Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, in vitro and in vivo approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results demonstrate that in mammalian CPNs, axonal mitochondria do not play a major role in ATP supply, despite playing other functions critical to regulating neurotransmission such as Ca2+ buffering.

PDZD8-FKBP8 tethering complex at ER-mitochondria contact sites regulates mitochondrial complexity.

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

A highly sensitive label-free electrochemical aptasensor for interferon-gamma detection based on graphene controlled assembly and nuclease cleavage-assisted target recycling amplification.

We report here a highly sensitive and label-free electrochemical aptasensing technology for detection of interferon-gamma (IFN-γ) based on graphene controlled assembly and enzyme cleavage-assisted target recycling amplification strategy. In this work, in the absence of IFN-γ, the graphene could not be assembled onto the 16-mercaptohexadecanoic acid (MHA) modified gold electrode because the IFN-γ binding aptamer was strongly adsorbed on the graphene due to the strong π-π interaction. Thus the electronic transmission was blocked (eT OFF). However, the presence of target IFN-γ and DNase I led to desorption of aptamer from the graphene surface and further cleavage of the aptamer, thereby releasing the IFN-γ. The released IFN-γ could then re-attack other aptamers on the graphene, resulting in the successive release of the aptamers from the graphene. At the same time, the "naked" graphene could be assembled onto the MHA modified gold electrode with hydrophobic interaction and π-conjunction, mediating the electron transfer between the electrode and the electroactive indicator. Then, measurable electrochemical signals were generated (eT ON), which was related to the concentration of the IFN-γ. By taking advantages of graphene and enzyme cleavage-assisted target recycling amplification, the developed label-free electrochemical aptasensing technology showed a linear response to concentration of IFN-γ range from 0.1 to 0.7 pM. The detection limit of IFN-γ was determined to be 0.065 pM. Moreover, this aptasensor shows good selectivity toward the target in the presence of other relevant proteins. Our strategy thus opens new opportunities for label-free and amplified detection of other kinds of proteins.

A label-free electrochemical assay for methyltransferase activity detection based on the controllable assembly of single wall carbon nanotubes.

A sensitive label-free "signal-on" electrochemical approach for detection of methyltransferases (MTase) activity is developed based on the signal transduction and amplification of single wall carbon nanotubes (SWCNTs). In this method, the oligonucleotide I is first self-assembled on the electrode via Au-S bonding. After hybridization with its complement ssDNA (oligonucleotide II), duplex strand DNA (dsDNA) probes containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I is then formed on the electrode. In the presence of Dam MTase and Dpn I, the dsDNA probes are methylated and subsequently cleaved into two dsDNA fragments. After heating, the remained dsDNA fragments on the electrode melted into ssDNA fragments. Then the SWCNTs can be controllably assembled on the ssDNA fragments remained on the electrode, mediating efficient electron transfer between the electrode and electroactive species. It generates measurable current signal (eT ON), which is related to the concentration of the Dam MTase. The resulting change in electron transfer efficiency is readily measured by differential pulse voltammetry at Dam MTase concentrations as low as 0.04 U/mL. This method does not need electroactive molecules labeling on the methylation-responsive DNA probes. The linear response of the developed facile signal-on electrochemical sensing system for Dam MTase is in the range of 0.1-1.0 U/mL. In addition, such a SWCNTs based electrochemical assay also has the ability to screen inhibitors for Dam MTase.

Ru(II) encapsulated phosphorylate-terminated silica nanoparticles-based electrochemiluminescent strategy for label-free assay of protein kinase activity and inhibition.

A highly sensitive and simple label-free electrochemiluminescent (ECL) sensing strategy has been developed for assay of protein kinase A (PKA) activity and inhibition by taking advantage of zirconium cation (Zr(4+)) mediated signal transition and signal amplification of Ru(II) encapsulated phosphorylate-terminated silica nanoparticles (R-PSiNPs). In the protocol, an N-terminally cysteine-containing peptide (S-peptide) is self-assembled onto the gold electrode via Au-S bonding and used as substrate for PKA. The R-PSiNPs are chosen as the signal indicator by virtue of the intrinsic phosphate groups on the surface of the silica nanoparticles and the high loading of Ru(II) markers for ECL signal generation and amplification. The substrate peptide on the electrode is phosphorylated by PKA in the presence of ATP. The phosphorylated peptide (P-peptide) is subsequently linked with the R-PSiNPs by Zr(4+). The R-PSiNPs then can be grafted to the surface of Au electrode and generate high ECL signal. The ECL intensity is proportional to the activity of PKA. Due to the high loading of Ru(II) markers in a single phosphorylate-terminated silica nanoparticle, this strategy can be employed to assay PKA activity with a low detection limit of 0.005 U/mL. The linear range of the assay for PKA was 0.01 U/mL to 1 U/mL. Furthermore, the interferences experiments of CK2 and PKA inhibition have been also studied by using this strategy. This selective and sensitive method does not require labeling of the substrate peptide with ECL molecules, which provides a diversified platform for kinase activity and inhibition assay.