Movement and structure of mitochondria in oligodendrocytes and their myelin sheaths.

Mitochondria play several crucial roles in the life of oligodendrocytes. During development of the myelin sheath they are essential providers of carbon skeletons and energy for lipid synthesis. During normal brain function their consumption of pyruvate will be a key determinant of how much lactate is available for oligodendrocytes to export to power axonal function. Finally, during calcium-overload induced pathology, as occurs in ischemia, mitochondria may buffer calcium or induce apoptosis. Despite their important functions, very little is known of the properties of oligodendrocyte mitochondria, and mitochondria have never been observed in the myelin sheaths. We have now used targeted expression of fluorescent mitochondrial markers to characterize the location and movement of mitochondria within oligodendrocytes. We show for the first time that mitochondria are able to enter and move within the myelin sheath. Within the myelin sheath the highest number of mitochondria was in the cytoplasmic ridges along the sheath. Mitochondria moved more slowly than in neurons and, in contrast to their behavior in neurons and astrocytes, their movement was increased rather than inhibited by glutamate activating NMDA receptors. By electron microscopy we show that myelin sheath mitochondria have a low surface area of cristae, which suggests a low ATP production. These data specify fundamental properties of the oxidative phosphorylation system in oligodendrocytes, the glial cells that enhance cognition by speeding action potential propagation and provide metabolic support to axons.

Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA.

Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

Isolation of mitochondria from tissue culture cells.

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ∼109 cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation.

Isolation of mitochondria from animal tissue.

Rat or mouse liver is the most frequently used tissue for mitochondrial preparations because it is readily available, easy to homogenize, and replete with mitochondria. A motor-driven Teflon and glass Potter-Elvehjem homogenizer is the best choice for homogenizing liver, but if one is not available, this tissue is soft enough that a Dounce homogenizer with a loose (A) pestle can also be used. The yield and purity of the mitochondrial preparation will be influenced by the method and speed of preparation and the age and physiological condition of the animal.

Purification of mitochondria by sucrose step density gradient centrifugation.

Mitochondrial fractions isolated from tissue culture cells or tissue such as liver after differential centrifugation can be purified further by density gradient centrifugation. Here we describe the use of sucrose for this purpose because it is commonly used and inexpensive and the resulting mitochondria preparations are useful for many purposes.

Isolation of mitochondria from cells and tissues.

Mitochondria are complex organelles at the center of cellular metabolism, apoptosis, and signaling. They continue to be the subject of intense basic investigation to understand their composition and function, but they have also captivated the attention of clinical researchers because of the growing knowledge of the (sometimes unexpected) roles of mitochondria in human diseases and aging. A full understanding of these intriguing organelles often requires their purification from cells or tissues under specific physiological or pathological conditions. Here we provide some introductory considerations for those interested in purifying mitochondria for subsequent downstream biophysical, structural, and functional analysis.

Correlative photoactivated localization and scanning electron microscopy.

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.

Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.

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.

Approaches toward super-resolution fluorescence imaging of mitochondrial proteins using PALM.

Mitochondria are difficult targets for microscopy because of their small size and highly compartmentalized, membranous interior. Super-resolution fluorescence microscopy methods have recently been developed that exceed the historical limits of optical imaging. Here we outline considerations and techniques in preparing to image the relative location of mitochondrial proteins using photoactivated localization microscopy (PALM). PALM and similar methods have the capacity to dramatically increase our ability to image proteins within mitochondria, and to expand our knowledge of the location of macromolecules beyond the current limits of immunoEM.

Native R-loops persist throughout the mouse mitochondrial DNA genome.

Mammalian mtDNA has been found here to harbor RNA-DNA hybrids at a variety of locations throughout the genome. The R-loop, previously characterized in vitro at the leading strand replication origin (OH), is isolated as a native RNA-DNA hybrid copurifying with mtDNA. Surprisingly, other mitochondrial transcripts also form stable partial R-loops. These are abundant and affect mtDNA conformation. Current models regarding the mechanism of mammalian mtDNA replication have been expanded by recent data and discordant hypotheses. The presence of stable, nonreplicative, and partially hybridized RNA on the mtDNA template is significant for the reevaluation of replication models based on two-dimensional agarose gel analyses. In addition, the close association of a subpopulation of mtRNA with the DNA template has further implications regarding the structure, maintenance, and expression of the mitochondrial genome. These results demonstrate that variously processed and targeted mtRNAs within mammalian mitochondria likely have multiple functions in addition to their conventional roles.

Initiation and beyond: multiple functions of the human mitochondrial transcription machinery.

Mitochondria contain their own DNA (mtDNA) that is expressed and replicated by nucleus-encoded factors imported into the organelle. Recently, the core human mitochondrial transcription machinery has been defined, comprising a bacteriophage-related mtRNA polymerase (POLRMT), an HMG-box transcription factor (h-mtTFA), and two transcription factors (h-mtTFB1 and h-mtTFB2) that also serve as rRNA methyltransferases. Here, we describe these transcription components as well as recent insights into the mechanism of human mitochondrial transcription initiation and its regulation. We also discuss novel roles for the mitochondrial transcription machinery beyond transcription initiation, including priming of mtDNA replication, packaging of mtDNA, coordination of ribosome biogenesis, and coupling of transcription to translation.

