Intracellular Spatial Dynamics of Metal Transcription Factor 1
Disciplines
Biotechnology | Cell Biology
Abstract (300 words maximum)
Metal Transcription Factor 1 (MTF-1) is a cytoplasmic transcription factor protein involved in cellular response to metal ions and stress. During cellular stress or heavy metal exposure, MTF-1 proteins translocate to the nucleus and bind DNA metal response elements (MRE), initiating the transcription of genes associated with intracellular metal regulation (e.g., zinc transporter (ZnTs), Zrt-/Irt-like protein (ZIP), metallothioneins). MTF-1 is positioned as a key sensor of cellular processes that trigger the need for bio-metals such as zinc. Therefore, monitoring its spatial distribution in cells can provide insight into what cellular conditions cause its intracellular movement. To visualize MTF-1 eukaryotic cellular location, chemically competent E. Coli (strain: DH5-α) cells were transformed with an MTF-1 plasmid containing Green Fluorescent Protein (GFP) and ampicillin (amp) resistance inserts. Transformed cells were selected using amp+ agar plates. Visible colonies were harvested then grown in amp+ Lennox Broth (LB) broth for amplification followed by plasmid purification. Purified endotoxin free plasmids were transfected into human embryonic kidney (HEK) 293T cells and imaged using fluorescent microscopy techniques. Control data demonstrated plasmid transformation, high yield purification, and efficient transfection. We aim to use this instrument to study intracellular MTF-1 trafficking during wound and regeneration and to determine if its nuclear translocation is regulated by reactive oxygen species.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Eric A. Albrecht
Intracellular Spatial Dynamics of Metal Transcription Factor 1
Metal Transcription Factor 1 (MTF-1) is a cytoplasmic transcription factor protein involved in cellular response to metal ions and stress. During cellular stress or heavy metal exposure, MTF-1 proteins translocate to the nucleus and bind DNA metal response elements (MRE), initiating the transcription of genes associated with intracellular metal regulation (e.g., zinc transporter (ZnTs), Zrt-/Irt-like protein (ZIP), metallothioneins). MTF-1 is positioned as a key sensor of cellular processes that trigger the need for bio-metals such as zinc. Therefore, monitoring its spatial distribution in cells can provide insight into what cellular conditions cause its intracellular movement. To visualize MTF-1 eukaryotic cellular location, chemically competent E. Coli (strain: DH5-α) cells were transformed with an MTF-1 plasmid containing Green Fluorescent Protein (GFP) and ampicillin (amp) resistance inserts. Transformed cells were selected using amp+ agar plates. Visible colonies were harvested then grown in amp+ Lennox Broth (LB) broth for amplification followed by plasmid purification. Purified endotoxin free plasmids were transfected into human embryonic kidney (HEK) 293T cells and imaged using fluorescent microscopy techniques. Control data demonstrated plasmid transformation, high yield purification, and efficient transfection. We aim to use this instrument to study intracellular MTF-1 trafficking during wound and regeneration and to determine if its nuclear translocation is regulated by reactive oxygen species.