The contractile vacuole in Ca2+-regulation in Dictyostelium: its essential function for cAMP-induced Ca2+-influx

Malchow, Dieter; Lusche, Daniel F.; Schlatterer, Christina; Lozanne, Arturo De; Müller‐Taubenberger, Annette

doi:10.1186/1471-213x-6-31

Cited by 38 publications

(26 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Samples were subjected to SDS-PAGE and Western blot analysis. 45 Ca 2ϩ Overlay Assay- 45 Ca 2ϩ overlay assays were carried out as described previously (32). Recombinant DdCAD-1 proteins were blotted onto nitrocellulose membrane using a slot blot apparatus (Bio-Rad).…”

Section: Construction Of Ddcad-1-gfp N-gfp and C-gfp Expression Vecmentioning

confidence: 99%

“…The membrane was washed for 1 h in four changes with the overlay buffer (60 mM KCl, 5 mM MgCl 2 , and 10 mM imidazole, pH 6.8). The blot was then incubated with 10 ml of 45 Ca 2ϩ (10 Ci/ml) in the overlay buffer for 30 min at room temperature on a platform shaker, followed by three 10-min washes with deionized water. Autoradiography was carried out by exposure of the air-dried membrane to Bioflex-MRI Films (Clonex Corp.) for 24 h at Ϫ70°C.…”

Section: Construction Of Ddcad-1-gfp N-gfp and C-gfp Expression Vecmentioning

confidence: 99%

“…6B). Their ability to bind Ca 2ϩ was assessed using the 45 Ca 2ϩ overlay assay (Fig. 6C) failed to enter the contractile vacuole, whereas His 6 -SIII was imported into the contractile vacuole at a level similar to that of the wild-type protein (Fig.…”

Section: Figure 5 Import Of Ddcad-1 Fusion Proteins Into Contractilementioning

confidence: 99%

“…The arrow indicates the position of His 6 -DdCAD-1, and the arrowhead indicates the position His 6 -N. B, Coomassie-stained gel of purified His-tagged wild-type (WT) and mutant DdCAD-1 containing mutations in one of the Ca 2ϩ -binding sites (SI, SII, SIII, and S(I ϩ II)). C, binding of 45 Ca 2ϩ to His 6 -DdCAD-1 and Ca 2ϩ -binding site mutant DdCAD-1. Protein samples were blotted onto nitrocellulose membrane.…”

Section: Figure 5 Import Of Ddcad-1 Fusion Proteins Into Contractilementioning

confidence: 99%

“…Protein samples were blotted onto nitrocellulose membrane. The blot was incubated with 10 ml of 45 Ca 2ϩ (10 Ci/ml) for 30 min at room temperature. After washing extensively with water, the blot was exposed to x-ray film at Ϫ70°C for 24 h. D, in vitro import of His-tagged wild-type (WT) and mutant (SI, SII, SIII, and S(I ϩ II)) DdCAD-1 into contractile vacuoles.…”

Section: Figure 5 Import Of Ddcad-1 Fusion Proteins Into Contractilementioning

confidence: 99%

See 4 more Smart Citations

Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

Sriskanthadevan

Lee

Lin

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

The cadA gene in Dictyostelium encodes a Ca 2؉ -dependent cell adhesion molecule DdCAD-1 that contains two ␤-sandwich domains. DdCAD-1 is synthesized in the cytoplasm as a soluble protein and then transported by contractile vacuoles to the plasma membrane for surface presentation or secretion. DdCAD-1-green fluorescent protein (GFP) fusion protein was expressed in cadA-null cells for further investigation of this unconventional protein transport pathway. Both morphological and biochemical characterizations showed that DdCAD-1-GFP was imported into contractile vacuoles. Time-lapse microscopy of transfectants revealed the transient appearance of DdCAD-1-GFP-filled vesicular structures in the lumen of contractile vacuoles, suggesting that DdCAD-1 could be imported by invagination of contractile vacuole membrane. To assess the structural requirements in this transport process, the N-terminal and C-terminal domains of DdCAD-1 were expressed separately in cells as GFP fusion proteins. Both fusion proteins failed to enter the contractile vacuole, suggesting that the integrity of DdCAD-1 is required for import. Such a requirement was also observed in in vitro reconstitution assays using His 6 -tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca 2؉ -binding sites were mutated, indicating a role for Ca 2؉ in the import process. Spectral analysis showed that mutations in the Ca 2؉ -binding sites resulted in subtle conformational changes. Indeed, proteins with altered conformation failed to enter the contractile vacuole, suggesting that the import signal is somehow integrated in the three-dimensional structure of DdCAD-1.

