Blystone Lab Research
The
focus of the lab is on understanding how integrin receptors on circulating
cells (platelets, lymphocytes, leukocytes) maintain their inactive state in the
circulation and how they become activated during hemostatic crisis or immune
challenge. This work involves the use of primary cells and transfection models,
mutational analysis, protein chemistry, small animal models, several types of
microscopy, and numerous molecular and cell biological techniques.
You can take a look at Blystone lab publications at PUBMED
Integrins are heterodimeric transmembrane receptors present on all cells except RBCs. Integrins are the primary receptor family responsible for cell-substrate adhesion and also participate in cell-cell adhesion events, particularly in the immune system. Some entry-level (and outdated) information on integrins can be found on The Integrin Page. As this site is not on our server, we cannot be responsible for errors. In recent years, the complexity of integrin function has been realized. Complex signaling processes simultaneously control and are initiated by integrins. Numerous excellent reviews of integrin signalling can be found in the medical literature. Several interesting questions in integrin biology occupy much of the lab efforts, including the regulation of integrin activation, communication between integrins on the same cell, and signaling processes controlling integrin-mediated behavior of cells.
The lab occupies
several adjoining rooms. Students and postdocs have an office next door,
Scott’s office is at the front of the lab. Common equipment is located on the
opposite hallway and the microscope room is one floor above. Tissue culture is
done in a room that opens into the lab. You can view a list of Equipment in the Lab and some more pictures.
Ongoing Projects
Beta-3 Integrin Tyrosine Phosphorylation
Several years ago, we discovered
that the cytoplasmic tail of the beta-3 integrin becomes phosphorylated upon a
tyrosine residue when the receptor binds one of its many ligands. Mutation of
that tyrosine prevents adhesion in hematopoietic cells, but has little effect
on non-hematopoietic cells such as fibroblasts that exhibit constitutive
adhesion. Modulating beta-3 integrin activation has important consequences in
vascular and cancer biology. Several members of the lab work on projects
related to pursuing this observation. These include searches for binding
partners of the tyrosine phosphorylated beta-3 tail by phage expression
screening, direct peptide affinity chromatography, and yeast 2-hybrid analysis.
We are also extending our initial observations in transfection models to
primary cells through the use of virally reconstituted mutant beta-3 integrins
expressed in leukocytes from beta-3 deficient mice. Additionally, we are
comparing phosphorylation of beta-3 when partnered either with the ubiquitously
expressed alpha-v chain or the platelet-selective alphaIIb chain. Finally, we are
tracing the signalling pathways that lead to and from the
tyrosine-phosphorylated integrin. 
Integrin Crosstalk
Others and we have demonstrated that different integrins communicate with one another on the same cell in order to establish a hierarchy of signals to affect an appropriate cellular phenotype. In our system, the beta-3 integrins regulate the high affinity functions of alpha-5/beta1 integrins. The first level of competition occurs through CamKII. Currently, we are attempting to map the signalling cascades that lead from CamKII regulation to the beta-1 integrin.
Cytoplasmic Integrin Activation
Complete activation of integrins results in cell spreading and firm adhesion to substrate. Spreading and firm adhesion require interaction of the integrin both with the extracellular environment and the actin cytoskeleton. We have initiated a series of studies to understand the cytoplasmic activation of integrin, i.e. the processes which permit association with actin. To quantitate integrin activation, we have developed an assay of actin polymerization from purified integrin tails. This assay permits four levels of modulation: 1) we can alter the ligand on the outside of the integrin, 2) we can mutate the integrin in a transfected cell prior to integrin harvest, 3) we can stimulate the cell prior to integrin harvest, and 4) we can add and delete components to the purified integrins prior to assaying actin polymerization. This assay has yielded some very exciting results to date and promises to be a mainstay of the lab in the near future. We are currently determining proteins necessary in conjunction with the integrin to initiate actin polymerization, critical motifs in the integrin required for cytoplasmic activation, and the signalling pathways which induce integrin interaction with the actin cyoskeleton.
Actin Nucleation at Adhesion Contacts
As an outgrowth of our studies on cytoplasmic integrin activation, we have localized and examined the regulation of numerous proteins involved in reorganization of the actin cytoskeleton. Several of these proteins are important in beta-3 integrin adhesion and their recruitment to adhesive contacts is dependent upon tyrosine phosphorylation of the beta-3 integrin. Organization of the actin cytoskeleton is critical for cell adhesion and migration during normal and pathologic events. Thus we have become interested in the ultimate molecules in this process – those that directly nucleate actin into filamentous structures – chiefly the Arp2/3 complex and members of the formin protein family. We are exploring ROCK (Rho-associated kinase) regulation of Arp2/3 localization. We have also cloned the formins present in mononuclear phagocytes and are examining their activation, regulation, localization and role in leukocyte adhesion and migration. This work is dually funded by two new NIH grants and a large share of the experimentation is being performed on a new TIRF microscope. TIRF microscopy permits visualization of fluorescent molecules at the interface between the cell surface and the substrate with extremely good resolution. We have numerous cell populations expressing fluorescently tagged integrins and integrin-signaling molecules for live cell TIRF microscopy analysis.
Other
There are always numerous small projects getting underway in the lab. Some are cancer related and some are vascular cell related. We try to pursue new observations regardless of their import for hematopoietic cell types or integrins in general. As a result, we are fairly adept at developing new technologies.
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