Carol A. Cooke

Identification and characterization of a functional mitochondrial angiotensin system

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

Structure of the human centromere at metaphase

Until recently the centromere was thought to be a relatively homogeneous region of densely packed heterochromatin with a single differentiated domain--the kinetochore--at its surface, representing the point of attachment of the mitotic spindle. We now know that the centromere of higher eukaryotes is composed of several domains that have been identified using antibody probes. Somewhere within the domains are located both the factor(s) that control the disjunction of sister chromatids and the molecular motor responsible for chromosome movement towards the spindle poles.

Ultrastructure and Regulation of Lateralized Connexin43 in the Failing Heart

Rationale: Gap junctions mediate cell-to-cell electric coupling of cardiomyocytes. The primary gap junction protein in the working myocardium, connexin43 (Cx43), exhibits increased localization at the lateral membranes of cardiomyocytes in a variety of heart diseases, although the precise location and function of this population is unknown. Objective: To define the subcellular location of lateralized gap junctions at the light and electron microscopic level, and further characterize the biochemical regulation of gap junction turnover. Methods and Results: By electron microscopy, we characterized gap junctions formed between cardiomyocyte lateral membranes in failing canine ventricular myocardium. These gap junctions were varied in structure and appeared to be extensively internalizing. Internalized gap junctions were incorporated into multilamellar membrane structures, with features characteristic of autophagosomes. Intracellular Cx43 extensively colocalized with the autophagosome marker GFP-LC3 when both proteins were exogenously expressed in HeLa cells, and endogenous Cx43 colocalized with GFP-LC3 in neonatal rat ventricular myocytes. Furthermore, a distinct phosphorylated form of Cx43, as well as the autophagosome-targeted form of LC3 (microtubule-associated protein light chain 3) targeted to lipid rafts in cardiac tissue, and both were increased in heart failure. Conclusions: Our data demonstrate a previously unrecognized pathway of gap junction internalization and degradation in the heart and identify a cellular pathway with potential therapeutic implications.

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

During the shaping of mitotic chromosomes, KIF4 and condensin work in parallel to promote lateral chromatid compaction and in opposition to topoisomerase IIα, which shortens the chromatid arms.

Cardiac nitric oxide synthase-1 localization within the cardiomyocyte is accompanied by the adaptor protein, CAPON

The mechanism(s) regulating nitric oxide synthase-1 (NOS1) localization within the cardiac myocyte in health and disease remains unknown. Here we tested the hypothesis that the PDZ-binding domain interaction between CAPON (carboxy-terminal PDZ ligand of NOS1), a NOS1 adaptor protein and NOS1, contribute to NOS1 localization in specific organelles within cardiomyocytes. Ventricular cardiomyocytes and whole heart homogenates were isolated from sham and post-myocardial infarction (MI) wild-type (C57BL/6) and NOS1(-/-) female mice for quantification of CAPON protein expression levels. NOS1, CAPON, xanthine oxidoreductase and Dexras1, a CAPON binding partner, were all present and enriched in isolated cardiac sarcoplasmic reticulum (SR) fractions. CAPON co-immunoprecipitated with the mu and alpha isoforms of NOS1 in whole heart lysates, and co-localization of CAPON and NOS1 was demonstrated in the SR and mitochondria with dual immuno-gold electron microscopy. Following MI, CAPON and NOS1 both redistributed to caveolae and colocalized with caveolin-3. In addition, following MI, expression level of CAPON remained unchanged and Dexras1 was reduced, CAPON binding to xanthine oxidoreductase was augmented and the plasma membrane calcium ATPase (PMCA) increased. In NOS1 deficient myocytes, CAPON abundance in the SR was reduced, and redistribution to caveolae and PMCA binding after MI was absent. Together these findings support the hypothesis that NOS1 redistribution in injured myocardium requires the formation of a complex with the PDZ adaptor protein CAPON.

