Serial block face SEM visualization of unusual plant nuclear tubular extensions in a carnivorous plant (Utricularia, Lentibulariaceae)

Background and Aims In Utricularia nelumbifolia, the nuclei of placental nutritive tissue possess unusually shaped projections not known to occur in any other flowering plant. The main aim of the study was to document the morphology and ultrastructure of these unusual nuclei. In addition, the literature was searched to find examples of nuclear tubular projections in other plant groups, and the nuclei of closely related species of Utricularia (i.e. sects Iperua, Orchidioides, Foliosa and Utricularia) were examined. Methods To visualize the complexity of the nuclear structures, transmission electron microscopy (TEM) was used, and 3-D ultrastructural reconstructions were made using the serial block face scanning electron microscopy (SBEM) technique. The nuclei of 11 Utricularia species, i.e. U. nelumbifolia, U. reniformis, U. cornigera, U. nephrophylla (sect. Iperua), U. asplundii, U. alpina, U. quelchii (sect. Orchidioides), U. longifolia (sect. Foliosa), U. intermedia, U. minor and U. gibba (sect. Utricularia) were examined. Key Results Of the 11 Utricularia species examined, the spindle-like tubular projections (approx. 5 \mu m long) emanating from resident nuclei located in placental nutritive tissues were observed only in U. nelumbifolia. These tubular nuclear extensions contained chromatin distributed along hexagonally shaped tubules. The apices of the projections extended into the cell plasma membrane, and in many cases also made contact at the two opposing cellular poles, and with plasmodesmata via a short cisterna of the cortical endoplasmic reticulum. Images from the SBEM provide some evidence that the nuclear projections are making contact with those of neighbouring cells. Conclusions The term chromatubules (chromatin-filled tubules) for the nuclear projections of U. nelumbifolia placental tissue was proposed here. Due to the apparent association with the plasma membrane and plasmodesmata, it was also speculated that chromatubules are involved in nucleus-cell-cell communication. However, further experimental evidence is required before any functional hypothesis can be entertained

Store-operated calcium entry contributes to abnormal Ca²⁺ signalling in dystrophic mdx mouse myoblasts

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca2+ homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca2+ concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease

Confocal microscopy and wide-field microscopy, two approaches to the live study

Doświadczenia na żywych organizmach od lat były podstawą badań w naukach biologicznych. Pozwalają one na bezpośrednią obserwację mechanizmów biologicznych zachodzących w różnego rodzaju komórkach, mikrobach czy nawet w żywych zwierzętach. Dzięki rozwojowi różnego rodzaju technik fluorescencyjnych i wykorzystaniu tradycyjnej mikroskopii fluorescencyjnej czy mikroskopii konfokalnej badania przyżyciowe prowadzi się obecnie w laboratoriach na całym świecie. Rozwój technik obrazowania przyżyciowego na wielu płaszczyznach technologicznych, zaczynając od różnego rodzaju komponentów optycznych, mechanicznych czy elektronicznych, poprzez koncepcyjne podejścia do tego typu badań, a kończąc na tworzeniu nowych znaczników fluorescencyjnych, których właściwości chemiczne są dostosowane do konkretnych doświadczeń, pozwolił na obserwacje i śledzenie nawet pojedynczych molekuł czy badanie interakcji pomiędzy białkami. Artykuł ten przybliża podstawowe techniki badań przyżyciowych z wykorzystaniem mikroskopu szerokiego pola oraz mikroskopu konfokalnego, jak również zalety i wady stosowania obu rodzajów mikroskopów.Experiments on living organisms has always been one of the most important research in the life sciences. They allow direct observation of biological mechanisms occurring in different types of cells, microbes, or even in living animals. With the development of the various techniques for measuring fluorescence signals, using either wide field or confocal microscopy, are by now extremely well established and routine in many laboratories in the world. This resulted in development of live techniques in many areas of technology, starting with the various components (optical components, mechanical or electronic) by a conceptual approach to this type of research, ending with the creation of new fluorescent dyes which chemical properties are adjusted to the specific experiments. Thanks to the current available techniques, it is possible to observe and track even single molecules or study the interactions between proteins. This article introduces the basic techniques of live experiments using a wide field microscopy and confocal microscopy as well as the advantages and disadvantages of those two microscopes

