Cloning vectors for the production of proteins in Dictyostelium discoideum

Manstein, Dietmar J.; Schuster, Hans−Peter; Morandini, Piero; Hunt, Deborah M.

doi:10.1016/0378-1119(95)00351-6

Cited by 197 publications

(142 citation statements)

References 21 publications

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“…After KpnI and BpiI digestion, the fragment was subcloned into the pDXA-M761 vector replacing its KpnI-BpiI motor domain coding fragment 25 . The N-terminal sequence of Md was modified to MDGTP where P corresponds to P3 of the native sequence.…”

Section: Methodsmentioning

confidence: 99%

Myosin cleft movement and its coupling to actomyosin dissociation

Conibear

Bagshaw

Fajer

et al. 2003

Nat Struct Mol Biol

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In recent years the structural basis of the actomyosin crossbridge cycle 1 has been defined in increasing detail by X-ray crystallography and electron microscopy 2-9 . 2 However, while crystal structures of myosin motor domains have been obtained for a number of nucleotide-bound states, the nature of the actin binding interaction has not been observed directly to date 2,3,8,10,11 . Residues involved in actin binding were identified by fitting the envelope of the myosin crystal structure into the electron micrograph density of decorated actin filaments. Initial docking attempts suggested that the cleft between the upper and lower 50K myosin domains might close on forming the rigor state 12 . This approach has been refined to the point of identifying a potential hinge point around switch 1 at the myosin nucleotide site that allows the upper 50K domain to rotate towards the lower 50K domain 7 . Recent crystallographic studies support this concept 6,8,9 . While structural evidence is mounting in favour of the cleft closure model, it is important to test this idea in solution. Structural data alone provides only a static picture and the proposed transitions between states have been made by inference. We have introduced cysteine residues at position 416 and 537 across the cleft (Dictyostelium discoideum, Dd, myosin II sequence) in a cysteine-deficient 13 myosin background (Fig 1a,b). Labelling these cysteine residues with N-1-pyrene

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Section: Methodsmentioning

confidence: 99%

Myosin cleft movement and its coupling to actomyosin dissociation

Conibear

Bagshaw

Fajer

et al. 2003

Nat Struct Mol Biol

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“…To express a fusion protein of Phg2 with the green fluorescent protein (Phg2-GFP), the full coding sequence of PHG2 preceded by the coding sequence of GFP was introduced in the pDXA-3C expression vector (Manstein et al, 1995). This vector was then introduced in phg2 mutant cells and clones expressing Phg2-GFP were isolated.…”

Section: Microscopymentioning

confidence: 99%

Phg2, a Kinase Involved in Adhesion and Focal Site Modeling inDictyostelium

Gebbie

Benghezal

Cornillon

et al. 2004

MBoC

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The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.

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“…AJ507828). PHG1b was subcloned into a derivative of pDXA-3C (Manstein et al, 1995;Cornillon et al, 2000) to obtain the pSC3B vector that upon transfection into D. discoideum cells drives overexpression (actin 15 promoter) of Phg1b protein.…”

Section: Nucleotide Sequences and Plasmidsmentioning

confidence: 99%

“…The pSC4 plasmid was produced by introducing into pDXA-3C (Manstein et al, 1995), two fragments of the PHG1b gene: XbaI/ClaI DNA fragment and a polymerase chain reaction DNA fragment amplified with the two following oligonucleotides, 5Ј-GGTACAGTTGGTTTCTAC-3Ј and 5Ј-ACCCTATTTG-TAGCACCA-3Ј.…”

Section: Phg1cmentioning

confidence: 99%

Synergistic Control of Cellular Adhesion by Transmembrane 9 Proteins

Benghezal¹,

Cornillon²,

Gebbie³

et al. 2003

MBoC

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The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface.

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Cloning vectors for the production of proteins in Dictyostelium discoideum

Cited by 197 publications

References 21 publications

Myosin cleft movement and its coupling to actomyosin dissociation

Myosin cleft movement and its coupling to actomyosin dissociation

Phg2, a Kinase Involved in Adhesion and Focal Site Modeling inDictyostelium

Synergistic Control of Cellular Adhesion by Transmembrane 9 Proteins

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