probing opioid signalosome on neuroblastoma原子力显微镜对阿片类signalosome对神经母细胞瘤课件

deflection (nm) = sensitivity (nm/V) deflection(V) force = (cantilever spring constant) (deflection) Functionalization of AFM tips NHS/EDCAntibody solution SH CH3 Thiols+ + OH SH O C CT(PEG12)MT(PEG4) Au S O C OH S O C OH S CH3 S CH3 Au S O C O O N O S CH3 S CH3 S O C O O N O Au S CH3 S CH3 NH S O C NH S O C Au antibody Detection of antibody-receptor recognition - SHSY5Y cell line stably expresses the MOPR tagged with T7. SHSY5Y neuroblastoma cell line T7T7 Anti-T7 - The T7-tagged receptor can be identified by AFM probe modified with T7-antibody approach contact retract Force measurements by AFM Forces between the tip and receptor were monitored by continuously indenting and retracting the tip at constant lateral positions. Force curve Three typical force curves demonstrating the interaction events between the tip and the receptor specific unbinding interaction nonspecific unbinding interaction no interaction deflection (nm) = sensitivity (nm/V) deflection(V) force = (cantilever spring constant) (deflection) Fuzzy logic is an approach to computing based on “degrees of truth“ rather than the usual “true or false“ (1 or 0) Boolean logic. Fuzzy logic includes 0 and 1 as extreme cases of truth but also includes the various states of truth in between so that, for example, someone whose age is 60 might be assigned a membership of 0.25 in the fuzzy set of old people. Reading the Force volume file and calibrating the force curves Loading a new force curve Detection of all the vertical segments on force curve Analysis of each force curve Higher than the threshold? Fuzz logic analysis of the corresponding FC segment Attribution of a grading to the event Building the histogram YES Has the whole FC been analyzed? NO NO YES Flow chart of detecting events algorithm Convolution nThe integral of two functions f and g. nThe resulting amount overlap that occurs when g is shifted over f is the convolution of f and g. nf * g f ( ) g (t )d nConvolution between force curves and the filter (difference) Convolution with different filters The convolution of the FC with three different filters gives three new curves that present peaks at locations where steep vertical segments, right angles, or v shapes exist on the original FC curve. (-0.03,-0.03, 0.5, 0) Unbinding force distribution histograms 123.1， 195.3 Determining elastic modulus Elastic modulus is the measure of a substances tendency to be deformed elastically when a force is applied to it. E=stress/strain The Hertz Model Indentation = z-x The data obtained by indentation experiments are usually plots of force against piezo displacement rather than tip sample separation. In applying the Hertz model, curves should eb converted to Force indentaion curve Elastic Modulus nwave E n0 241.46 n7 298.3 n15 259.37 n23 313.26 n31 332.96 n39 276.49 n47 158.73 n55 275.02 n63 294.34 nAverage 267.0838 Results Poisson statistical method: The number of receptor-antibody pairs (n) is finite and follows a Poisson distribution. n= n2 F= measured force for n pairs, F = nFs , Fs= single pair force F= nFs, F2 = n2 Fs2, Fs = F2 / F If there are nonspecific interactions (Fn), F= nFs + Fn, F2 = n2 Fs2 = (F- Fn) Fs= F Fs - Fn Fs n1 n2 F = F1+ F2 F1 F2 Variance versus mean of the unbinding forces Mean Force (pN) Force variance (pN2) N 149.4417355.42188 178.375410596.7293 190.964611350.1291 158.10899043.40893 100.1809868.3233107 126.67183337.373106 Fs= 123 pN Fn= 93 pN Computed forces based on Poisson Statistical method Conclusion nBased on the Poisson statistical method, the force required to unbind a single ligand and its pair is 123 pN and the nonspecific unbinding force is 93 pN. The unbinding force calculated is consistent with those obtained for antigen-antibody pairs by others. nThe potential of the sensor for localizing individual receptor sites can be used in future simultaneous studies on the effect of agonist stimulation on the receptor distribution and its association to changes in biophysical properties. Appendix Opiod receptor (MOPR) major target of pain treatment RECEPTORS interact with specific ligands to produce changes in the function of the system. permits the encounter of the receptors, ligands, membrane proteins and effectors initiating events leading to any of ligands effect. LIGANDS are Chemica