AS-30D hepatoma as a model to study on insulin resistance in vitro

Studies on insulin resistance of liver cells are often performed with the use of various hepatoma cell lines. Such an approach allows investigating selected biochemical pathways at the cellular level. However, possible modifications of metabolic processes due to the neoplastic nature of such cells must be considered. Expanding the diversity of hepatoma cell lines used in metabolic studies could deliver new data for comparison with those obtained for other cell lines and should reduce the risk of misleading conclusions. In this study rat hepatoma AS-30D cells were tested as a potential model for studies on palmitate-induced insulin resistance. It was found that insulin-induced Akt kinase phosphorylation was substantially reduced in cells incubated with palmitate at a concentration as low as 75 µM. This effect was not accompanied by excessive reactive oxygen species (ROS) generation or increased Jun N-terminal kinase (JNK) phosphorylation. Moreover, preincubation of AS-30D cells with rosiglitazone, an antidiabetic agonist of peroxisome proliferator-activated receptor gamma (PPARγ), efficiently prevented the palmitate-induced insulin resistance. We conclude that AS-30D hepatoma cells may be used as a model sensitive to insulin and vulnerable to palmitate-induced insulin resistance

The regulatory role of mitochondria in capacitative calcium entry

AbstractCapacitative regulation of calcium entry is a major mechanism of Ca2+ influx into electrically non-excitable cells, but it also operates in some excitable ones. It participates in the refilling of intracellular calcium stores and in the generation of Ca2+ signals in excited cells. The mechanism which couples depletion of intracellular calcium stores located in the endoplasmic reticulum with opening of store-operated calcium channels in the plasma membrane is not clearly understood. Mitochondria located in close proximity to Ca2+ channels are exposed to high Ca2+ concentration, and therefore, they are able to accumulate this cation effectively. This decreases local Ca2+ concentration and thereby affects calcium-dependent processes, such as depletion and refilling of the intracellular calcium stores and opening of the store-operated channels. Finally, mitochondria modulate the intensity and the duration of calcium signals induced by extracellular stimuli. Ca2+ uptake by mitochondria requires these organelles to be in the energized state. On the other hand, Ca2+ flux into mitochondria stimulates energy metabolism. To sum up, mitochondria couple cellular metabolism with calcium homeostasis and signaling

CB1 Cannabinoid Receptor Expression in the Barrel Field Region Is Associated with Mouse Learning

We found previously that fear conditioning by combined stimulation of a row B facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) leads to expansion of the cortical representation of the “trained” row, labeled with 2-deoxyglucose (2DG), in the layer IIIb/IV of the adult mouse the primary somatosensory cortex (S1) 24 h later. We have observed that these learning-dependent plastic changes are manifested by increased expression of somatostatin, cholecystokinin (SST+, CCK+) but not parvalbumin (PV+) immunopositive interneurons We have expanded this research and quantified a numerical value of CB1-expressing and PV-expressing GABAergic axon terminals (CB1+ and PV+ immunopositive puncta) that innervate different segments of postsynaptic cells in the barrel hollows of S1 cortex. We used 3D microscopy to identify the CB+ and PV+ puncta in the barrel cortex “trained” and the control hemispheres CS+UCS group and in controls: Pseudoconditioned, CS-only, UCS-only, and naive animals. We have identified that (i) the association between whisker-shock “trained” barrel B hollows and CB1+, but not PV+ puncta expression remained significant after Bonferroni correction, (ii) CS+UCS has had a significant increasing effect on expression of CB1+ but not PV+ puncta in barrel cortex “trained” hemisphere, and (iii) the pseudoconditioning had a significant decreasing effect on expression of CB1+, but not on PV+ puncta in barrel cortex, both trained and untrained hemispheres. It is correlated to disturbing behaviors. The results suggest that CB1+ puncta regulation is specifically linked with mechanisms leading to learning-dependent plasticity in S1 cortex