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0 Q&A 976 Views Apr 5, 2022

Ultra-precise stimulation solely to individual mitochondria, without any influence to the whole cell, is quite difficult by traditional biochemical reagents. In mitophagy research, the mitochondria and even the whole cell usually suffer irreversible and great damage caused by treatment with potent chemicals. In this protocol, we present the technical procedures of our developed noninvasive ultra-precise laser stimulation (UPLaS) technology, which introduces precise stimulation to individual mitochondria, to excite mitochondrial Ca2+ (mitoCa2+) oscillations, with little perturbation to mitochondrial membrane potential (MMP), or mitochondrial reactive oxygen species (mitoROS). The mitoCa2+ oscillation by UPLaS was able to initiate the PINK1/Parkin pathway for mitophagy. This protocol has good potential to benefit researches on mitophagy and mitochondrial diseases.

Graphic abstract:

Figure 1. Flowchart of the UPLaS technology.

The femtosecond laser (1030 nm, 1 MHz, 220 fs) can stimulate individual mitochondria (1 μm2) for a short period (0.1 s), whereas confocal microscopy (CM) provides continuous cell imaging to monitor molecular dynamics in real time, before and after UPLaS.

0 Q&A 3708 Views Mar 20, 2021

Synaptic vesicles (SVs) are clustered in the presynaptic terminals and consistently trafficking along axons. Based on their release features, SVs are classified into different “pools”. Imaging of SVs that are traveling among multiple presynaptic terminals has helped define a new pool named “SV super-pool”. Here we describe a Fluorescent Recovery After Photobleaching (FRAP) approach to elucidate the relationship between SVs from the super-pool with SV clusters at presynaptic terminals. This method is powerful to investigate SV mobility regulation mechanisms.

0 Q&A 6327 Views Feb 20, 2020
Direct protein-protein interactions are known to regulate a wide range of cellular activities. To understand these contacts one can employ various experimental methods like Dynamic Light Scattering (DLS), Fluorescence Resonance Energy Transfer (FRET), Isothermal titration calorimetry (ITC), Chemical crosslinking, Co-immunoprecipitation (Co-IP), Surface Plasmon Resonance (SPR) and many more. Among these, SPR stands out as a quick, label-free, reliable, and accurate quantitation technique. We have used SPR to elucidate the linkage between 14-3-3 Protein 3 (EhP3) and the actin cytoskeleton in the protist pathogen Entamoeba histolytica. It allowed us to screen EhP3 binding with several actin-binding/actin regulatory proteins (Coactosin, Actophorin, Twinfilin, Profilin, and Filamin). Our screening results suggested Coactosin as an important interacting partner of EhP3. A complete kinetic analysis indeed confirmed that EhCoactosin binds EhP3 with an affinity constant of 3 μM.

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