HaloTag p65 translocation

Layman's notes:

* These are cultured human cells living on a glass slide.
* The large black circles are the nuclei (plural of nucleus). This is where the chromosomes, made of DNA, are.
* A gene is DNA sequence that includes the code for a protein, plus regulatory sequences on each side.
* The red dots are proteins called transcription factors (or TFs) that have been tagged with a fluorescent dye.
* A TF can bind to a specific regulatory DNA sequence of a gene, and turn that gene on or off, like a switch.
* The two sides are the same image, but in the left side, the white light has been turned off so you can see the fluorescent dye better.
* At the beginning of this video, these TFs are mainly in the cytoplasm of the cells.
* When the cells are washed with a stimulus - either a chemical or another protein - the cells respond by moving the TFs into the nuclei, so that they can find their target DNA sequence and turn on their genes.
* Once the stimulus is removed, the TFs aren't needed anymore, so they recycle back to the cytoplasm.
* This is an example of how cells regulate their activity depending on environmental cues.

More technical notes:

* A p65-HaloTag fusion construct was transfected into HeLa cells to do real-time imaging of protein translocation events.

Review NFkB (NF-kappa-B) pathway:
* NFkB complex is composed of heterodimer of p65 and p50.
* IkB (I-kappa-B) p65 has a nuclear localization sequence (NLS) that is masked by IkB (Inhibitor of NFkB).
* In response to a stimulus such as TNF-alpha, LPS, or UV light, the NF-kappa-B pathway is activated.
* IkB is phosphorylated, ubiquitinated and targeted for degradation. This unmasks the NLS of the p50/p65 complex, allowing it to translocate into the nucleus where it regulates gene expression.
* We used this pathway as a model for translocation studies.

* This video shows time-lapsed imaging showing the response of p65 when challenged with TNF-alpha
* HeLa cells expressing p65-HaloTag™ protein fusion, labeled with HaloTag™ TMR Ligand (5 mM, 15 min, 37oC), and challenged with TNF (20 ng/ml).
* Live cell time lapse imaging was performed on the Olympus FV500 confocal microscope (5 min/frame; 120 min).
* By fusing to HaloTag, we can track the real time movement of p65 from the cytoplasm, to the nucleus, and then back to the cytoplasm again.
* P65-HaloTag fusion behaves as expected for p65. HaloTag does not appear to interfere with normal protein activity.
* This video spans a 2 hour time period. Live cell imaging can be done over longer periods of time too.

Category:

Science & Technology

Tags:

• NFkB
• cell
• protein
• trafficking
• in
• vivo
• promega
• halotag

[bllackbird]

how long were the cells exposed to the stimulus for the translocation to be completed?