Osmoregulation inParamecium: in situ ion gradients permit water to cascade through the cytosol to the contractile vacuole

In vivo K+, Na+, Ca2+ and Cl-activities in the cytosol and the contractile vacuole fluid of Paramecium multimicronucleatum were determined in cells adapted to a number of external osmolarities and ionic conditions by using ion-selective microelectrodes. It was found that: (1) under standardized saline conditions K+ and Cl- were the major osmolytes in both the cytosol and the contractile vacuole fluid; and (2) the osmolarity of the contractile vacuole fluid, determined from K+ and Cl- activities only, was always more than 1.5 times higher than that of the cytosol. These findings indicate that excess cytosolic water crosses the contractile vacuole complex membrane osmotically. Substitution of choline or Ca2+ for K+ in the external solution or the external application of furosemide caused concomitant decreases in the cytosolic K+ and Cl- activities that were accompanied by a decrease in the water segregation activity of the contractile vacuole complex. This implies that the cytosolic K+ and Cl- are actively coimported across the plasma membrane. Thus, the osmotic gradients across both the plasma membrane and the membrane of the contractile vacuole complex ensure a controlled cascade of water flow through the cell that can provide for osmoregulation as well as the possible extrusion of metabolic waste by the contractile vacuole complex.

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The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein

Journal of Cell Science ◽

10.1242/jcs.112.22.3995 ◽

1999 ◽

Vol 112 (22) ◽

pp. 3995-4005 ◽

Cited By ~ 8

Author(s):

D. Gabriel ◽

U. Hacker ◽

J. Kohler ◽

A. Muller-Taubenberger ◽

J.M. Schwartz ◽

...

Keyword(s):

Plasma Membrane ◽

Transmembrane Protein ◽

Fluid Phase ◽

Endocytic Pathway ◽

Contractile Vacuole ◽

Specific Marker ◽

Sharp Separation ◽

Cell Fractions ◽

Entire Network

The contractile vacuole system is an osmoregulatory organelle composed of cisternae and interconnecting ducts. Large cisternae act as bladders that periodically fuse with the plasma membrane, forming pores to expel water. To visualize the entire network in vivo and to identify constituents of the vacuolar complex in cell fractions, we introduced a specific marker into Dictyostelium cells, GFP-tagged dajumin. The C-terminal, GFP-tagged region of this transmembrane protein is responsible for sorting to the contractile vacuole complex. Dajumin-GFP negligibly associates with the plasma membrane, indicating its retention during discharge of the bladder. Fluorescent labeled cell-surface constituents are efficiently internalized by endocytosis, while no significant cycling through the contractile vacuole is observed. Endosomes loaded with yeast particles or a fluid-phase marker indicate sharp separation of the endocytic pathway from the contractile vacuole compartment. Even after dispersion of the contractile vacuole system during mitosis, dajumin-GFP distinguishes the vesicles from endosomes, and visualizes post-mitotic re-organization of the network around the nucleus. Highly discriminative sorting and membrane fusion mechanisms are proposed to account for the sharp separation of the contractile vacuole and endosomal compartments. Evidence for a similar compartment in other eukaryotic cells is discussed.

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WATER PERMEABILITY OF ROCK MASS

САВРЕМЕНА ТЕОРИЈА И ПРАКСА У ГРАДИТЕЉСТВУ ◽

10.7251/stp1813642z ◽

2018 ◽

Vol 13 (1) ◽

Author(s):

Marin Zelenika

Keyword(s):

Numerical Modeling ◽

Rock Mass ◽

Water Flow ◽

Water Permeability ◽

Natural State ◽

Rock Masses ◽

Flow Through

The presence of water in the rock masses, or water filtering, has an important role in theconstruction of engineering facilities (dams, tunnels, ...), as well as in formation of rockslopes, caused by the implementation of various projects. The rock mass, that we find innature (in-situ), is heterogeneous, anisotropic, jointed and is in the natural state of stress.On numerical modeling of planned geotechnical project, in addition to other inputparameters, it is necessary, as more precisely, to define the water permeability of the rockmass. The paper describes the mechanisms of water flow through the rock mass and themethodology for defining the waterpermeability parameter of such a geologicalenvironment.

