Abstract
Purpose: :
Thirteen-lined ground squirrels serve as a novel and fascinating physiological model in which to study mitochondrial dynamics and mitophagy because they hibernate. In doing so, they transition from a period of severely lowered oxygen consumption and core body temperature to their baseline awake conditions without suffering any damage secondary to the global decrease in metabolic rate or the reperfusion associated with awakening. We are investigating changes in mitochondrial morphology and proteins associated with fusion, fission and mitophagy in hibernating and awake animals, searching for adaptive changes that enable photoreceptors to survive these metabolically-challenged conditions.
Methods: :
We performed Western Blots on whole cell lysates and on fractionated and carbonate-extracted samples which isolate the mitochondrial membranes. We screened for changes in proteins known to be associated with mitochondrial fusion, including optic atrophy 1 (OPA1), OPA3, mitofusin1 (MFN1) and MFN2. We also assessed LC3-II, the post-translational form of a mammalian protein known to associate with autophagosomes, and PINK1 and Parkin, proteins essential to mitochondrial quality control. Immunohistochemistry and confocal microscopy were used to evaluate mitochondrial density and morphology. We are developing a novel strategy for culturing retinal tissue and will transfect the tissue with photoactivatable GFP in order to isolate single mitochondria for study.
Results: :
Preliminary results reveal a novel variation of MFN1, a GTPase associated with the fusion of mitochondrial outer membranes, that migrates on SDS gels more slowly. This variation is more prevalent in hibernating animals and segregates with non-membrane-bound proteins. MFN1 may be behaving like other well-known proteins which are redirected to alternative locations in hibernating animals as a mechanism for regulating activity (i.e. Plasma membrane Ca2+ ATPase, Ribbon protein - ribeye). Some percentage of the MFN1 protein could be shunted from its native position to a less active cytosolic form, thereby lowering mitochondrial fusion rates as demonstrated by the increased mitochondrial fragmentation in hibernating animals.
Conclusions: :
Hibernating animals may induce decreased mitochondrial fusion by redirecting MFN1 proteins from the mitochondrial outer membrane to an inactive state in the cytosol, thus reducing a given mitochondria’s ability to fuse with its neighbor.
Keywords: mitochondria • photoreceptors • protein modifications-post translational