Apical Endoplasmic Reticulum Research Articles

Highly localized intracellular Ca2+ signals promote optimal salivary gland fluid secretion.

Salivary fluid secretion involves an intricate choreography of membrane transporters to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are largely based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels, present on the apical and basolateral plasma membrane, respectively. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to fully propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. A new mathematical model, incorporating these data was constructed to probe how salivary secretion can be optimally stimulated by apical Ca2+ signals.

A mathematical model of intricate calcium dynamics and modulation of calcium signalling by mitochondria in pancreatic acinar cells

• The model is divided into two sub domains: the apical cytosolic sub domain, and the mitochondrial sub domain. However, these two sub domains are connected with the Ca2+. • The proposed model is Class II model. It includes the dynamics of IP3, we take into account the Ca2+ modulation of IP3 generation through PLC, and Ca2+ activated IP3 removal by IP3K. • The biphasic regulation of mitochondrial Ca2+ uptake by apical Ca2+ concentration, and the rapid mode of Ca2+ uptake through mitochondria are considered. • In this model the three types of mechanism by Ca2+ have been well considered. Firstly, the time dependent gating of IPR by Ca2+. Secondly, the Ca2+ enhances the production of IP3. Subsequently, due to the 3- kinase stimulated by Ca2+, degradation of IP3. Thirdly, the gating of uniporter by Ca2+: i.e. Ca2+ activated CU uptake, and subsequently it inactivates the uniporter for further up taking Ca2+. Thus, a [Ca2+] concentration is required for the activation and the inactivation of CU. • How these two sub domain: apical sub domain and the mitochondrial sub domain combine together into a well- mixed model. • The bifurcation analysis is performed to identify the oscillatory structures depending on the different agonist strength to produce complex dynamics in the apical region of the cell. The complex bifurcation points like hopf, period doubling, torus, saddle node bifurcation are studied. The pancreatic acinar cells display wide varieties of Ca 2+ oscillations within the cell. The study of these calcium signals/oscillations are of great interest because they are physiologically important. The Ca 2+ release from the endoplasmic reticulum via inositol trisphosphate receptors (IPR) and the ryanodine receptors (RyR) creates a high cytosolic Ca 2+ signal in the acinar cell. The pancreatic acinar cells are highly polarized cells, and the Ca 2+ response is quite extensive in the apical (peri-granular) region than the basal region of the cell. The Ca 2+ signals which originated in the apical region control the exocytosis process and triggered peri-granular mitochondria (Mito) to uptake Ca 2+ . The peri-granular mitochondria play a critical role in confining cytosolic Ca 2+ elevation by acting as a Ca 2+ buffer barrier. They also participate in shaping the Ca 2+ response by restricting the Ca 2+ signals into the granular region of the acinar cells. They sequestered Ca 2+ very rapidly. Then they release Ca 2+ very slowly to provide Ca 2+ supply to cause Ca 2+ oscillations in the apical region. Thus, herein we proposed a Class II compartmental mathematical model which systematically simulates the complex Ca 2+ oscillations in the apical region, and the peri-granular mitochondria matrix of the pancreatic acinar cells. The model assumes the functional coupling between the IPR and the RyR through the mechanism of calcium induced calcium release (CICR). In the proposed compartmental model of Ca 2+ dynamics, the Ca 2+ dependent production and degradations of (1,4,5) inositol-trisphosphate (IP 3 ) along with the apical parameters, apical endoplasmic reticulum (ER), and the peri-granular mitochondria are considered. The bifurcation analysis of the model is performed which explores the intricate dynamic behaviour of the apical region of the acinar cells, and hence derive the complex Ca 2+ oscillations in both the apical region and the peri-granular mitochondria. The model predicts the different patterns of Ca 2+ oscillation from the regular to sinusoidal to raised-baseline to sustained spikes in the apical region of the acinar cells.

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H2O2 in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.

Cystic Fibrosis Airway Epithelial Ca2+i Signaling: THE MECHANISM FOR THE LARGER AGONIST-MEDIATED Ca2+i SIGNALS IN HUMAN CYSTIC FIBROSIS AIRWAY EPITHELIA

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca(2+) stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y(2)-R activation-dependent Ca(2+)(i) responses, which reproduced the increased Ca(2+)(i) signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca(2+)(i) signals elicited by apical P2Y(2)-R activation in CF airway epithelia is an expansion of the apical ER Ca(2+) stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca(2+)(i) signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.

The endoplasmic reticulum as one continuous Ca(2+) pool: visualization of rapid Ca(2+) movements and equilibration.

We investigated whether the endoplasmic reticulum (ER) is a functionally connected Ca(2+) store or is composed of separate subunits by monitoring movements of Ca(2+) and small fluorescent probes in the ER lumen of pancreatic acinar cells, using confocal microscopy, local bleaching and uncaging. We observed rapid movements and equilibration of Ca(2+) and the probes. The bulk of the ER at the base was not connected to the granules in the apical part, but diffusion into small apical ER extensions occurred. The connectivity of the ER Ca(2+) store was robust, since even supramaximal acetylcholine (ACh) stimulation for 30 min did not result in functional fragmentation. ACh could elicit a uniform decrease in the ER Ca(2+) concentration throughout the cell, but repetitive cytosolic Ca(2+) spikes, induced by a low ACh concentration, hardly reduced the ER Ca(2+) level. We conclude that the ER is a functionally continuous unit, which enables efficient Ca(2+) liberation. Ca(2+) released from the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.

A radioautographic comparison of glycerol-H3 and galactose-H3 uptake during intestinal glyceride synthesis.

AbstractRadioautography was used to compare the in vivo incorporation of glycerol‐H3 and galactose‐H3 in intestinal absorptive cells in relation to glyceride synthesis. Each labeled compound was injected, either singly or mixed with linoleic acid chyme, directly into ligated segments of rat upper jejunum. The segments were removed at 5 and 20 minutes and prepared for light and electron microscopic radioautography. Following glycerol‐H3 and chyme injections, label is rapidly incorporated in the vicinity of newly formed fat droplets in the apical endoplasmic reticulum. Later, labeled droplets accumulate in dilated Golgi cisternae and intercellular spaces. Galactose label is initially found in the Golgi region and later in the apical cytoplasm. Glycerol labeling is considerably reduced in the cells when the fat is extracted prior to radioautography, or when glycerol alone is absorbed. Galactose labeling is not affected by these procedures. The results indicate a significant incorporation of glvcerol label into newly synthesized glycerides, as previously also shown for glucose label. Galactose label follows a different metabolic pathway not related to glyceride synthesis.