title: Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy.
abstract: Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. One of the principal determinants of morbidity and mortality following TBI is traumatic axonal injury (TAI). Current hypotheses on the pathogenesis of TAI involve activation of apoptotic cascades secondary to TBI. While a number of studies have demonstrated direct evidence for the activation of apoptotic cascades in TAI, the precise pathway by which these cascades are initiated remains a subject of intense investigation. As axolemmal disruption with the subsequent intra-axonal influx of large molecular weight species has been demonstrated to occur in relation to local axonal breakdown, attention has focused on cascades that may occur as a result of loss of ionic homeostasis. One proposed pathway by which this has been hypothesized to occur is the Ca(2+)-mediated activation of calmodulin and subsequent activation of the phosphatase calcineurin with dephosphorylation of a protein known as BAD, leading to a proapoptotic interaction between BAD and the mitochondrial protein Bcl-xL. While this pathway is an intriguing route for traumatic axonal pathogenesis, neither conventional immunocytochemical/histochemical nor ultrastructural approaches have had the capacity to shed insight on whether BAD and Bcl-xL interact in TAI in vivo. We describe the implementation of confocal and two-photon excitation fluorescence resonance energy transfer (FRET) microscopy techniques through which we demonstrate interaction between the proapoptotic protein BAD and the prosurvival protein Bcl-xL within TAI following TBI. Further, we report on a method to reliably detect protein interactions within aldehyde fixed tissue sections through conventional immunohistochemical approaches.
authors.affiliation: University of Virginia Health Sciences Center, Department of Neurosurgery, Charlottesville, Virginia 22908, USA.
authors.forename: James D
authors.initials: JD
authors.lastname: Mills
authors.forename: James R
authors.initials: JR
authors.lastname: Stone
authors.forename: David G
authors.initials: DG
authors.lastname: Rubin
authors.forename: David E
authors.initials: DE
authors.lastname: Melon
authors.forename: David O
authors.initials: DO
authors.lastname: Okonkwo
authors.forename: Ammasi
authors.initials: A
authors.lastname: Periasamy
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Helm
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism* / Q000473:pathology; D001921:Brain / Q000378:metabolism / Q000473:pathology; D001930:Brain Injuries / Q000209:etiology / Q000378:metabolism* / Q000473:pathology*; D002352:Carrier Proteins / Q000378:metabolism*; D020833:Diffuse Axonal Injury / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016489:Head Injuries, Closed / Q000150:complications / Q000378:metabolism / Q000473:pathology; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051381:Rats; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
chemical_list: C494821:Bad protein, rat; C494813:Bcl2l1 protein, rat; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein