Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1

Abstract

The mitochondrial signaling complex PKA/AKAP1 protects neurons against mitochondrial fragmentation and cell death by phosphorylating and inactivating the mitochondrial fission enzyme Drp1.

Allosteric Modulation of Drp1 Mechanoenzyme Assembly and Mitochondrial Fission by the Variable Domain

“…1 cover glasses (20 mm 2 chamber, Nalgen Nunc, Thermo Fisher) and transfected using Lipofectamine 2000 (Invitrogen). Cells were fixed with 4% paraformaldehyde in PBS and subjected to immunofluorescence staining with primary antibodies to cytochrome c oxidase II (COX2, Neomarkers) and GFP (Abcam), and Alexa Fluor 555-and 488-coupled secondary antibodies (Invitrogen) as reported (22). Images were captured at 1000ϫ magnification using a Leica epifluorescence microscope and processed for local contrast enhancement (contrast-limited adaptive histogram equalization) using ImageJ software.…”

“…Images subjected to red/green colocalization analysis underwent a second processing step involving iterative two-dimensional deconvolution (macro code and algorithm parameters are available upon request). To quantify mitochondrial morphology, mitochondria channel images were analyzed using a custom ImageJ macro described previously (22,23). Shape metrics reported here are aspect ratio (long axis divided by short axis of a best fit ellipse) and form factor (perimeter 2 /(4 ϫ ⌸ ϫ area)), both of which have a minimum value of 1 for perfectly circular mitochondria.…”

“…Fluorescence Recovery after Photobleaching (FRAP)-FRAP of GFP-Drp1 in HeLa cells was carried out as described (22). Diffusion coefficients of cytosolic GFP-Drp1 were approximated using the equation 0.224 ϫ r 2 /t1 ⁄ 2 , where r is the radius of the photobleached area and t1 ⁄ 2 is the time of half-recovery obtained by curve fitting (25,26).…”

“…Drp1 VD Is Autoinhibitory Domain-To assess the mitochondria-fragmenting activity of the Drp1⌬VDs, transfected HeLa cells were immunofluorescently labeled for COX2 to stain mitochondria, and epifluorescence micrographs were subjected to digital morphometry (22,23). Mitochondrial shape changes were quantified as aspect ratio and form factor, both of which have a minimum value of 1 for perfectly circular objects.…”

“…Phosphorylation Modulates Drp1 Assembly-Drp1 cycles between the cytosol and the OMM, and Drp1 turnover at the OMM correlates with mitochondrial fission rates (22,30). Because some hypoactive Drp1⌬VD mutants, such as ⌬VD5, form mitochondria-localized puncta, we reasoned that slow turnover may contribute to their functional impairment.…”