Funding: University of Pretoria (ResCom)
Collaborators: Prof D.A. Cromarty
Degree: Bachelor of Science Honours (B.Sc. Hons)
Nuclear Factor Kappa B (NF-κB) is a family of transcription factors that play a central role in regulating numerous cellular processes, including inflammation, immune responses, cell proliferation, and apoptosis. NF-κB activation is a tightly controlled process that allows the transcription factors to enter the cell nucleus and initiate the expression of target genes involved in various cellular functions. NF-κB exists as a homo- or heterodimeric complex of proteins, with the most common form being a heterodimer composed of two subunits, typically p50 and p65 (also known as RelA). In the cytoplasm, NF-κB is sequestered in an inactive form by a family of inhibitory proteins known as Inhibitor of κB (IκB). The IκB proteins bind to NF-κB, masking its nuclear localization signals and preventing its entry into the nucleus. NF-κB activation is typically triggered by various extracellular signals, such as pro-inflammatory cytokines (e.g., tumor necrosis factor-alpha, interleukins), microbial pathogens (e.g., lipopolysaccharide), and stress-related stimuli (e.g., reactive oxygen species). These stimuli induce the activation of the IκB kinase (IKK) complex, which phosphorylates IκB proteins, targeting them for degradation by the proteasome. As IκB proteins are degraded, the NF-κB dimers are released and become free to translocate to the nucleus. Importins and nuclear pore complexes facilitate the transport of NF-κB dimers across the nuclear membrane. Once inside the nucleus, NF-κB binds to specific DNA sequences known as κB sites within the promoter regions of target genes.
Upon binding to the κB sites, NF-κB recruits co-activators and the transcriptional machinery, initiating the transcription of target genes. These genes encode a variety of proteins involved in immune responses, inflammatory pathways, cell survival, and other critical cellular processes. The duration and intensity of NF-κB activation are tightly regulated to prevent excessive inflammation and ensure proper cellular responses. After completing its transcriptional function, NF-κB is deactivated by a negative feedback loop, leading to the resynthesis of IκB proteins and sequestration of NF-κB in the cytoplasm, ready for future activation. As such, NF-κB activation and nuclear translocation are crucial processes that regulate gene expression and mediate various cellular responses, making them essential components of immune and inflammatory pathways. Dysregulation of NF-κB signalling has been associated with numerous diseases, including chronic inflammation, autoimmune disorders, and cancer, making it an area of significant interest for medical research and therapeutic interventions. Flow cytometry is a powerful technique used to analyse and quantify characteristics of individual cells in heterogeneous populations. When studying transcription factors, flow cytometry is particularly useful for the quantification of transcription factor activation, the detection of intracellular transcription factors, and time course studies.
To develop methods to study NF-kB activation and nuclear translocation in cultured monocytes using flow cytometry and SDS-PAGE by:
transforming U-937 cells into adherent monocytes with PMSF.
stimulation cells with LPS from E. coli.
optimising reagents used for fixation and permeation.
optimising the detection of activated NF-kB by polyclonal antibody.
U-937 cells will be cultured under standard conditions in flasks until a 60% confluence is reached. Hereafter cells will be stimulated for 48h with PMSF to induce differentiation into adherent monocytes. Once adherence is achieved, cells will be stimulated with LPS from E. coli for 2, 4, and 8 hours. Initially, cells from the control and stimulus groups will be lysed and protein extracts quantitated with a Bradford assay on a spectrometer followed by electrophoretic separation by SDS-PAGE to visualise differences in the protein profile for the expected bands for NF-kB and confirm activation by phosphorylation. Hereafter cells will be fixed in methanol, permeated with three different surfactants and incubated with an Alexa-Fluor 488 conjugated rabbit polyclonal antibody for activated NF-kB. Levels of NF-kB activation at the various time points in response to LPS will then be quantitated on a Beckman Coulter Flow cytometer.