Organellar Volume Research Articles

Solute Transport Controls Membrane Tension and Organellar Volume.

The regulation of cellular volume in response to osmotic change has largely been studied at the whole cell level. Such regulation occurs by the inhibition or activation of ionic and organic solute transport pathways at the cell surface and is coincident with remodelling of the plasma membrane. However, it is only in rare instances that osmotic insults are experienced by cells and tissues. By contrast, the relatively minute luminal volumes of membrane-bound organelles are constantly subject to shifts in their solute concentrations as exemplified in the endocytic pathway where these evolve alongside with maturation. In this review, we summarize recent evidence that suggests trafficking events are in fact orchestrated by the solute fluxes of organelles that briefly impose osmotic gradients. We first describe how hydrostatic pressure and the resultant tension on endomembranes can be readily dissipated by controlled solute efflux since water is obliged to exit. In such cases, the relief of tension on the limiting membrane of the organelle can promote its remodelling by coat proteins, ESCRT machinery, and motors. Second, and reciprocally, we propose that osmotic gradients between organellar lumens and the cytosol may persist or be created. Such gradients impose osmotic pressure and tension on the endomembrane that prevent its remodelling. The control of endomembrane tension is dysregulated in lysosomal storage disorders and can be usurped by pathogens in endolysosomes. Since trafficking and signaling pathways conceivably sense and respond to endomembrane tension, we anticipate that understanding how cells control organellar volumes and the movement of endocytic fluid in particular will be an exciting new area of research.

A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast.

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here, we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells, we observe drastic changes in organellar volume, leading to crowding on the micrometer scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.

Resolution of macropinosomes, phagosomes and autolysosomes: Osmotically driven shrinkage enables tubulation and vesiculation.

Macropinosomes, phagosomes and autolysosomes are comparatively large, quasi-spherical organelles that play essential functions in immunity and homeostasis. These vacuolar organelles are relatively short-lived, promptly fragmenting into smaller structures. Vacuolar resolution is mediated by tubulation and vesiculation, processes orchestrated by protein complexes that are recruited to highly curved membranes. Importantly, the surface-to-volume ratios of the tubules and vesicles generated during the resolution process are considerably larger than that of the parental vacuole. Because membranes under high hydrostatic tension resist deformation, an active, concomitant loss of volume is required to sustain the resolution process and may even initiate tubulation and vesiculation. Despite its fundamental role in membrane remodeling, the mechanisms that account for organellar volume loss are poorly understood, but are likely to involve the export of solutes followed by osmotically obliged water. In this review, we describe the principles and possible mechanisms underlying the resolution of organelles, with particular attention paid to the osmolytes they contain and the pathways mediating their exit.

The size-wise nucleus: nuclear volume control in eukaryotes

Eukaryotic cells have an “awareness” of their volume and organellar volumes, and maintain a nuclear size that is proportional to the total cell size. New studies in budding and fission yeast have examined the relationship between cell and nuclear volumes. It was found that the size of the nucleus remains proportional to cell size in a wide range of genetic backgrounds and growth conditions that alter cell volume and DNA content. Moreover, in multinucleated fission yeast cells, Neumann and Nurse (see p. 593 of this issue) found that the sizes of individual nuclei are controlled by the relative amount of cytoplasm surrounding each nucleus. These results highlight a role of the cytoplasm in nuclear size control.

Optimal observability of sustained stochastic competitive inhibition oscillations at organellar volumes

When molecules are present in small numbers, such as is frequently the case in cells, the usual assumptions leading to differential rate equations are invalid and it is necessary to use a stochastic description which takes into account the randomness of reactive encounters in solution. We display a very simple biochemical model, ordinary competitive inhibition with substrate inflow, which is only capable of damped oscillations in the deterministic mass-action rate equation limit, but which displays sustained oscillations in stochastic simulations. We define an observability parameter, which is essentially just the ratio of the amplitude of the oscillations to the mean value of the concentration. A maximum in the observability is seen as the volume is varied, a phenomenon we name system-size observability resonance by analogy with other types of stochastic resonance. For the parameters of this study, the maximum in the observability occurs at volumes similar to those of bacterial cells or of eukaryotic organelles.

The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.

Identification of a Mitochondrial Na+/H+Exchanger

The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.

Morphometric Assessment of Pulmonary Toxicity in the Rodent Lung

An overview of the epithelial and interstitial composition of rat respiratory airways shows complexity and variability. Airway epithelium varies in 1) different airway levels; 2) the types and ultrastructure of cells present; and 3) the abundance, type, and composition of stored secretory product. Unbiased sampling of airways is done using airway microdissection with a specific binary numbering system for airway generation. Vertical sections of selected airways are used to sample epithelium and interstitium. We determine the ratios of the volume of epithelial or interstitial cells to the total epithelial or interstitial volume (Vv). The surface of the epithelial basal lamina to the total epithelial or interstitial volume (Sv) is determined using point and intersection counting with a cycloid grid. Using the selector method on serial plastic sections, we determine the number of epithelial or interstitial cells per volume (Nv) of total epithelium or interstitium. We calculate the number of epithelial or interstitial cells per surface of epithelial basal lamina (Ns) by dividing Nv by Sv where the volumes are the same compartment. We calculate average cell volumes (v) for specific epithelial and interstitial cells by dividing the absolute nuclear volume by the ratio of the nucleus to cell volume (Vv). By multiplying the average cell volume (v) by the ratio of organellar volume to cell volume (Vv), we calculate the average organellar volume per cell. These unbiased stereological approaches are critical in a quantitative evaluation of toxicological injury of rat tracheobronchial airways.

Morphometric analysis of cucurbit mitochondria: The relationship between chondriome volume and DNA content

Electron micrographs of cucurbit seedling cells were subjected to morphometric analysis in order to determine organellar volumes. Serial sections revealed that for watermelon, zucchini squash, cucumber and muskmelon there were hundreds of small, ovoid mitochondria in cotyledon, leaf and hypocotyl cells, but only about 7 or 8 large, branching mitochondrial reticula in apical meristem cells of watermelon and muskmelon. For a given tissue there was little species to species difference in the volume of the cells, nuclei, total plastids and total mitochondria (the chondriome), although for each species the volumes were widely variable for cells and plastids, and somewhat variable for nuclei and the chondriome. Despite our previously determined 7- to 8-fold range in mitochondrial DNA content among cucurbit species, there was no variation among species in the volume of the chondriome for any of the four tissues analyzed. We conclude that for both individual, ovoid mitochondria and the entire chondriome, there is no correlation between volume and DNA content, as has been sometimes reported for the nucleus. Calculations show that in cells of differentiated tissues, the number of ovoid mitochondria may be larger than the number of mitochondrial genomes.

A modified grid apparatus for stereological analysis of light and electron micrographs.

A simplified and inexpensive grid apparatus for use with the differential point counting technique of stereological analysis of cellular and organellar volumes is described.