A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes

We describe a method for the isolation of conditional knockouts of essential multiply spliced genes in which the entire body of the gene downstream of the ATG start codon is left untouched but can be switched off rapidly and completely by adding tetracycline to the culture medium. The approach centers on a “promoter-hijack” strategy in which the genes promoter is replaced with a minimal promoter responsive to the tetracycline-repressible transactivator (tTA). Elsewhere in the genome, a cloned fragment of the genes promoter is used to drive expression of a tTA. Thus, the gene is essentially regulated by its own promoter but through the intermediary tTA. Using this strategy, we generated a conditional knockout of chromokinesin KIF4A, an important mitotic effector protein whose mRNA is multiply spliced and whose cDNA is highly toxic when overexpressed in cells. We used chicken DT40 cells, but the same strategy should be applicable to ES cells and, eventually, to mice.

Impaired mitochondrial degradation by autophagy in the skeletal muscle of the aged female interleukin 10 null mouse

Mitochondrial dysfunction, chronic inflammation and muscle aging are closely linked. Mitochondrial clearance is a process to dampen inflammation and is a critical pre-requisite to mitobiogenesis. The combined effect of aging and chronic inflammation on mitochondrial degradation by autophagy is understudied. In interleukin 10 null mouse (IL-10(tm/tm)), a rodent model of chronic inflammation, we studied the effects of aging and inflammation on mitochondrial clearance. We show that aging in IL-10(tm/tm) is associated with reduced skeletal muscle mitochondrial death signaling and altered formation of autophagosomes, compared to age-matched C57BL/6 controls. Moreover, skeletal muscles of old IL-10(tm/tm) mice have the highest levels of damaged mitochondria with disrupted mitochondrial ultrastructure and autophagosomes compared to all other groups. These observations highlight the interface between chronic inflammation and aging on altered mitochondrial biology in skeletal muscles.

Nuclear structure and chromosome segregation in Drosophila male meiosis depend on the ubiquitin ligase dTopors.

In many organisms, homolog pairing and synapsis at meiotic prophase depend on interactions between chromosomes and the nuclear membrane. Male Drosophila lack synapsis, but nonetheless, their chromosomes closely associate with the nuclear periphery at prophase I. To explore the functional significance of this association, we characterize mutations in nuclear blebber (nbl), a gene required for both spermatocyte nuclear shape and meiotic chromosome transmission. We demonstrate that nbl corresponds to dtopors, the Drosophila homolog of the mammalian dual ubiquitin/small ubiquitin-related modifier (SUMO) ligase Topors. We show that mutations in dtopors cause abnormalities in lamin localizations, centriole separation, and prophase I chromatin condensation and also cause anaphase I bridges that likely result from unresolved homolog connections. Bridge formation does not require mod(mdg4) in meiosis, suggesting that bridges do not result from misregulation of the male homolog conjunction complex. At the ultrastructural level, we observe disruption of nuclear shape, an uneven perinuclear space, and excess membranous structures. We show that dTopors localizes to the nuclear lamina at prophase, and also transiently to intranuclear foci. As a role of dtopors at gypsy insulator has been reported, we also asked whether these new alleles affected expression of the gypsy-induced mutation ct6 and found that it was unaltered in dtopors homozygotes. Our results indicate that dTopors is required for germline nuclear structure and meiotic chromosome segregation, but in contrast, is not necessary for gypsy insulator function. We suggest that dtopors plays a structural role in spermatocyte lamina that is critical for multiple aspects of meiotic chromosome transmission.

Molecular cloning and characterization of human centromeric autoantigen CENP-C: a component of the inner kinetochore plate

Chromosomes move in mitosis as a result of interactions between the centromere region and microtubules of the mitotic spindle. It is currently believed that a specialized chromosomal substructure located at the surface of the centromere is responsible for these movements. This structure, the kinetochore, is typically described as a three-layered disk composed of two dense plates with a space between them (Rieder, 1982). The inner dense plate is congruent with the surface of the centromeric heterochromatin, and under some circumstances is not clearly resolved from the surrounding chromatin. In human the three layers are all about 35 nm thick and 430 nm in diameter (diameter estimated from Rieder (1982) assuming 20 microtubules per chromosome).