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Membrane-induced bundling of actin filaments

Membrane-induced bundling of actin filaments. Nature Physics. Porgress Report. Mutant in the N-terminus of ParA1 scoe Double labeling (YFP-ParA and CyPet-ParB ) Co-localization of the ParA foci with ParB Photobleach experiment ParA1 dynamic movement in/on nucleoid

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Membrane-induced bundling of actin filaments

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  1. Membrane-induced bundling of actin filaments Nature Physics

  2. Porgress Report

  3. Mutant in the N-terminus of ParA1scoe • Double labeling (YFP-ParA and CyPet-ParB) • Co-localization of the ParA foci with ParB • Photobleach experiment • ParA1 dynamic movement in/on nucleoid • Bacteria twoHybrid • Role of ParA1-NTD in protein-protein interaction

  4. Mutants in the N-terminal domain YFP DAPI merge DIC R8E R19E R26E R19E+R26E Q29E R31E

  5. Localization in the presence of ParB Plac-yfp::parA1-parB_silent mutation on parS merge YFP DAPI DIC Plac-yfp::parA1-B merge YFP DAPI DIC The bright spots could be the nucleation of ParA1 on the parS-ParB complex

  6. YFP::PARA-PARB::CYPET

  7. FRAP, Fluorescence Recover After Photobleach

  8. FRAP, data processing Raw data ROI1 ROI Tot BG Tot ROI3 ROI2 ROI1 BG Correction X normalization factor [ROI(t)-BG(t)]/[Tot(t)-BG(t)] X [Tot(t0)-BG(t0)]/[ROI(t0)-BG(t0)]

  9. FRAP, kinetic plot and fitting AIM, fitting Molecular dynamics, I=I0 - I1x e -t/τ IΔ Fitting I1 I0 t1/2 I1 IΔ IΔ IΔ not not I1 + IΔ I0 I1 + IΔ I0

  10. Difference between the growth stages Bleach regions Inter or intra nucleoid movement Different chemical interactions between cytoplasm and nucleoid localization Number of molecules (is this meaningful in an inducible system?)

  11. considerations Thin cellular extensions  63X 1.4NA oil immersion objective Maximize the difference between bleaching and imaging mode  100-fold higher Short wavelengths, high-intensity illuminations  antioxidants Bleach on the optical axis  Pinhole/ low NA objective Lower zooms to monitor neighboring control cells Good spatial and temporal resolution are achieved at high zooms Timing  bleaching time < 1/10 T1/2 ; data collection >10-50X T1/2 Offset  slightly greater than zero Gain  few or no pixels are saturated Increase the effective dynamic range of measurements  12-bit images (4096 gray values)

  12. Results Intranucleoid exchange of ParA1scoe is much faster than inter-nucleoid exchange

  13. Interaction of ParA1scoe-NTD with ParA1scoe, ParA1scoeNC, and ParA1scoe-NTD 1) There is no ParA1scoe-NTD interaction with other parts of ParA1scoe detectable in B2H experiment. 2) Intermolecular association between ParA1scoe is very strong.

  14. NEXT PROJECT • parSA1B on miniF-sopABC--functional assay • B2H experiments --learn the interplay between ParA-ParANC, ParA-ParB • In vitro experiments --learn the interplay between parS-ParA-ParB • Gel shift • Biacore

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