Gene replacement in Dictyostelium: generation of myosin null mutants.

Manstein, Dietmar J.; Titus, Margaret A.; Lozanne, Arturo De; Spudich, James A.

doi:10.1002/j.1460-2075.1989.tb03453.x

Cited by 289 publications

(204 citation statements)

References 44 publications

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“…discoideum wild-type and mutant cells were cultivated in petri dishes using liquid AX2 nutrient medium (provided by the Max-Planck-Institut fü r Biochemie, Martinsried, Germany). The wild-type AX2 and the myosin null mutant HS2205 [29] were transfected to express fluorescent GFP-atubulin (cell lines HG1668 and HG1671 [30,31]). For each experiment, the cells were taken from axenic culture.…”

Section: Preparation Of Cellsmentioning

confidence: 99%

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule–actin crosstalk

Heinrich

Sackmann²

2006

Acta Biomaterialia

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The micro-viscoelasticity of the intracellular space of Dictyostelium discoideum cells is studied by evaluating the intracellular transport of magnetic force probes and their viscoelastic responses to force pulses of 20-700 pN. The role of the actin cortex, the microtubule (MT) aster and their crosstalk is explored by comparing the behaviour of wild-type cells, myosin II null mutants, latrunculin A and benomyl treated cells. The MT coupled beads perform irregular local and long range directed motions which are characterized by measuring their velocity distributions (P(v)). The correlated motion of the MT and the centrosome are evaluated by microfluorescence of GFP-labelled MTs. P(v) can be represented by log-normal distributions with long tails and it is determined by random sweeping motions (v $ 0.5 lm/s) of the MTs (caused by tangential forces on the filament ends coupled to the actin cortex) and by intermittent bead transports parallel to the MTs (v max $ 1.5 lm/s). The tails are due to spontaneous filament deflections (with speeds up to 10 lm/s) attributed to pre-stressing of the MT by local cortical tensions, generated by dynactin motors generating plus-end directed forces in the MTs. The viscoelastic responses are strongly non-linear and are mostly directed opposite or perpendicular to the force, showing that the cytoplasm behaves as an active viscoplastic body with time and force dependent drag coefficients. Nano-Newton loads exerted on the soft MT are balanced by traction forces arising at the MT ends coupled to the actin cortex and the centrosome, respectively. The mechanical coupling between the soft microtubules and the viscoelastic actin cortex provides cells with high mechanical stability despite the softness of the cytoplasm.

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Section: Preparation Of Cellsmentioning

confidence: 99%

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule–actin crosstalk

Heinrich

Sackmann²

2006

Acta Biomaterialia

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“…For the developmental study, cells were washed twice in 12 mM Na/K phosphate buffer and resuspended at 7 ϫ 10 7 cells/ml and plated at various densities on agar plates. Manstein et al (1989). Because there are two copies of SPKA genes, spkA null cells were generated in JH10 cells by using two targeting constructs containing a Thy cassette and a Bsr cassette, respectively.…”

Section: Cell Culture and Development Of Dictyostelium Cellsmentioning

confidence: 99%

“…The spkA null mutant was obtained by a gene replacement technique based on that of Manstein et al (1989). Because there are two copies of SPKA genes, spkA null cells were generated in JH10 cells by using two targeting constructs containing a Thy cassette and a Bsr cassette, respectively.…”

Section: Generation Of Spka Null Cells and Cells Expressing Wildtype mentioning

confidence: 99%

DictyosteliumStress-activated Protein Kinase α, a Novel Stress-activated Mitogen-activated Protein Kinase Kinase Kinase-like Kinase, Is Important for the Proper Regulation of the Cytoskeleton

