Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

Prolonged depolarization induces a slow inactivation process in some K⁺ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K⁺ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K⁺ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.     

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.     

Segregation-distortion in the B-chromosome system ofTettigidea lateralis (say) (Orthoptera: Tetrigidae)

A marginal population ofTettigidea lateralis was found to be polymorphic with respect to a large, mitotically stable supernumerary (B) chromosome. Male and female individuals may carry one or two B-chromosomes. In the male sex the frequency of individuals with one B was 33.8% whereas that of 2B-carriers was 2.9 %. A comparison with a small sample of female individuals suggests similar frequencies of B-chromosome carriers in the two sexes. The pycnocity cycle of the B's is virtually identical to that of the X chromosome which is always distinguishable by virtue of its larger size and other structural details. Persistent heterochromatic associations between the B and the X, which may last until metaphaseanaphase I, lead to a preferential migration of the B with the X to the same pole in male carriers of a single supernumerary. This distortional segregation of the B-chromosome may produce a differential transmission of the supernumerary to the two sexes if the various types of gametes are equally functional. Achiasmate, persistent B-B associations in 2B individuals can also cause segregation-distortion. The two supernumeraries segregate to the same pole in approximately 1/3 of the spermatocytes, but their poleward movement relative to that of the X is random. Both –B and +B individuals show only a single chiasma per individual bivalent. However, the presence of a single B raises very significantly the frequency at which the chiasma forms at the extreme distal ends of the L₁-L₂ and M₃-M₄ autosomes. The effect on recombination exerted by the supernumeraries and the possible implications of the segregation-distortion system ofT. lateralis are discussed in the light of recent studies on comparable B-chromosome polymorphisms.Research supported by N.R.C. of Canada.     

ConfocalVR: Immersive Visualization for Confocal Microscopy

ConfocalVR is a virtual reality (VR) application created to improve the ability of researchers to study the complexity of cell architecture. Confocal microscopes take pictures of fluorescently labeled proteins or molecules at different focal planes to create a stack of two-dimensional images throughout the specimen. Current software applications reconstruct the three-dimensional (3D) image and render it as a two-dimensional projection onto a computer screen where users need to rotate the image to expose the full 3D structure. This process is mentally taxing, breaks down if you stop the rotation, and does not take advantage of the eye's full field of view. ConfocalVR exploits consumer-grade VR systems to fully immerse the user in the 3D cellular image. In this virtual environment, the user can (1) adjust image viewing parameters without leaving the virtual space, (2) reach out and grab the image to quickly rotate and scale the image to focus on key features, and (3) interact with other users in a shared virtual space enabling real-time collaborative exploration and discussion. We found that immersive VR technology allows the user to rapidly understand cellular architecture and protein or molecule distribution. We note that it is impossible to understand the value of immersive visualization without experiencing it first hand, so we encourage readers to get access to a VR system, download this software, and evaluate it for yourself. The ConfocalVR software is available for download at http://www.confocalvr.com, and is free for nonprofits.     

Reduced mitochondrial Ca2+ transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase

Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca²⁺ homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca²⁺ uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca²⁺ homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.Mitochondrial disorders include a wide range of pathological conditions characterized by defects in organelle homoeostasis and energy metabolism, in particular in the electron transport chain (ETC) complexes. They are mostly caused by mutations in nuclear- or mtDNA-encoded genes of the respiratory chain complexes leading to a variety of clinical manifestations, ranging from lesions in specific tissues, such as in Leber''s hereditary optic neuropathy, to complex multisystem syndromes, such as myoclonic epilepsy with ragged-red fibers, Leigh syndrome or the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS).^{1, 2} Despite the detailed knowledge of the molecular defects in these diseases, their pathogenesis remains poorly understood. The heterogeneity of signs and symptoms depends on the diversity of the genetic background and on patient-specific compensatory mechanisms. Several studies investigated the consequences of nuclear DNA mutations on intracellular organelle physiology and Ca²⁺ homeostasis.^{3, 4} Here we analyzed a cohort of patients with mutations in the mtDNA-encoded ND5 subunit of NADH dehydrogenase in order to correlate the clinical phenotype with relevant intracellular parameters involved in mitochondrial physiology, such as the rate of autophagy and mitophagy fluxes, mitochondrial Ca²⁺ dynamics, mitochondrial membrane potential and their functional relationship.Mitochondrial Ca²⁺ is a key regulator of organelle physiology, and impairment of cation homeostasis is a general feature of many pathological conditions, including mitochondrial diseases.⁵ In addition, Ca²⁺ uptake in this organelle has recently been demonstrated to be a fundamental regulator of autophagy.^{6, 7} Autophagy is involved in physiological organelle turnover and in the removal of damaged or non-functional mitochondria by autophagy (called ‘mitophagy'')^{8, 9, 10, 11} and is critical for organelle quality control. Given the pivotal role of mitochondrial Ca²⁺ in the adaptation of adenosine triphosphate (ATP) production to cellular energy demand, the recent identification of the channel responsible for Ca²⁺ entry into the organelle, the mitochondrial Ca²⁺ uniporter (MCU), is instrumental for the understanding of the regulation of mitochondrial Ca²⁺ transport in both physiological and pathological conditions. MCU was identified in 2011,^{12, 13} and in the following years, molecular insight on its complex regulatory mechanism was obtained. The pore region is composed of MCU, its isoform MCUb¹⁴ and essential MCU regulator (EMRE).¹⁵ The channel is gated by the Ca²⁺-sensitive proteins mitochondrial Ca²⁺ uptake 1 (MICU1) and MICU2^{16, 17, 18, 19} and further regulated by the SLC25A23 protein.²⁰ As to its cellular function, mitochondrial Ca²⁺ has been shown to stimulate ATP production by positive regulation of three key dehydrogenases of the tricarboxylic acid cycle²¹ and of the ETC.²² In parallel, unregulated and sustained organelle Ca²⁺ overload can also lead to the opening of the mitochondrial permeability transition pore,^{23, 24} with consequent dissipation of mitochondrial membrane potential (ΔΨ_mt), release of caspase cofactors and activation of the apoptotic cascade.⁵ Despite the significant molecular understanding of all these cellular processes, their role in the pathogenesis of mitochondrial diseases is still poorly understood. Here we investigated the interplay of these pathways and the possibility of their contribution to determine the severity of the pathology in a cellular model consisting of fibroblasts from patients carrying mutations in the mitochondrial ND5 gene.