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

1999 ◽  
Vol 112 (22) ◽  
pp. 3995-4005 ◽  
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.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

Acidification of vasopressin-induced endosomes in toad urinary bladder

1994 ◽  
Vol 267 (1) ◽  
pp. F106-F113
Author(s):  
F. Emma ◽  
H. W. Harris ◽  
K. Strange
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Apical Membrane ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Toad Urinary Bladder ◽  
Acidic Ph ◽  
Terminal Event ◽  
Ultimate Fate

It is well established that water channels (WC) are removed from the apical membrane of vasopressin-sensitive epithelia by endocytosis. The processing and the ultimate fate of endocytosed WC is, however, incompletely understood. In many cells, endosome acidification plays an important role in the processing and sorting of endocytosed proteins. Endosome acidification in the toad urinary bladder was therefore examined in vivo by fluorescence ratio video microscopy after induction of endocytosis by vasopressin removal and transepithelial water flow in the presence of the pH-sensitive fluid phase marker 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-dextran. Fifteen minutes after induction of endocytosis, the majority of endosomes had a neutral or slightly acidic pH. The number of acidic endosomes increased progressively with time. Two hours after endocytosis began, 98% of the endosomes had a pH < 6.0. Bafilomycin completely blocked endosome acidification, indicating that H+ transport is mediated by a vacuolar H(+)-adenosinetriphosphatase. Bafilomycin had no effect on transepithelial water flow in bladders repetitively stimulated by vasopressin. These findings, as well as the work of other investigators, suggest that if WC recycling occurs, it is not dependent on acidification of the endosomal compartment. Acidification of vasopressin-induced endosomes most likely represents a terminal event in the endocytic pathway.

scholarly journals Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo

2014 ◽  
Vol 306 (5) ◽  
pp. C441-C449 ◽  
Author(s):  
Polly E. Mattila ◽  
Venkatesan Raghavan ◽  
Youssef Rbaibi ◽  
Catherine J. Baty ◽  
Ora A. Weisz
Keyword(s):  
Proximal Tubule ◽  
Primary Cultures ◽  
Fluid Phase ◽  
Chloride Ions ◽  
Endocytic Pathway ◽  
Kidney Cortex ◽  
Glomerular Filtrate ◽  
Electron Microscopy Analysis ◽  
Pt Cell

The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosphate, glucose, water, and plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell (PTC) function, the organization of the endocytic pathway in these cells remains poorly understood. We have used immunofluorescence and live-cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PTCs isolated from mouse kidney cortex. Cells from the S1 segment could be distinguished from those from more distal PT segments by their robust uptake of albumin and comparatively low expression of γ-glutamyltranspeptidase. Rab11a in these cells is localized to variously sized spherical compartments that resemble the apical vacuoles observed by electron microscopy analysis of PTCs in vivo. These Rab11a-positive structures are highly dynamic and receive membrane and fluid-phase cargo. In contrast, fluid-phase cargoes are largely excluded from Rab11a-positive compartments in immortalized kidney cell lines. The unusual morphology and sorting capacity of Rab11a compartments in primary PTCs may reflect a unique specialization of these cells to accommodate the functional demands of handling a high endocytic load.

scholarly journals Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

1996 ◽  
Vol 109 (6) ◽  
pp. 1479-1495 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Rodriguez-Paris ◽  
J.M. Bush ◽  
L. Zhang ◽  
J.A. Cardelli
Keyword(s):  
Morphological Changes ◽  
Specific Inhibitor ◽  
Fluid Phase ◽  
Contractile Vacuole ◽  
Dependent Manner ◽  
Concanamycin A ◽  
Latex Beads ◽  
And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

scholarly journals Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants

1996 ◽  
Vol 109 (3) ◽  
pp. 663-673 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Bush ◽  
M.D. Peterson ◽  
K.D. Novak ◽  
M.A. Titus ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Double Mutant ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Contractile Vacuole ◽  
C Cell ◽  
Myosin I ◽  
Lysosomal System

The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, alpha-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes alpha-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface.

scholarly journals A Myosin I Is Involved in Membrane Recycling from Early Endosomes

