Visualizing CREB family transcription factor activation in living cells

Friedrich, Michael (2007): Visualizing CREB family transcription factor activation in living cells. Dissertation, LMU München: Fakultät für Biologie

Abstract

Many transcription factors integrate a variety of cellular stimuli to produce a transcriptional response. There is increasing evidence that the timing and kinetics of activation are crucial in determining specificity and strength of gene expression, however so far only few tools are available to address these questions in live cells and these have severe drawbacks, like very low signal strength, complicated handling, irreversibility and lack of good targeting properties.The Ca 2+- and cyclic adenosine monophosphat responsive element-binding protein (CREB) and the related ATF-1 and CREM are stimulus inducible transcription factors that link certain forms of cellular activity to changes in gene expression and are involved in differentiation, cancer, survival and neuronal plasticity. Using fluorescence resonance energy transfer (FRET) we here develop genetically encoded indicators that enable imaging activation of CREB family transcription factors due to phosphorylation of the critical serine 133 and subsequent recruitment of a coactivator in single live cells. The indicator for CREB activation due to phosphorylation (ICAP) consitsts of the kinase inducible domain (KID) of CREB fused together with the KIX of CREB binding protein (CBP) via a flexible linker, sandwiched between a cyan and a yellow variant of the green fluorescent protein. The specificity and reliability of ICAP as a measure for CREB activation was demonstrated first in the cuvette and then in the nucleus and mitochondria of HeLa cells. After that, we analyzed the properties of ICAP in primary hippocampal neurons, where we characterize different signaling pathways with distinct kinetics that lead to CREB activation. Furthermore, combining the imaging of CREB activation with calcium imaging we see a summation of CREB activation in neurons that can be achieved by appropriately timed depolarizing stimuli and occurs even when individual stimulations are separated by hours. Finally, sensors for the activation of ATF-1, CREM and the recruitment of P300, were introduced and preliminarily characterized. On the whole, these array of biosensors complement the toolbox for the investigation of the activation of the CREB family of transcription factors in living cells and organisms.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: CREB, GFP, Imaging
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 2. Juli 2007
• 1. Bericht­erstatter:in: Borst, Alexander