Single Molecule Fluorescence

Transcription

1 Single Molecule Fluorescence Joseph Irudayaraj Founder Professor of Bioengineering Micro and Nanotechnology Laboratory & Beckman Institute University of Illinois at Urbana Champaign

2 STED

3 Lifetime imaging and photon correlation studies resolve MBD2/MBD3 stochiometry Behavior of MBD3 unraveled by single-molecule tools: 1. Low chromatin binding affinity in G1-phase 2. Homodimer to heterodimer transition in S-phase 3. Further recruitment of MBD2 & DNMT1 for methylation maintenance in G2-phase NAR (2015)

4 Real-time dynamics of MBD3 in DNA demethylation by FCS

5 Live Cells Experimental setup for kinase activity monitoring EGFR EGF Integrin Oxidative stress Src sensor Src Syk Syk sensor Cortactin Abl N-WASP lasers SPAD1 SPAD2 Kinase sensor with delivery tag

6 Multiplex Abl-Src Dasatinib drug response Abl-EGF 10 ng/ml 25 min Src-EGF 10 ng/ml 25 min Src-EGF 10 ng/ml 25 min Abl-EGF 10 ng/ml 25 min Abl-EGF 10 ng/ml imatinib 1mM /15 min Src-EGF 10 ng/ml imatinib 1mM /15 min Src-EGF 10 ng/ml Dasatinib 1mM /15 min Abl-EGF 10 ng/ml Dasatinib 1mM /15 min 0 ns Fluorescence Lifetime 4 ns

7 Real time Multiplex Abl and Src in MDA-MB 231 cell upon EGF stimulation video Abl-Tat A-488/EGF 20 min Src-Tat Cy5/EGF 20 min

8 Quantitative Analysis 150 average lifetime per-pixel t average MATLAB 150 t averagemulti-exponential fitting Amplitude of short and long lifetime per-pixel SPAD TAC ADC Photon detector Digital converter Damayanti, Parker, Irudayaraj, under preparation, 2014

9 Quantitative Analysis 1. Average Lifetime histogram

10 Quantitative Analysis 2. Population of short/long lifetime histogram Abl kinase 0 min Src kinase 0 min Real time video Abl kinase 10 min 10 min 25 min 25 min Src kinase 30 min 30 min

11 Optogenetics

12 Identification of optogenetic tools for targeted bio-molecular engineering PhyB-PIF6 optogenetic pair CIB1-CRY2 optogenetic pair CIB1-EGFP CRY2-mCherry Overlap PhyB, a photoreceptive phytochrome isolated from Arabidopsis thaliana activates at 65o nm (red) through photoisomerzation with chromophores as Phycocyanobilin (PCB). Activated PhyB binds directly to a downstream transcription factor, phytochrome interaction factor (e.g. PIF3/PIF6), translocates to the nucleus as a heterodimer and directly modulates the transcription of response genes. They further dissociates at 750 nm(infra-red). 8, 9 Genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. Researchers have demonstrated the utility of this system by inducing protein translocation, transcription and Cre recombinase mediated DNA recombination using light. 10

13 Optogenetic regulation by loci-specific targeting - Telomere

14 Optogenetic interactions querried by Single molecule FLIM-FRET

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16 Differential methylation status at the Ascl1 promoter Achaete-Scute homolog 1 (Ascl1 or Mash1 in mammal) is an important proneural genes, known to promote cell cycle exit and neuronal differentiation. Mash1 initiates the neuroblast differentiation from neuroepithelial cells and also protects the neuroblasts from damages through delta protein mediated lateral inhibition machinery during nervous system development. Hypermethylation at Ascl1 promoter in Dorsal Root Ganglion (DRG) cells Hypomethylation at Ascl1 promoter in Striatal (STR) neuronal cells The occurrence of global hypermethylation with concomitant upregulation of DNMTs have been reported Excitatory neurons sometimes exhibits clustered hypomethylation at their candidate gene promoters. Targeting loci specific epigenetic changes at the Ascl1 promoter EF-1a TAL-TF CIB1 mcherry 2A WPRE TALE target EF-1a TET1/DNMT-CD CRY2PHR EGFP 2A WPRE TSS Exon Target CpG sites were within 250 bp, upstream of the Ascl1 promoter site. CpG-1 is 41bp, and CpG-4 is 73 bp away from the TAL-TF binding site

17 Induced methylation at the Ascl1 promoter in striatal neurons Determining methylation level with pyrosequencing The mrna level decreased by ½ fold in the cell population, co-transfected along with blue light illumination. Elevated expression level of Ascl1 Methylation level incresed by 10% at CpG-1, 13% at CpG-2, 14% at CpG-3, and 14% at CpG-4 in the cells co-transfected and illuminated (COL)