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.

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.

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.

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 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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Differences in nuclear gene expression between cells containing monomer and dimer mitochondrial genomes.

It is known that point mutations and rearrangements (deletions and duplications) of mammalian mitochondrial DNA (mtDNA) can result in mitochondrial dysfunction and human disease. Very little attention has been paid to mtDNA circular dimers (a complex form consisting of two genomes joined head-to-tail) despite their close association with human neoplasia. MtDNA dimers are frequently found in human leukemia, but the clinical relevance of their presence remains unknown.