Isolation and properties of calmodulin from Dictyostelium discoideum

Clarke, Margaret; Bazari, W L; Kayman, Samuel C.

doi:10.1128/jb.141.1.397-400.1980

Cited by 84 publications

(23 citation statements)

References 12 publications

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“…Dictyostelium discoideum strain AX2 and its fluorescent derivatives were cultured at 228C in HL5 [Clarke et al, 1980]. As appropriate, 5-10 lg/ml G418 and/or 10 lg/ml blasticidin were included for maintenance of plasmid(s) expressing fluorescent fusion proteins.…”

Section: Cells Plasmids and Culture Conditionsmentioning

confidence: 99%

Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol‐(3,4,5)‐trisphosphate and phosphatidylinositol‐(3,4)‐bisphosphate

Giorgione

Clarke

2008

Cell Motil. Cytoskeleton

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Latex beads are the preferred phagocytic substrate in biochemical studies of phagosome composition and maturation. Using living Dictyostelium cells and fluorescent probes, we compared the properties of phagosomes formed to ingest latex beads or digestible prey. Significant differences were found during the initial steps of phagocytosis. During uptake of bacteria or yeast, PHcrac-GFP, a probe that binds to membranes enriched in PI(3,4,5)P(3) and PI(3,4)P(2), always labeled the nascent phagosome and faded shortly after it sealed. However, labeling of bead-containing phagosomes was highly variable. Beads were engulfed by phagosomes either lacking or displaying the PHcrac-GFP label, and that label, if present, often persisted for many minutes, revealing that early trafficking steps for bead-containing phagosomes are quite heterogeneous. Later stages of the endocytic pathway appeared more similar for phagosomes containing prey and latex beads. Both types of phagosomes fused with acidic endosomes while undergoing transport along microtubules, both acquired the V-ATPase and lost it prior to exocytosis, and both bound the late endosome marker vacuolin B, which was transferred to the plasma membrane upon exocytosis. We conclude that caution is needed in extrapolating results from latex bead phagosomes to phagosomes containing physiological substances, especially in early stages of the endocytic pathway.

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Section: Cells Plasmids and Culture Conditionsmentioning

confidence: 99%

Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol‐(3,4,5)‐trisphosphate and phosphatidylinositol‐(3,4)‐bisphosphate

Giorgione

Clarke

2008

Cell Motil. Cytoskeleton

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“…Dictyostelium discoideum strain AX2 and its fluorescent derivatives were cultured at 22∞C in HL5 (Clarke et al, 1980). Cells expressing one of the following fluorescent markers were employed as indicated: GFP-a-tubulin (Neujahr et al, 1998), coronin-GFP (Maniak et al, 1995), calnexin-GFP (Müller-Taubenberger et al, 2001), GFP-calreticulin (Müller-Taubenberger et al, 2001), GFP-HDEL (Monnat et al, 2000) and VatM-GFP (Clarke et al, 2002a).…”

Section: Strainsmentioning

confidence: 99%

Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae

Clarke

2005

Cellular Microbiology

110

105

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SummaryThe natural hosts of the bacterial pathogen Legionella pneumophila are amoebae and protozoa. In these hosts, as in human macrophages, the pathogen enters the cell through phagocytosis, then rapidly modifies the phagosome to create a compartment that supports its replication. We have examined L. pneumophila entry and behaviour during early stages of the infection of Dictyostelium discoideum amoebae . Bacteria were labelled with a red fluorescent marker, and selected proteins and organelles in the host were labelled with GFP, allowing the dynamics and interactions of L. pneumophila -containing phagosomes to be tracked in living cells. These studies demonstrated that entry of L. pneumophila is an actin-mediated process, that the actin-binding protein coronin surrounds the nascent phagosome but dissociates immediately after internalization, that ER membrane is not incorporated into a phagosome during uptake, that the newly internalized phagosome is rapidly transported about the cell on microtubules, that association of ER markers with the phagosome occurs in two steps that correlate with distinct changes in phagosome movement, and that the vacuolar H( + + + + )-ATPase does not associate with mature replication vacuoles. These studies have clarified certain aspects of the infection process and provided new insights into the dynamic interactions between the pathogen and its host.

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“…However, existing Western blot methods were unable to detect calmodulin in Dictyosteliurn cell lysates, because of the poor blotting properties of calmodulin and the low calmodulin content of Dictyostelium cells. Calmodulin is only 0.04% of the total protein in Dictyostelium cells [Clarke et al, 1980;Bazari and Clarke, 19821, in contrast to levels of 0.2 to 0.5% found in vertebrate tissues [Klee and Vanaman, 19821. We therefore undertook to raise new, monoclonal antibodies against Dictyostelium calmodulin and to develop methods that would permit rigorous testing of the specificity of both the monoclonal and polyclonal antibodies.…”

Section: Introductionmentioning

confidence: 99%

Production and specificity of monoclonal antibodies against calmodulin from dictyostelium discoideum

Hulen

Baron

Salisbury

et al. 1991

Cell Motil. Cytoskeleton

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Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For the purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were used of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin-specific antibodies were identified; most of these reacted preferentially with the calcium-containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross-reacted with calmodulin from bovine brain.

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Isolation and properties of calmodulin from Dictyostelium discoideum

Cited by 84 publications

References 12 publications

Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol‐(3,4,5)‐trisphosphate and phosphatidylinositol‐(3,4)‐bisphosphate

Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol‐(3,4,5)‐trisphosphate and phosphatidylinositol‐(3,4)‐bisphosphate

Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae

Production and specificity of monoclonal antibodies against calmodulin from dictyostelium discoideum

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