2000 ◽  
Vol 150 (5) ◽  
pp. 1013-1026 ◽  
Author(s):  
Eva M. Neuhaus ◽  
Thierry Soldati
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Surface Proteins ◽  
Endocytic Pathway ◽  
Membrane Recycling ◽  
Myosin I ◽  
Early Endosomes ◽  
Butanedione Monoxime ◽  
Microscopy Method ◽  
Membrane Components

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin–myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA−/B−, and myoB− cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA−/B− cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.

scholarly journals Domains of the Rsp5 Ubiquitin-Protein Ligase Required for Receptor-mediated and Fluid-Phase Endocytosis

2001 ◽  
Vol 12 (2) ◽  
pp. 421-435 ◽  
Author(s):  
Rebecca Dunn ◽  
Linda Hicke
Keyword(s):  
Plasma Membrane ◽  
Point Mutations ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Temperature Sensitive ◽  
C2 Domain ◽  
Ww Domains ◽  
Amino Terminal ◽  
Regulatory Event

Yeast Rsp5p and its mammalian homologue, Nedd4, arehect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast α-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on α-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.

Traffic to the endocytic compartment of plants

1989 ◽  
Vol 47 ◽  
pp. 754-755
Author(s):  
L. R. Griffing ◽  
R. D. Record ◽  
H. H. Mollenhauer
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fluid Phase ◽  
Multivesicular Body ◽  
Endocytic Pathway ◽  
Cationized Ferritin ◽  
Plant Protoplasts ◽  
Whole Cells ◽  
Membrane Bound ◽  
And Function

The endocytic pathway of plants has been identified and partially characterized using nonspecific membrane-bound and fluid phase probes . The function of endocytosis in plants is, however, unknown. We shall describe how ultrastructural histochemistry, immunocytochemical analyses and fluorescence imaging have been used to explore the physiology and function of the endocytic pathway in plant protoplasts and whole cells.Cationized ferritin (CF) can be used as a marker of plasma membrane uptake in plant protoplasts. Several different organelles become labeled upon exposure of protoplasts to CF: clathrin-coated vesicles (CV), the partially coated reticulum (PCR), the Golgi complex (GC), the multivesicular body (MVB), and the vacuole (V). These organelles also participate in the pathways of secretion and delivery of protein to the lysosome (vacuole). What are the sites of overlap/divergence among the secretory, endocytic and lysosomal pathways in these cells?

An LI and ML motif in the cytoplasmic tail of the MHC-associated invariant chain mediate rapid internalization

1994 ◽  
Vol 107 (7) ◽  
pp. 2021-2032 ◽  
Author(s):  
B. Bremnes ◽  
T. Madsen ◽  
M. Gedde-Dahl ◽  
O. Bakke
Keyword(s):  
Plasma Membrane ◽  
Mhc Class Ii ◽  
Transmembrane Protein ◽  
Point Mutations ◽  
Cytoplasmic Tail ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Chimeric Proteins ◽  
Endosomal Pathway

Invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Two regions of the 30 residue cytoplasmic tail of Ii contain sorting information able to direct Ii to the endocytic pathway. The full-length cytoplasmic tail of Ii and the two tail regions were fused to neuraminidase (NA) forming chimeric proteins (INA). Ii is known to form trimers and when INA was transfected into COS cells it assembled as a tetramer like NA. The INA molecules were targeted to the endosomal pathway and cotransfection with Ii showed that both molecules appeared in the same vesicles. By labelling the INA fusion proteins with iodinated antibody it was found that molecules with either endocytosis signal were expressed at the plasma membrane and internalized rapidly. Point mutations revealed that an LI motif within the first region of the cytoplasmic tail and an ML motif in the second region were essential for efficient internalization. The region containing the LI motif is required for Ii to induce large endosomes but a functional LI internalization motif was not fundamental for this property. The cytoplasmic tail of Ii is essential for efficient targeting of the class II molecules to endosomes and the dual LI and ML motif may thus be responsible for directing these molecules to the endosomal pathway, possibly via the plasma membrane.

scholarly journals The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3

2013 ◽  
Vol 201 (1) ◽  
pp. 97-111 ◽  
Author(s):  
Arun T. John Peter ◽  
Jens Lachmann ◽  
Meenakshi Rana ◽  
Madeleine Bunge ◽  
Margarita Cabrera ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Sorting ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Gtpase Activating Protein ◽  
Early Endosomes ◽  
Lysosome Related Organelles

Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.

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