Sun

Firtel

2003

MBoC

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Mitogen-activated protein kinase cascades regulate various cellular functions, including growth, cell differentiation, development, and stress responses. We have identified a new Dictyostelium kinase (stress-activated protein kinase [SAPK]␣), which is related to members of the mixed lineage kinase class of mitogen-activated protein kinase kinases. SAPK␣ is activated by osmotic stress, heat shock, and detachment from the substratum and by a membrane-permeable cGMP analog, a known regulator of stress responses in Dictyostelium. SAPK␣ is important for cellular resistance to stresses, because SAPK␣ null cells exhibit reduced viability in response to osmotic stress. We found that SAPK␣ mutants affect cellular processes requiring proper regulation of the actin cytoskeleton, including cell motility, morphogenesis, cytokinesis, and cell adhesion. Overexpression of SAPK␣ results in highly elevated basal and chemoattractant-stimulated F-actin levels and strong aggregation and developmental defects, including a failure to polarize and chemotax, and abnormal morphogenesis. These phenotypes require a kinase-active SAPK␣. SAPK␣ null cells exhibit reduced chemoattractant-stimulated F-actin levels, cytokinesis, developmental and adhesion defects, and a motility defect that is less severe than that exhibited by SAPK␣-overexpressing cells. SAPK␣ colocalizes with F-actin in F-actin-enriched structures, including membrane ruffles and pseudopodia during chemotaxis. Although SAPK␣ is required for these F-actin-mediated processes, it is not detectably activated in response to chemoattractant stimulation. INTRODUCTIONThe actin cytoskeleton plays a vital role in numerous cellular processes, such as cell locomotion, phagocytosis, cytokinesis, cell adhesion, cell shape changes, and stress responses. During these processes, the actin cytoskeleton is dynamically changed, a process that involves F-actin polymerization and depolymerization and the reorganization of existing filament networks. Mutations of signaling molecules or structural elements involved in these changes affect cellular processes to various extents.Eukaryotic cells are constantly exposed to stress conditions. To survive, cells adopt various mechanisms to adapt to environmental changes. Mitogen-activated protein kinase (MAPK) pathways regulate cellular responses to external stress in various organisms, including yeast, plants, and mammals (Canman and Kastan, 1996;Jonak et al., 1996;Gustin et al., 1998;Hirt, 2000;Kyriakis and Avruch, 2001;O'Rourke et al., 2002). In Saccharomyces cerevisiae, the wellcharacterized HOG1 MAPK pathway is activated by osmotic stress and leads to gene expression and glycerol production, which is critical for yeast cells to withstand osmotic stress (Gustin et al., 1998;O'Rourke et al., 2002). In mammalian cells, the c-Jun NH 2 -terminal kinase/stress-activated protein kinase (JNK/SAPK1) pathway and the p38/SAPK2 MAP kinase pathway are both activated by diverse stimuli including UV, osmotic, and thermal stresses (Canman and Kastan, 1996;Chang and Kari...

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“…Dictyostelium amoebae are haploid, and many genes, including calmodulin (Goldhagen and Clarke, 1986), that are present in multiple copies in mammalian cells are single-copy in Dictyostelium. Thus, for several Dictyostelium proteins, it has been possible to manipulate gene expression by deleting or altering the appropriate genomic sequences via homologous recombination (DeLozanne and Spudich, 1987;Witke et al, 1987;Manstein et al, 1989;Jung and Hammer, 1990;Doring et al, 1991;Kumagai et al, 1991;Sun and Devreotes, 1991;Cox et al, 1992). Reduction of mRNA levels by the introduction of vectors producing antisense RNA also has proved effective.…”

Section: Introductionmentioning

confidence: 99%

Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis.

Liu

Williams

Clarke

1992

MBoC

100

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The single gene encoding calmodulin in the eukaryotic microorganism Dictyostelium discoideum was cloned and sequenced. The gene was found to contain three introns, one lying immediately after the translation initiation codon. The deduced amino acid sequence indicated that Dictyostelium calmodulin contains 19 amino acid differences from vertebrate calmodulin, including extensions at both termini. Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells. A complete copy of the calmodulin cDNA was prepared, and an 87-base pair fragment complementary to the 5'-end of the calmodulin mRNA was subcloned into the Dictyostelium transformation vector pVEII, such that expression of the antisense transcript was driven by the discoidin I gamma promoter. Transformed cells were selected and maintained at low cell density, a condition resulting in minimal activity of the discoidin I promoter. High level expression was induced by allowing the transformants to reach high cell density or by growing them in the presence of medium conditioned by high density cells. Under these conditions, in which calmodulin mRNA and protein levels were reduced about twofold, the calmodulin antisense transformants lost the ability to complete cytokinesis. A contractile ring formed and constricted, but the midbody linking daughter cells failed to break. The resulting cell population contained multinucleated cells and networks of cells connected by cytoplasmic bridges. Normal cell division was restored when the cells were diluted to low density. These observations have identified a new point at which calmodulin may regulate cell cleavage.

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Gene replacement in Dictyostelium: generation of myosin null mutants.

Cited by 289 publications

References 44 publications

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule–actin crosstalk

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule–actin crosstalk

DictyosteliumStress-activated Protein Kinase α, a Novel Stress-activated Mitogen-activated Protein Kinase Kinase Kinase-like Kinase, Is Important for the Proper Regulation of the Cytoskeleton

Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis.

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