How does the contractile vacuole of Paramecium multimicronucleatum expel fluid? Modelling the expulsion mechanism

1997 ◽  
Vol 200 (4) ◽  
pp. 713-721 ◽  
Author(s):  
Y Naitoh ◽  
T Tominaga ◽  
M Ishida ◽  
A Fok ◽  
M Aihara ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Actin Polymerization ◽  
Time Course ◽  
Cytochalasin B ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Effective Inhibitor ◽  
Vacuole Membrane ◽  
Discharge Mechanism

To examine the forces needed for discharge of the fluid contents from the contractile vacuole of Paramecium multimicronucleatum, the time course of the decrease in vacuole diameter during systole (the fluid-discharging period) was compared with that of various vacuole discharge models. The observed time course did not fit that predicted by a model in which contraction of an actin­myosin network surrounding the vacuole caused discharge nor that predicted by a model in which the surface tension of the lipid bilayer of the vacuole caused discharge. Rather, it fitted that predicted by a model in which the cell's cytosolic pressure was responsible for discharge. Cytochalasin B, an effective inhibitor of actin polymerization, had no effect on the in vivo time course of systole. An injection of a monoclonal antibody raised against the proton pumps of the decorated spongiomes (now known to be the locus of fluid segregation in P. multimicronucleatum) disrupted the decorated spongiomes and reduced the rate of fluid segregation, whereas it did not alter the time course of systole. We conclude that in P. multimicronucleatum the internal pressure of the contractile vacuole is caused predominantly by the cytosolic pressure and that the fluid-segregation mechanism does not directly affect the fluid-discharge mechanism. Elimination of this cytosolic pressure by rupturing the cell revealed the presence of a novel fluid-discharge mechanism, apparently centered in the vacuole membrane. The involvement of tubulation of the vacuole membrane as the force-generating mechanism for fluid discharge in disrupted cells is discussed.

Periodic tension development in the membrane of the in vitro contractile vacuole of Paramecium multimicronucleatum: modification by bisection, fusion and suction

2000 ◽  
Vol 203 (2) ◽  
pp. 239-251 ◽  
Author(s):  
T. Tani ◽  
R.D. Allen ◽  
Y. Naitoh
Keyword(s):  
Cytochalasin B ◽  
Contractile Vacuole ◽  
Cycle Period ◽  
Membrane Tension ◽  
Tension Development ◽  
Vacuole Membrane ◽  
Filling Phase ◽  
Membrane Bound

The contractile vacuole of the freshwater protozoan Paramecium multimicronucleatum is a membrane-bound exocytotic vesicle that expels excess cytosolic water. The in vitro contractile vacuole isolated from P. multimicronucleatum along with a small amount of cytosol and confined under mineral oil showed periodic rounding and slackening at fairly regular intervals. Activity lasted for over 30 min at room temperature (24–27 degrees C). The rounding of the in vitro contractile vacuole corresponded to the increased membrane tension of the in vivo contractile vacuole that occurs immediately before fluid expulsion. Unlike the in vivo contractile vacuole, the in vitro contractile vacuole did not expel fluid, since it lacked a mechanism to form a pore. The subsequent slackening of the in vitro contractile vacuole corresponded to the fluid-filling phase of the in vivo contractile vacuole that occurs at decreased membrane tension. Fluid filling occurred in the in vitro contractile vacuole only when it was isolated together with its radial arms. In vitro membrane-bound vesicles obtained by ‘bisecting’ (although the two parts were not always identical in size) an in vitro contractile vacuole established their own independent rounding-slackening cycles. In vitro contractile vacuole vesicles could fuse again when the vesicles slackened. The fused vesicle then showed a rounding-slackening cycle with a period closer to that of the vesicle that exhibited the shorter cycle period. An additional rounding phase of the in vitro contractile vacuole could be induced by applying suction to a portion of its membrane with a micropipette when the contractile vacuole was in its slackened phase. This suggests that maximum tension development in the contractile vacuole membrane can be triggered when tension is increased in any part of the contractile vacuole membrane. The time from the start of an extra rounding phase to the next spontaneous rounding and for subsequent rounding-slackening cycles was nearly the same as that before the extra rounding phase. This implies that there is no master pacemaker to control the rounding-slackening cycle in the contractile vacuole membrane. Severed radial arms also became vesiculated and, like contractile vacuole membranes, these in vitro vesicles showed independent rounding-slackening cycles and vesicle-vesicle fusions. Thus, membrane derived from the radial arm seems to be identical in its tension-developing properties with the contractile vacuole membrane. ATP was found to be required for contractile vacuole rounding but inhibitors of actin or tubulin polymerization, such as cytochalasin B and Nocodazole, had no effect on the in vitro contractile vacuole's rounding-slackening cycle.

Complex Influence of Cytochalasin B on Actin Polymerization

1979 ◽  
Vol 34 (7-8) ◽  
pp. 555-557 ◽  
Author(s):  
P. Dancker ◽  
I. Low
Keyword(s):  
Light Scattering ◽  
Actin Polymerization ◽  
Time Course ◽  
Cytochalasin B ◽  
Degree Of Polymerization ◽  
Viscosity Measurements ◽  
Low Concentrations ◽  
Scattering Measurements ◽  
Final Length ◽  
Complex Influence

Abstract In the presence of very low concentrations (about 2 x 10-7 ᴍ) of cytochalasin B (CB) the time course of actin polymerization is much more sigmoidal when followed by viscosity measurements than when followed by light scattering measurements. This suggests that under these conditions actin polymers do not immediately reach their final length but only via short ”bent“ polymers which can be detected only by light scattering but not by viscosity measurements. At higher CB concentrations (about equimolar to those of actin) CB reduces the average degree of polymerization and favors the nucleation step necessary for polymerization.

