CUREs for high-level Galectin-3 expression.

Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds ß-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl ß-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.

CALM supports clathrin-coated vesicle completion upon membrane tension increase.

The most represented components of clathrin-coated vesicles (CCVs) are clathrin triskelia and the adaptors clathrin assembly lymphoid myeloid leukemia protein (CALM) and the heterotetrameric complex AP2. Investigation of the dynamics of AP180-amino-terminal-homology (ANTH) recruitment during CCV formation has been hampered by CALM toxicity upon overexpression. We used knock-in gene editing to express a C-terminal-attached fluorescent version of CALM, while preserving its endogenous expression levels, and cutting-edge live-cell microscopy approaches to study CALM recruitment at forming CCVs. Our results demonstrate that CALM promotes vesicle completion upon membrane tension increase as a function of the amount of this adaptor present. Since the expression of adaptors, including CALM, differs among cells, our data support a model in which the efficiency of clathrin-mediated endocytosis is tissue specific and explain why CALM is essential during embryogenesis and red blood cell development.

Strengthening bonds via RyR2 inhibition helps immune suppression.

Foxp3-expressing Tregs employ multiple suppressive mechanisms to curtail conventional T cell (Tconv) responses and establish tissue homeostasis. How Foxp3 coordinates Treg contact-dependent suppressive function is not fully resolved. In this issue of the JCI, Wang and colleagues revealed that Foxp3-mediated inhibition of ryanodine receptor 2 (RyR2) led to strong Treg-DC interactions and enhanced immunosuppression. RyR2 depletion in Tconvs phenocopied this effect and equipped Tconvs with Treg-like suppressive function in multiple inflammatory or autoimmune contexts. This study provides molecular and therapeutic insights underlying how cell-cell contact limits immune reactivity.

Microscopy approaches to study extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) have drawn the attention of both biological researchers and clinical physicians due to their function in mediating cell-to-cell communication and relevance as potential diagnostic markers. Since their discovery, the small size and heterogeneity of EVs has posed a hindrance to their characterization as well as to the definition of their biological significance. SCOPE OF THE REVIEW: Recent technological advances have considerably expanded the tools available for EV studies. In particular, the combination of novel microscope setups with high resolution imaging and the flexibility in EV labelling allows for the precise detection and characterization of the molecular composition of single EVs. Here we will review the microscopy techniques that have been applied to unravel the mechanism of EV-mediated intercellular communication and to study their molecular composition. MAJOR CONCLUSIONS: Microscopy technologies have largely contributed to our understanding of molecular processes, including EV biology. As we discuss in this review, careful experimental planning is necessary to identify the most appropriate technique to use to answer a specific question. GENERAL SIGNIFICANCE: The considerations regarding microscopy and experimental planning that are discussed here are applicable to the characterization of other small structures, including synthetic nanovectors and viruses.

Time-Dependent Fluorescence Spectroscopy to Quantify Complex Binding Interactions.

Measuring the binding affinity for proteins that can aggregate or undergo complex binding motifs presents a variety of challenges. In this study, fluorescence lifetime measurements using intrinsic tryptophan fluorescence were performed to address these challenges and to quantify the binding of a series of carbohydrates and carbohydrate-functionalized dendrimers to recombinant human galectin-3. Collectively, galectins represent an important target for study; in particular, galectin-3 plays a variety of roles in cancer biology. Galectin-3 binding dissociation constants (K D) were quantified: lactoside (73 ± 4 µM), methyllactoside (54 ± 10 µM), and lactoside-functionalized G(2), G(4), and G(6)-PAMAM dendrimers (120 ± 58 µM, 100 ± 45 µM, and 130 ± 25 µM, respectively). The chosen examples showcase the widespread utility of time-dependent fluorescence spectroscopy for determining binding constants, including interactions for which standard methods have significant limitations.