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Remodeling Membrane Binding by Mono-Ubiquitylation

Biomolecules ◽

10.3390/biom9080325 ◽

2019 ◽

Vol 9 (8) ◽

pp. 325 ◽

Cited By ~ 1

Author(s):

Neta Tanner ◽

Oded Kleifeld ◽

Iftach Nachman ◽

Gali Prag

Keyword(s):

Crystal Structure ◽

Plasma Membrane ◽

Live Cell Imaging ◽

Cell Imaging ◽

Membrane Binding ◽

Cyclic Activity ◽

E Coli

Ubiquitin (Ub) receptors respond to ubiquitylation signals. They bind ubiquitylated substrates and exert their activity in situ. Intriguingly, Ub receptors themselves undergo rapid ubiquitylation and deubiquitylation. Here we asked what is the function of ubiquitylation of Ub receptors? We focused on yeast epsin, a Ub receptor that decodes the ubiquitylation signal of plasma membrane proteins into an endocytosis response. Using mass spectrometry, we identified lysine-3 as the major ubiquitylation site in the epsin plasma membrane binding domain. By projecting this ubiquitylation site onto our crystal structure, we hypothesized that this modification would compete with phosphatidylinositol-4,5-bisphosphate (PIP2) binding and dissociate epsin from the membrane. Using an E. coli-based expression of an authentic ubiquitylation apparatus, we purified ubiquitylated epsin. We demonstrated in vitro that in contrast to apo epsin, the ubiquitylated epsin does not bind to either immobilized PIPs or PIP2-enriched liposomes. To test this hypothesis in vivo, we mimicked ubiquitylation by the fusion of Ub at the ubiquitylation site. Live cell imaging demonstrated that the mimicked ubiquitylated epsin dissociates from the membrane. Our findings suggest that ubiquitylation of the Ub receptors dissociates them from their products to allow binding to a new ubiquitylated substrates, consequently promoting cyclic activity of the Ub receptors.

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How external osmolarity affects the activity of the contractile vacuole complex, the cytosolic osmolarity and the water permeability of the plasma membrane in Paramecium multimicronucleatum

Journal of Experimental Biology ◽

10.1242/jeb.204.2.291 ◽

2001 ◽

Vol 204 (2) ◽

pp. 291-304 ◽

Cited By ~ 5

Author(s):

C. Stock ◽

R.D. Allen ◽

Y. Naitoh

Keyword(s):

Plasma Membrane ◽

Water Permeability ◽

External Solution ◽

Contractile Vacuole ◽

Hypertonic Solution ◽

Hypotonic Solution ◽

Natural Conditions ◽

Short Term ◽

Short Term Exposure ◽

Narrow Concentration Range

The rate of fluid expulsion, R(CVC), from the contractile vacuole complex (CVC) of Paramecium multimicronucleatum was estimated from the volume of the contractile vacuoles (CVs) immediately before the start of fluid discharge and from the time elapsing between discharges. The R(CVC) increased when the cell was exposed to a strongly hypotonic solution and decreased in a weakly hypotonic solution. When the cell was exposed to an isotonic or a hypertonic solution, R(CVC) fell to zero. The time constant, tau, used to describe the change in R(CVC) in response to a change in external osmolarity shortened after a short-term exposure to a strongly hypotonic solution and lengthened after a short-term exposure to a less hypotonic solution. A remarkable lengthening of tau occurred after a short-term exposure to isotonic or hypertonic solution. Under natural conditions, mechanisms for controlling R(CVC) are effective in maintaining the cytosolic osmolarity hypertonic within a narrow concentration range despite changes in the external osmolarity, which is normally hypotonic to the cytosol. Cells exposed to an isotonic or hypertonic solution resumed CV activity when left in the solution for 12 h. The cytosolic osmolarity was found to increase and to remain hypertonic to the external solution. This will permit cells to continue to acquire water. The increase in the cytosolic osmolarity occurred in a stepwise fashion, rather than linearly, as the external osmolarity increased. That is, the cytosolic osmolarity first remained more-or-less constant at an increased level until the external osmolarity exceeded this level. Thereupon, the cytosolic osmolarity increased to a new higher level in 12 h, so that the cytosol again became hypertonic to the external solution and the cells resumed CV activity. These results imply that the cell needs to maintain water segregation activity even after it has been exposed to an isotonic or hypertonic environment. This supports the idea that the CVC might be involved not only in the elimination of excess cytosolic water but also in the excretion of some metabolic waste substances.

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In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163903 ◽

1996 ◽

Vol 54 ◽

pp. 288-289

Author(s):

Greg V. Martin ◽

Ann L. Hubbard

Keyword(s):

Three Dimensional ◽

Integral Membrane Proteins ◽

Polarized Secretion ◽

Microscope Images ◽

Computer Aided ◽

Intracellular Organelles ◽

Cytoskeletal Elements ◽

Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

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