Chemistry graduate student Yun Zhang (Salaita Group) received an award for “Best Poster” at the 2015 Gordon Research Conference “Fibronectin, Integrins & Related Molecules.” The conference, a key scientific meeting for those interested in integrins and the interactions between cells with the extracellular matrix in development, homeostasis and disease, took place February 10th – 15th at the Ventura Beach Marriott, Ventura, California.
Zhang’s poster was presented as part of the session “”Mechanobiology and Matrix Dynamics.” The full abstract for the poster appears below:
Mapping integrin forces with high spatial and temporal resolution during platelet activation
Yun Zhang, Yongzhi Qui, Wilbur Lam, Khalid Salaita
Platelets play an essential role in hemostatic response by forming blood clots that seal the damaged vessel sites and promote vascular healing. Through the coordination of physical-chemical interaction between integrin aIIbb3, actin, and myosin, platelets adhere and generate forces to strengthen their adhesion to the exposed extracellular matrix and fibrin mesh. Recent work suggests that platelets sense the stiffness of their extracellular matrix and respond by tuning their activation levels. Nonetheless, a fundamental question pertaining to the role of mechanics in platelet activation is whether the aIIbb3 integrins experience mechanical tension during activation and its precise magnitude, and spatial and temporal distribution during platelet activation. To address this question, we first immobilized integrin ligands onto a supported lipid membrane and found that laterally mobility markedly dampens the activity of ligands for aIIbb3 integrins. Next, we developed DNA-based fluorescence tension probes to visualize piconewton scale forces during platelet activation cascades. These probes provided the highest resolution force maps associated with platelet activation to date. Interestingly, we found that platelets exert force through filopodia nearly instantaneously when activated by contact with the sensor surface. As filopodia coalesced to form circumferential lamellae, the tension was greatly increased and specifically accumulated at the cell periphery and the center of the cell area. The tension at the center of the platelet was highly influenced by myosin contractility. Finally, By comparing the tension signal associated with a small library of integrin ligands that bind aIIbb3, we found that platelet-generated forces are highly dependent on the chemical identify of the ligands. For example, cyclic RGD ligands experience over 19 pN of tension, whereas the HHLGGAKQAGD ligand experience values of tension greater than 2.4 pN but lower than 4.2 pN. Taken together, this data demonstrates that mechanical forces play a critical role in platelet activation and newly emerging molecular tension probes provide a power approach to elucidate the role of integrin tension in platelet activation. This may potentially guide new hemostatic or antithrombotic treatments.
The Department of Chemistry is pleased to congratulate Dr. Omar Villanueva on successfully defending his thesis, “Design and Development of Novel Bis(amidophenyl)amine Redox-active Ligands to Promote Novel Reactivity at First-row Transition Metal Centers.”
The Drefyus award supports the research and teaching careers of talented young faculty in the chemical sciences. According to the Dreyfus Foundation, they seek “Teacher-Scholars who demonstrate leadership in research and education. Nominations must provide compelling evidence of the advance of important knowledge in the chemical sciences by the nominee.” The award provides a $75,000 unrestricted research grant, $7,500 of which is allocated towards departmental expenses associated with research and education.
The NSF Early CAREER Award is the organization’s most prestigious award in support of junior faculty. The award recognizes “Faculty who exemplify the role of teacher-scholars through outstanding research, excellent education and the integration of education and research within the context of the mission of their organizations.”
The Emory University Department of Chemistry is pleased to recognize Dr. Salaita’s achievements as an accomplished researcher and an engaged member of the Emory community.
The Atlanta Area Molecular and Cellular Biophysics Symposium was held at Emory University on December 7th, 2013. This networking event brought together graduate students, postdocs, and faculty from the greater Atlanta area with interests in biophysics, biological chemistry, bioengineering, and cell and molecular biology. Khalid Salaita, Laura Finzi (Emory physics), and JC Gumbart (GATech physics) organized the symposium.
Last spring, Assistant Professor Khalid Salaita‘s lab was awarded a grant from the National Institute of General Medical Science (part of the NIH) to study the Notch signaling pathway and develop techniques to look at the forces applied at the interface of cell membranes. Originally named for its role in the formation of notched Drosophila wings, the Notch receptor play a crucial role in cell to cell communication, cell development and differentiation. Mutations in this transmembrane protein result in dysfunction of the entire pathway, which can lead to various types of cancers including T-cell acute lymphoblastic leukemia (T-ALL). Because Notch is so crucial in cell differentiation, having too much or too little present in the membrane can also cause tumor growth.
Notch’s ligand-binding domain exists outside the cell membrane and when it locates a ligand molecule on the surface of an adjacent cell they bind, and the signal pathway begins. Once the ligand is bound, a protease comes in and snips off the extracellular domain from the transmembrane domain. However, the site that gets cut is buried within the folded protein, suggesting there must be a conformational change to allow access to the site. The Salaita Lab hypothesizes this conformational change occurs via a mechanical force; the cell pulling back on Notch, exposing the cleavage site. Their lab is working on tagging ligands with chromophores and quenchers so they can use fluorescence to see the protein stretching as it is being pulled by the cell. By calibrating the fluorescence of a given chromophore/quencher pair to the amount of force being applied to stretch them apart, they can quantitatively look at mechanical force exerted by the cell.
The Notch receptor with a green fluorescent protein tag on the intracellular domain is overexpressed in mammalian cells and seeded onto a membrane surface functionalized with the ligand (DLL4-mCherry). The two proteins bind and the extracellular domain gets snipped, the domain inside the cell can then be cut and act as a transcription factor, starting the signaling pathway. (Fig. 1)
By labeling the ligand and Notch with fluorescence tags they can not only establish that the two are binding based on the overlap (Fig 2), but by studying the intensity of the fluorescence, they can determine the density of molecules at the interface and their binding stoichiometry.
Unlike other membrane proteins that have been more heavily studied, only parts of the Notch structure are known by either NMR or X-ray crystallography, which makes it even more difficult to work with. The Salaita lab is up for the challenge though: “Having a five year grant allows you to take a breath and really dive in to solving some hard problems,” said Salaita.
Notch receptor mechanotransduction could be just the tip of the iceberg, there could be thousands of other receptors where a similar mechanism could be in play. Developing the methodology to explore these forces will open up new avenues for understanding and ultimately controlling membrane proteins and the diseases to which they contribute.
Graduate student Yoshie Narui (Salaita Group) attended the 63rd Lindau Nobel Laureates Meeting in Germany. Once every year, 30–40 Nobel Laureates convene at Lindau to meet the next generation of leading scientists: undergraduates, PhD students, and post-doc researchers from all over the world. The Lindau Nobel Laureate Meetings foster the exchange among scientists of different generations, cultures, and disciplines.