Genesis and wanderings: origins and migrations in asymmetrically replicating mitochondrial DNA.

Mammalian mitochondria maintain a small circular genome that encodes RNA and polypeptides that are essential for the generation of ATP through oxidative phosphorylation. The mechanism of replication of mammalian mitochondrial DNA (mtDNA) has recently been a topic of controversy. New evidence has led to a modified strand-displacement model that reconciles much of the current data. This revision stems from a new appreciation for alternative light-strand origins. We consider here some of the potential mechanisms for light-strand origin initiation. We also consider further the susceptibility of branch migration within replicating mtDNA molecules. The existence of alternative light-strand origins and a propensity for branch migration in replicating mtDNA molecules exposes a new array of possible configurations of mtDNA. The assortment and assignment of these forms is relevant to the interpretation of experimental data and may also yield insight into the molecular basis of replication errors.

Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism.

The established strand-displacement model for mammalian mitochondrial DNA (mtDNA) replication has recently been questioned in light of new data using two-dimensional (2D) agarose gel electrophoresis. It has been proposed that a synchronous, strand-coupled mode of replication occurs in tissues, thereby casting doubt on the general validity of the "orthodox," or strand-displacement model. We have examined mtDNA replicative intermediates from mouse liver using atomic force microscopy and 2D agarose gel electrophoresis in order to resolve this issue. The data provide evidence for only the orthodox, strand-displacement mode of replication and reveal the presence of additional, alternative origins of lagging light-strand mtDNA synthesis. The conditions used for 2D agarose gel analysis are favorable for branch migration of asymmetrically replicating nascent strands. These data reconcile the original displacement mode of replication with the data obtained from 2D gel analyses.

A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient mouse hearts.

We performed global gene expression analyses in mouse hearts with progressive respiratory chain deficiency and found a metabolic switch at an early disease stage. The tissue-specific mitochondrial transcription factor A (Tfam) knockout mice of this study displayed a progressive heart phenotype with depletion of mtDNA and an accompanying severe decline of respiratory chain enzyme activities along with a decreased mitochondrial ATP production rate. These characteristics were observed after 2 weeks of age and became gradually more severe until the terminal stage occurred at 10-12 weeks of age. Global gene expression analyses with microarrays showed that a metabolic switch occurred early in the progression of cardiac mitochondrial dysfunction. A large number of genes encoding critical enzymes in fatty acid oxidation showed decreased expression whereas several genes encoding glycolytic enzymes showed increased expression. These alterations are consistent with activation of a fetal gene expression program, a well-documented phenomenon in cardiac disease. An increase in mitochondrial mass was not observed until the disease had reached an advanced stage. In contrast to what we have earlier observed in respiratory chain-deficient skeletal muscle, the increased mitochondrial biogenesis in respiratory chain-deficient heart muscle did not increase the overall mitochondrial ATP production rate. The observed switch in metabolism is unlikely to benefit energy homeostasis in the respiratory chain-deficient hearts and therefore likely aggravates the disease. It can thus be concluded that at least some of the secondary gene expression alterations in mitochondrial cardiomyopathy do not compensate but rather directly contribute to heart failure progression.

Mitochondrial DNA replication: what we know.

Since the isolation and physical characterization of mammalian mitochondrial DNA (mtDNA) over 35 years ago, numerous works have been published that have examined its physical structure and properties, including its mode of replication and transcription. The established replication model posits that leading-strand replication of mammalian mtDNA begins at closely spaced, defined sites located downstream from a major transcription promoter and proceeds unidirectionally with displacement of the parental leading strand until approximately two-thirds of the closed circular mtDNA has been copied. As a consequence, the replication fork passes a major origin for lagging-strand synthesis, exposing it in single-stranded form. Displacement as a single-strand is thought to allow the characteristic secondary structure of this origin to occur, thereby permitting initiation of lagging-strand synthesis. A natural consequence of the separate and distinct locations of the two origins is that the two segregated progeny mtDNA circles are of two types: one a duplex circle with a newly synthesized leading strand and the other a gapped circle with a partial newly synthesized lagging strand. In each case, the final steps of synthesis and ligation result in closed circular mtDNA products. Recently, mammalian mtDNA isolates have been subjected to 2D-gel electrophoretic analysis in attempts to assign features to mtDNA molecules that, by virtue of their anomalous migration behavior, could infer them to be candidates for replication intermediates. This review will describe the essential features of the historical findings of mammalian mtDNA replication studies and integrate the more recent observations in developing a current model for this process.

Release of replication termination controls mitochondrial DNA copy number after depletion with 2',3'-dideoxycytidine.

Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O(H)) are prematurely terminated, defining the 3' boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to approximately 20% of normal levels by treating with 2',3'-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O(H) strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.