show abstract

Section: Construction Of Ddcad-1-gfp N-gfp and C-gfp Expression Vecmentioning

confidence: 99%

Section: Construction Of Ddcad-1-gfp N-gfp and C-gfp Expression Vecmentioning

confidence: 99%

Section: Figure 5 Import Of Ddcad-1 Fusion Proteins Into Contractilementioning

confidence: 99%

Section: Figure 5 Import Of Ddcad-1 Fusion Proteins Into Contractilementioning

confidence: 99%

Section: Figure 5 Import Of Ddcad-1 Fusion Proteins Into Contractilementioning

confidence: 99%

See 3 more Smart Citations

Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

Sriskanthadevan

Lee

Lin

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Copine A regulates the size and exocytosis of contractile vacuoles and postlysosomes in Dictyostelium

2020

View full text Add to dashboard Cite

Copines are a family of cytosolic proteins that associate with membranes in a calcium‐dependent manner and are found in many eukaryotic organisms. Dictyostelium discoideum has six copine genes (cpnA‐cpnF), and cells lacking cpnA(cpnA−) have defects in cytokinesis, chemotaxis, adhesion, and development. CpnA has also been shown to associate with the plasma membrane, contractile vacuoles (CV), and organelles of the endolysosomal pathway. Here, we use cpnA− cells to investigate the role of CpnA in CV function and endocytosis. When placed in water, cpnA− cells made abnormally large CVs that took longer to expel. Visualization of CVs with the marker protein GFP‐dajumin indicated that cpnA− cells had fewer CVs that sometimes refilled before complete emptying. In endocytosis assays, cpnA− cells took up small fluorescent beads by macropinocytosis at rates similar to parental cells. However, cpnA− cells reached a plateau sooner than parental cells and had less fluorescence at later time points. p80 antibody labeling of postlysosomes (PL) indicated that there were fewer and smaller PLs in cpnA− cells. In dextran pulse‐chase experiments, the number of PLs peaked earlier in cpnA− cells, and the PLs did not become as large and disappeared sooner as compared to parental cells. PLs in cpnA− cells were also shown to have more actin coats, suggesting CpnA may play a role in actin filament disassembly on PL membranes. Overall, these results indicate that CpnA is involved in the regulation of CV size and expulsion, and the maturation, size, and exocytosis of PLs.

show abstract

Biology of Human Pathogenic Trypanosomatids: Epidemiology, Lifecycle and Ultrastructure

Rodrigues

Godinho

Souza

2013

Subcellular Biochemistry

View full text Add to dashboard Cite

Leishmania and Trypanosoma belong to the Trypanosomatidae family and cause important human infections such as leishmaniasis, Chagas disease, and sleeping sickness. Leishmaniasis, caused by protozoa belonging to Leishmania, affects about 12 million people worldwide and can present different clinical manifestations, i.e., visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), and post-kala-azar dermal leishmaniasis (PKDL). Chagas disease, also known as American trypanosomiasis, is caused by Trypanosoma cruzi and is mainly prevalent in Latin America but is increasingly occurring in the United States, Canada, and Europe. Sleeping sickness or human African trypanosomiasis (HAT), caused by two sub-species of Trypanosoma brucei (i.e., T. b. rhodesiense and T. b. gambiense), occurs only in sub-Saharan Africa countries. These pathogenic trypanosomatids alternate between invertebrate and vertebrate hosts throughout their lifecycles, and different developmental stages can live inside the host cells and circulate in the bloodstream or in the insect gut. Trypanosomatids have a classical eukaryotic ultrastructural organization with some of the same main organelles found in mammalian host cells, while also containing special structures and organelles that are absent in other eukaryotic organisms. For example, the mitochondrion is ramified and contains a region known as the kinetoplast, which houses the mitochondrial DNA. Also, the glycosomes are specialized peroxisomes containing glycolytic pathway enzymes. Moreover, a layer of subpellicular microtubules confers mechanic rigidity to the cell. Some of these structures have been investigated to determine their function and identify potential enzymes and metabolic pathways that may constitute targets for new chemotherapeutic drugs.

show abstract

The contractile vacuole in Ca2+-regulation in Dictyostelium: its essential function for cAMP-induced Ca2+-influx

Cited by 38 publications

References 29 publications

Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

Copine A regulates the size and exocytosis of contractile vacuoles and postlysosomes in Dictyostelium

Biology of Human Pathogenic Trypanosomatids: Epidemiology, Lifecycle and Ultrastructure

Contact Info

Product

Resources

About