Superresolution Fluorescence Imaging of Mitochondrial Nucleoids Reveals Their Spatial Range, Limits, and Membrane Interaction

Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 12/2011; 31(24):4994-5010. DOI: 10.1128/MCB.05694-11
Source: PubMed


A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.

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    • "tion imaging 355 the image , and the axial sectioning capability is only ~1 μm . Epi - fluorescence PALM was used to image dendritic spines ( Izeddin et al . 2011 ) , microtubules ( Burnette et al . 2011 ; Cox et al . 2012 ) , actin bundles ( Hedde et al . 2009 ; Zhang et al . 2012 ) , desmin filaments ( Brodehl et al . 2012 ) , and mitochondria ( Brown et al . 2011 ) . In plant cells , localization microscopy is challenging due to autofluorescent compounds such as chlorophyll or secondary plant metabolites . Also , TIRF illumination is only applicable after protoplasting ( Hohenberger et al . 2011 ) because the thickness of the cell exceeds greatly the range of the evanescent wave . However , epi "
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    • "Contrary to the current paradigm, we have shown that a substantial nonmembrane-bound or loosely membrane-associated mtDNA–protein fraction also exists in mammalian mitochondria. A second recent super-resolution microscopy study has similarly suggested that not all nucleoids are in direct close contact with the inner mitochondrial membrane (38) and that consequently mtDNA–membrane interactions could be transient in nature. We can agree with these findings using a different, and in this case biochemical, approach, and show that transient interactions include association with Twinkle to facilitate mtDNA replication. "
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