Structure/function studies on cytoskeletal proteins in <i>Dictyostelium</i> amoebae as a paradigm

Schleicher, Michael; André, Bernadette; Andréoli, Christophe; Eichinger, Ludwig M.; Haugwitz, Michael; Hofmann, Andreas; Karakesisoglou, Jacques; Stöckelhuber, Mechthild; Noegel, Angelika A.

doi:10.1016/0014-5793(95)00579-x

Cited by 33 publications

(18 citation statements)

References 46 publications

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“…Since PIP 2 is known to inhibit actin filament severing and capping by gelsolin in vitro , the dominant gelsolin activity in vivo might be capping of the barbed ends rather than severing. Profilin, a ubiquitous 12 to 15 kDa protein, is also multifunctional, and may contribute to sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting polymerization when barbed ends are free (Carlier and Pantaloni, 1997;Schafer and Cooper, 1995;Schleicher et al, 1995). Profilin forms a complex with actin, named profilactin, which caps the barbed ends of the filaments.…”

Section: Initiation and Growthmentioning

confidence: 99%

Chemoattractant-induced lamellipod extension

Bailly

Condeelis

Segall

1998

Microsc. Res. Tech.

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Section: Initiation and Growthmentioning

confidence: 99%

Chemoattractant-induced lamellipod extension

Bailly

Condeelis

Segall

1998

Microsc. Res. Tech.

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“…Dictyostelium has many advantages as a model system for the study of the cytoskeleton (for reviews, see Schleicher and Noegel, 1992;Schleicher et al, 1995) and developmental processes (for reviews, see Firtel, 1996;Loomis, 1996). Like yeast, it offers the possibility of combining biochemical and genetic approaches, but in addition, it shows a full spectrum of cellular functions, including chemotaxis and multicellular development.…”

Section: Introductionmentioning

confidence: 99%

A Developmentally Regulated Kinesin-related Motor Protein fromDictyostelium discoideum

Hostos

McCaffrey²,

Sucgang

et al. 1998

MBoC

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The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive ␣-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 m/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells. INTRODUCTIONSince the prototype kinesin was purified from squid axoplasm (Lasek and Brady, 1985;Schroer et al., 1985;Vale et al., 1985), dozens of related proteins (here referred to as kinesins or kinesin-related proteins [KRPs]) have been identified in organisms ranging from fungi to mammals (Moore and Endow, 1996). KRPs share a conserved mechanochemical motor domain responsible for ATP hydrolysis and microtubule binding. Outside of the 350-amino acid-long motor domain, KRPs show great sequence diversity, but regions predicted to mediate homodimerization via formation of coiled-coil structures are often present (Goldstein, 1993). Sequence diversity among KRPs outside of the motor domain is thought to play a key role in their functional diversity by specifying which proteins and cargo associate with the motor (Goldstein, 1993). Kinesins have been shown to drive the movement of vesicles in fast axonal transport (Hall and Hedgecock, 1991;, the movement of vesicles between the Golgi † Corresponding Author. E-mail address: hostos@bioc.rice.edu. and the ER , and the movement of other organelles, including secretory vesicles, lysosomes, mitochondria, and nuclei (

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“…Schleicher et al (2) classified these binding proteins into three categories. The first group of proteins, such as profilin and cofilin, are those that bind to monomeric G-actin and thus reversibly remove polymerizable actin from the equilibrium with F-actin.…”

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confidence: 99%

A heterozygous mutation of β-actin associated with neutrophil dysfunction and recurrent infection

Nunoi

Yamazaki

Tsuchiya

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

105

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A human disorder caused by mutation in nonmuscle actin has not been reported. We report here a variant of nonmuscle actin in a female patient with recurrent infections, photosensitivity, and mental retardation. She also had abnormalities in neutrophil chemotaxis, superoxide production, and membrane potential response. Two-dimensional PAGE analysis of proteins from neutrophils and other cell types from this patient demonstrated a unique protein spot migrating at 42 kDa with pI shifted slightly to neutral relative to normal ␤-and ␥-actin. Digestion peptide mapping and Western blotting showed this spot to be an abnormal actin. A full-length cDNA library was constructed by using mRNA from patient's cells and cDNA encoding the mutant ␤-actin molecule was identified by an in vitro translation method. Sequencing of the clones demonstrated a G-1174 to A substitution, predicting a glutamic acid-364 to lysine substitution in ␤-actin and eliminating a HinfI DNase restriction site found in normal ␤-actin sequence. By HinfI digestion and by sequencing, the mutation in one allele of patient's genomic DNA was confirmed. Though no defect in cell-free polymerization of actin was detected, this defect lies in a domain important for binding to profilin and other actin-regulatory molecules. In fact, the mutant actin bound to profilin less efficiently than normal actin did. Heterozygous expression of mutant ␤-actin in neutrophils and other cells of this patient may act in a dominant-negative fashion to adversely affect cellular activities dependent on the function of nonmuscle actin.

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Structure/function studies on cytoskeletal proteins in Dictyostelium amoebae as a paradigm

Cited by 33 publications

References 46 publications

Chemoattractant-induced lamellipod extension

Chemoattractant-induced lamellipod extension

A Developmentally Regulated Kinesin-related Motor Protein fromDictyostelium discoideum

A heterozygous mutation of β-actin associated with neutrophil dysfunction and recurrent infection

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