Dynamics of the vacuolar H+-ATPase in the contractile vacuole complex and the endosomal pathway ofDictyosteliumcells

2002 ◽  
Vol 115 (14) ◽  
pp. 2893-2905 ◽  
Author(s):  
Margaret Clarke ◽  
Jana Köhler ◽  
Quyen Arana ◽  
Tongyao Liu ◽  
John Heuser ◽  
...  
Keyword(s):  
Time Course ◽  
Fluorescent Protein ◽  
Membrane System ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Gene Encoding ◽  
Endosomal Membrane ◽  
C Terminus ◽  
Direct Assembly ◽  
Green Fluorescent

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit enzyme that plays important roles in eukaryotic cells. In Dictyostelium, it is found primarily in membranes of the contractile vacuole complex, where it energizes fluid accumulation by this osmoregulatory organelle and also in membranes of endolysosomes, where it serves to acidify the endosomal lumen. In the present study, a fusion was created between vatM, the gene encoding the 100 kDa transmembrane subunit of the V-ATPase, and the gene encoding Green Fluorescent Protein (GFP). When expressed in Dictyostelium cells, this fusion protein, VatM-GFP, was correctly targeted to contractile vacuole and endolysosomal membranes and was competent to direct assembly of the V-ATPase enzyme complex. Protease treatment of isolated endosomes indicated that the GFP moiety, located on the C-terminus of VatM, was exposed to the cytoplasmic side of the endosomal membrane rather than to the lumenal side. VatM-GFP labeling of the contractile vacuole complex revealed clearly the dynamics of this pleiomorphic vesiculotubular organelle. VatM-GFP labeling of endosomes allowed direct visualization of the trafficking of vacuolar proton pumps in this pathway, which appeared to be entirely independent from the contractile vacuole membrane system. In cells whose endosomes were pre-labeled with TRITC-dextran and then fed yeast particles,VatM-GFP was delivered to newly formed yeast phagosomes with the same time course as TRITC-dextran, consistent with transfer via a direct fusion of endosomes with phagosomes. Several minutes were required before the intensity of the VatM-GFP labeling of new phagosomes reached the level observed in older phagosomes, suggesting that this fusion process was progressive and continuous. VatM-GFP was retrieved from the phagosome membrane prior to exocytosis of the indigestible remnants of the yeast particle. These data suggest that vacuolar proton pumps are recycled by fusion of advanced with newly formed endosomes.

scholarly journals WASP-interacting Protein Is Important for Actin Filament Elongation and Prompt Pseudopod Formation in Response to a Dynamic Chemoattractant Gradient

2006 ◽  
Vol 17 (10) ◽  
pp. 4564-4575 ◽  
Author(s):  
Scott A. Myers ◽  
Laura R. Leeper ◽  
Chang Y. Chung
Keyword(s):  
Actin Polymerization ◽  
Time Course ◽  
Leading Edge ◽  
Delayed Response ◽  
Dependent Manner ◽  
Actin Assembly ◽  
Interacting Protein

The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation.

scholarly journals Pharmacokinetic-Pharmacodynamic Modelling of Systemic IL13 Blockade by Monoclonal Antibody Therapy: A Free Assay Disguised as Total

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 519
Author(s):  
John Hood ◽  
Ignacio González-García ◽  
Nicholas White ◽  
Leeron Marshall ◽  
Vincent F. S. Dubois ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Clinical Data ◽  
Time Course ◽  
Subcutaneous Administration ◽  
Metabolic Clearance ◽  
Indirect Response Model ◽  
Target Time ◽  
Indirect Response ◽  
Over Time

A sequential pharmacokinetic (PK) and pharmacodynamic (PD) model was built with Nonlinear Mixed Effects Modelling based on data from a first-in-human trial of a novel biologic, MEDI7836. MEDI7836 is a human immunoglobulin G1 lambda (IgG1λ-YTE) monoclonal antibody, with an Fc modification to reduce metabolic clearance. MEDI7836 specifically binds to, and functionally neutralizes interleukin-13. Thirty-two healthy male adults were enrolled into a dose-escalation clinical trial. Four active doses were tested (30, 105, 300, and 600 mg) with 6 volunteers enrolled per cohort. Eight volunteers received placebo as control. Following single subcutaneous administration (SC), individual time courses of serum MEDI7836 concentrations, and the resulting serum IL13 modulation in vivo, were quantified. A binding pharmacokinetic-pharmacodynamic (PK-PD) indirect response model was built to characterize the exposure-driven modulation of the target over time by MEDI7836. While the validated bioanalytical assay specification quantified the level of free target (i.e., a free IL13 assay), emerging clinical data suggested dose-dependent increase in systemic IL13 concentration over time, indicative of a total IL13 assay. The target time course was modelled as a linear combination of free target and a percentage of the drug-target complex to fit the clinical data. This novel PK-PD modelling approach integrates independent knowledge about the assay characteristics to successfully elucidate apparently complex observations.

scholarly journals Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule.

1989 ◽  
Vol 93 (5) ◽  
pp. 885-902 ◽  
Author(s):  
R G Ye ◽  
L B Shi ◽  
W I Lencer ◽  
A S Verkman
Keyword(s):  
Proximal Tubule ◽  
Time Course ◽  
Apical Membrane ◽  
Water Channels ◽  
Fluorescence Signal ◽  
Proton Pumps ◽  
Osmotic Gradient ◽  
Proton Atpase ◽  
Osmotic Water

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP-driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped-flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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