ABSTRACT
We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.
Subject(s)
Biomedical Research , Databases, Bibliographic , Extracellular Vesicles/physiology , InternationalityABSTRACT
Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp)2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds.-Bertier, L., Boucherie, C., Zwaenepoel, O., Vanloo, B., Van Troys, M., Van Audenhove, I., Gettemans, J. Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.
Subject(s)
Cortactin/chemistry , Neoplasm Invasiveness , Podosomes/physiology , Single-Domain Antibodies/physiology , Cell Line, Tumor , Cloning, Molecular , Cortactin/metabolism , Epitopes , Gene Expression Regulation, Neoplastic , Humans , Indoles/pharmacology , Protein DomainsABSTRACT
Cortactin is a multidomain actin binding protein that activates Arp2/3 mediated branched actin polymerization. This is essential for the formation of protrusive structures during cancer cell invasion. Invadopodia are cancer cell-specific membrane protrusions, specialized at extracellular matrix degradation and essential for invasion and tumor metastasis. Given the unequivocal role of cortactin at every stage of invadopodium formation, it is considered an invadopodium marker and potential drug target. We used cortactin nanobodies to examine the role of cortactin domain-specific function at endogenous protein level. Two cortactin nanobodies target the central region of cortactin with high specificity. One nanobody interacts with the actin binding repeats whereas the other targets the proline rich region and was found to reduce EGF-induced cortactin phosphorylation. After intracellular expression as an intrabody, they are both capable of tracing their target in the complex environment of the cytoplasm, and disturb cortactin functions during invadopodia formation and extracellular matrix degradation. These data illustrate the use of nanobodies as a research tool to dissect the role of cortactin in cancer cell motility. This information can contribute to the development of novel therapeutics for tumor cell migration and metastasis.
Subject(s)
Actins/metabolism , Cell Surface Extensions/metabolism , Cortactin/metabolism , Extracellular Matrix/pathology , Neoplasm Invasiveness , Proline/metabolism , Single-Domain Antibodies/physiology , Carrier Proteins/metabolism , Cell Line, Tumor , Extracellular Matrix/metabolism , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Microfilament Proteins/metabolism , Neoplasms, Squamous Cell/metabolism , Neoplasms, Squamous Cell/pathology , Protein Binding , Protein DomainsABSTRACT
Neurostimulation is a promising treatment for refractory epilepsy. We studied the effect of cortical stimulation with different parameters in the rat motor cortex stimulation model. High intensity simulation (threshold for motor response--100 µA), high frequency (130 Hz) stimulation during 1 h decreased cortical excitability, irrespective of the interpulse interval used (fixed or Poisson distributed). Low intensity (10 µA) and/or low frequency (5 Hz) stimulation had no effect. Cortical stimulation appears promising for the treatment of neocortical epilepsy if frequency and intensity are high enough.