to make yeast cells that can detect light.

Professor Belinda Chang studies the cells which respond to light in the retina and send that response as a signal to the brain. Her lab focuses on the light-sensitive rhodopsin protein molecule. She and her graduate students look at how these molecules work in species as diverse as mice, snakes, and bats, and why they stop working in humans who are losing their sight.

One day over a pizza lunch, the Chang lab graduate students discussed how they were each focused on a different species, which made it hard to compare results between projects. Making rhodopsin protein and studying its effect on other proteins in response to light were also very labour intensive experiments. The students decided to invent a system to test many types of rhodopsin in the same organism, rapidly and accurately.

Professor Chang supervised their project and the students produced their rhodopsin proteins in cells of brewers’ yeast. This synthetic biology approach took advantage of the fact that yeast cells can sense nearby yeast through a protein molecule known as a GPCR. GPCR molecules are found in organisms from yeast to mammals, and rhodopsin is also a GPCR. After replacing the yeast GPCR with human rhodopsin, shining light on the engineered yeast turned on a synthetic gene that made the yeast fluorescent.

This techniques was used to address a practical problem: patients who are losing their vision may have mutations in rhodopsin, but the severity of future vision loss can’t be judged based on mutation information alone.

Led by graduate student Ben Scott, this technique determined that variants of rhodopsin found in people afflicted with retinitis pigmentosa were less responsive to light in yeast, and that more clinically severe variants had even less of a response. Sergei Plotnikov’s lab provided support in characterizing the appearance of mutant rhodopsin, which enhanced the diagnostic value of the yeast system. This exciting result was published in the peer-reviewed journal Genetics as “Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease.

This work formed part of Ben Scott’s PhD in Chang’s lab at CSB. “Synthetic biology approaches can create new tools to help answer fundamental biological questions,” says Scott. “That’s really where this work fits in. But synthetic biology is a research field in its own right, working to create entirely new biological pathways and systems to solve problems in medicine and industry.“

This paper was recently awarded the Editors’ Choice Award for outstanding Molecular Genetics articles published in Genetics in 2019.