Cells & Protocells
The Hammer laboratory develops quantitative tools to analyze or mimic biology. We have particular interest in the role of cell adhesion and motility in the immune response. Also, we are interested in developing synthetic tools to mimic biology, such as making artificial cells (protocells) from synthetic, tunable materials.
After leukocytes dock at inflammatory sites, they crawl to their targets. These amoeboid cells crawl quickly but exert small forces. We have used traction force microscopy (TFM) and micropost arrays, combined with microfluidic gradient chambers, to image the forces that leukocytes exert during chemotaxis and chemokinesis. We have found that neutrophils exert their largest stresses in the rear while dendritic cells exert pulling forces from the front. We are now exploring how the internal molecular machinery of these cells is related to the force generation using knock-out mice and engineered cell lines.
For an example of papers in this area, see:
Brendon G. Ricart, Michael T. Yang, Christopher A. Hunter, Christopher S. Chen, and Daniel A. Hammer, (2011) ”Measuring Traction Forces of Motile Dendritic Cells on Micropost Arrays”, Biophysical Journal; 101(1):2620-2628; http://dx.doi.org/10.1016/j.bpj.2011.09.022
Henry, Steven J., John C. Crocker and D.A. Hammer (2014). “Ligand density elicits a phenotypic switch in human neutrophils,”Integrative Biology 6; 348-356; http://dx.doi.org/10.1039/C3IB40225H
UPSTREAM MIGRATION OF LEUKOCYTES
It was recently discovered by a laboratory at University of Marseille (O. Théodoly) that T-lymphocytes can crawl upstream against the direction of flow. Upstream migration is mediated by the binding between the integrin LFA-1 and it’s cognate ligand, Mac-1. We have been studying this fascinating phenomenon. We found that T-lymphocytes can crawl upstream with only a small amount LFA-1/ICAM-1 interactions. We also found that T-lymphocytes can persist in the direction of flow after flow is turned off, but only if both LFA-1 and VLA-4 are both engaged, suggesting integrin crostalk is key for migrational memory.
We have also shown that other cell types can crawl upstream, including hematopoeitic stem cells and neutrophils. In the latter case, upstream migration requires blocking Mac-1, a competing receptor for ICAM-1.
For papers in this area, please see:
Kim, Sarah Hyun Ji and Daniel A. Hammer, (2020) “Integrin crosstalk allows CD4+ T lymphocytes to continue migrating in the upstream direction after flow,” Integrative Biology 11(10):384-393. doi.org/10.1093/intbio/zyz034.
Alex J. Buffone, Nicholas R. Anderson, and D.A. Hammer, (2019). “Human neutrophils will crawl upstream on ICAM-1 if Mac-1 is blocked,” Biophysical Journal 14(8): 1393-1404. doi.org/10.1016/j.bpj.2019.08.044.
Adhesive dynamics (AD) is a computational algorithm to simulate biological adhesion. It calculates the adhesive trajectory of a cell in response to externally applied forces through a balance of mechanical energy with engaged adhesion receptors. Over the years, AD has been used to calculate state diagrams for adhesion, to simulate the binding of viruses to cell surfaces, to understand how collisions between cells lead to changes in the dynamics of cell adhesion, and recently how intracellular signaling cascades affect the dynamics of cell adhesion and stopping. A good review is found here:
Daniel A. Hammer, (2014), “Adhesive Dynamics,” Journal of Biomechanical Engineering 136:2 Article Number: 021006; ncbi.nlm.nih.gov/pubmed/24384944
Current projects integrate signaling and adhesion, and make predictions about how changes in cell pathways affect cell binding. This work represents a paradigm for how we might reengineer cell behavior using gene editing (CRISPR/Cas9 editing) to add or delete key molecules in cell adhesion to reduce or enhance cell adhesion, homing, and the inflammatory response.
Nicholas R. Anderson, Dooyoung Lee, and Daniel A. Hammer, (2019). “Adhesive Dynamics simulations quantitatively predict effects of kindlin-3 deficiency on T-cell homing,” Integrative Biology 11(6):293-300. doi.org/10.1093/intbio/zyz024.
Dooyoung Lee, Jiyeon Kim, Gary A. Koretzky and Daniel A. Hammer, (2012). “Diacylglycerol kinase zeta negatively regulates CXCR4-stimulated T lymphocyte firm arrest to ICAM-1 under shear flow,” Integrative Biology 4:606-614; doi.org/10.1039/C2IB00002D.
We are making protocells – which are synthetic cells that can be engineered to do a variety of tasks.
The Hammer lab has has a long standing interest in making membranes from novel surfactants, extending back to the co-invention of polymersomes in 1999. Most recently, we have been making vesicles and other supramolecular assemblies (such as micelles, sheets, and fibers) entirely from recoimbinant proteins, through molecular engineering of naturally occurring surfactant proteins, such as oleosin. These materials allow us to use the tools of molecular biotechnology to make tailored, responsive materials of designed sequence with specific function and responsiveness. An example is:
Kevin B. Vargo, Ranganath Parthasarathy and Daniel A. Hammer, (2012). “Tunable Protein Suprastructures from Recombinant Oleosin”, Proceedings of the National Academy of Sciences USA 109 (29) 11657-11662; http://dx.doi.org/10.1073/pnas.1205426109
In collaboration with Daeyeon Lee (Penn) we recently made self-motile protocells that are adherent to a surface but can crawl. To achieve this feat, we encapsulated the enzyme catalase in polymersomes which were adherent to a surface. Upon the addition of hydrogen peroxide, catalase causes force generation, and the polymersomes crawl over the surface (see next page for videos). We are interested in which other enzymes can be used to generate forces can drive motility.
Woo-Sik Jang, Hyun Ji Kim, Chen Gao, Daeyeon Lee, D.A. Hammer,(2018) “Enzymatically powered surface-associated self-motile protocells,” Small 14; 1801715; doi.org/10.1002/smll.201801715.
In collaboration with Matt Good, we have been developing ways to make membraneless organelles that can be used as catalytic hubs in protocells. Our design of membraneless organelles currently revolves around the RGG subdomain of the coacervating protein Laf-1. We showed that dimers of RGG avidly coacervate, and that proteins can be incorporated within these droplets. We then showed that the assembly of organelles can be controlled by light, using a photocleavable domain tied to a solubilization tag that prevents coacervation; cleaving away the solubilization tag initiates the assembly of organelles in emulsion droplets and in yeast.
Benjamin S. Schuster, Ranganath Parthasarathy, Craig N. Jahnke, Ellen H. Reed, Matthew Good, and D.A. Hammer, (2018), “Controllable protein phase separation and modular recruitment to form responsive, membraneless organelles,” (2018) Nature Communications 9, No. 2985 (2018); 10.1038/s41467-018-05403-1.
Reed, Ellen H., Benjamin Schuster, Matthew C. Good, and Daniel A. Hammer, (2020), ”Light responsive membraneless organelles from recombinant proteins,” in press, ACS Synthetic Biology. doi.org/10.1021/acssynbio.9b00503