FRET Detection of Lymphocyte Function-Associated Antigen-1 Conformational Extension

Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation

Panorama of Undergraduate Research in Brazil: Profile, Scientific Production, and Perceptions

Undergraduate Research (UR) is an institutional program that introduces undergraduate students to scientific research. The program selects research projects proposed by advisors and students for execution. Despite the importance of knowing the stages of research activities in undergraduate research, only a few studies have evaluated data on this subject. Therefore, this study aims to outline an overview of UR in a Brazilian educational institution, considering the profiles of students and advisors, students’ scientific productions, and perceptions about the experience of both. The study was a mixed-approach case study conducted through a questionnaire and interviews. The sample consisted of 213 undergraduate students and 167 UR supervisors. The results show that the largest group of students were aged 21 and 22 (46.6%) and supervisors 33 to 38 years (38.9%). Regarding the scientific productions of students, those who participated twice or more in undergraduate research had higher indicators compared to those who were participating for the first time. Students’ perceptions of their evolution and perceptions of the advisors were mostly positive, with a greater number of responses classified as very good to good. Thus, the satisfaction of researchers in being part of this experience was perceived and the need to improve the scientific production indicators of students, mediated by the advisors stimulating the writing of articles, abstracts, and books, as well as participation in events and patent development, was shown. We conclude that undergraduate research activities promote the integral development of students’ academic, scientific, personal, and professional terms, which ultimately reflect critical and emancipatory actions in society

Active Site Formation, Not Bond Kinetics, Limits Adhesion Rate between Human Neutrophils and Immobilized Vascular Cell Adhesion Molecule 1

The formation of receptor ligand bonds at the interface between different cells and between cells and substrates is a widespread phenomenon in biological systems. Physical measurements of bond formation rates between cells and substrates have been exploited to increase our understanding of the biophysical mechanisms that regulate bond formation at interfaces. Heretofore, these measurements have been interpreted in terms of simple bimolecular reaction kinetics. Discrepancies between this simple framework and the behavior of neutrophils adhering to surfaces expressing vascular cell adhesion molecule 1 (VCAM-1) motivated the development of a new kinetic framework in which the explicit formation of active bond formation sites (reaction zones) are a prerequisite for bond formation to occur. Measurements of cells interacting with surfaces having a wide range of VCAM-1 concentrations, and for different durations of contact, enabled the determination of novel kinetic rate constants for the formation of reaction zones and for the intrinsic bond kinetics. Comparison of these rates with rates determined previously for other receptor-ligand pairs points to a predominant role of extrinsic factors such as surface topography and accessibility of active molecules to regions of close contact in determining forward rates of bond formation at cell interfaces

AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes

Affinity of integrin lymphocyte function-associated antigen 1 (LFA-1) is enhanced by conformational changes from the low-affinity closed form to the high-affinity (HA) open form of the ligand-binding inserted (I) domain as shown by work with purified I domains. However, affinity up-regulation of LFA-1 on the cell surface by physiological agonists such as chemokines has yet to be demonstrated by monovalent reagents. We characterize a mAb, AL-57 (activated LFA-1 clone 57), that has been developed by phage display that selectively targets the HA open conformation of the LFA-1 I domain. AL-57 discriminates among low-affinity, intermediate-affinity, and HA states of LFA-1. Furthermore, AL-57 functions as a ligand mimetic that binds only upon activation and requires Mg(2+) for binding. Compared with the natural ligand intercellular adhesion molecule-1, AL-57 shows a tighter binding to the open I domain and a 250-fold slower off rate. Monovalent Fab AL-57 demonstrates affinity increases on a subset (≈10%) of lymphocyte cell surface LFA-1 molecules upon stimulation with CXCL-12 (CXC chemokine ligand 12). Affinity up-regulation correlates with global conformational changes of LFA-1 to the extended form. Affinity increase stimulated by CXCL-12 is transient and peaks 2 to 5 min after stimulation