Cell motility and SCAR localisation in axenically growing Dictyostelium cells

Pollitt, Alice Y.; Blagg, Simone L.; Ibarra, Neysi; Insall, Robert H.

doi:10.1016/j.ejcb.2006.05.014

Cited by 21 publications

(23 citation statements)

References 35 publications

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“…We therefore tested macropinocytosis in null mutants of these two Ras genes. RasS-null cells have previously been reported to be defective in macropinocytosis yet strain-backgrounddependent inconsistencies (Pollitt et al, 2006) encouraged us to confirm this result with a different isolate (Ax2 background against DH1 background of the original isolate). The effect was smaller than in the original report yet still clear.…”

Section: Pi3-kinase-ras Interaction Mapsupporting

confidence: 67%

Two distinct functions for PI3-kinases in macropinocytosis

Hoeller

Bolourani

Clark

et al. 2013

Journal of Cell Science

159

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SummaryClass-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P 3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P 3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P 3 , with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.

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Section: Pi3-kinase-ras Interaction Mapsupporting

confidence: 67%

Two distinct functions for PI3-kinases in macropinocytosis

Hoeller

Bolourani

Clark

et al. 2013

Journal of Cell Science

159

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“…5). Previously it has been observed that in Dictyostelium, different genetic backgrounds use SCAR to a greater or lesser extent during vegetative growth (Pollitt et al, 2006). When we repeated these experiments with scar-null cells in an Ax2 genetic background [a kind gift from Jan Faix, Hannover Medical School, Hannover, Germany (Steffen et al, 2006)] we saw the same effect, with a large increase in multinucleate cells upon blebbistatin treatment as well as a disorganised, blebby morphology as the cells divided (supplementary material Fig.…”

Section: Scar Drives Myosin-ii-independent Cytokinesissupporting

confidence: 53%

“…S3) and we conclude that this probe gives an authentic representation of SCAR localisation. This enabled us for the first time to image SCAR dynamics in live Dictyostelium, giving a similar but much clearer localisation than previous images of fixed cells using a SCARspecific antibody (Pollitt et al, 2006) and with temporal resolution.…”

Section: Enhanced Migration and Activation Of Scar At Cytokinesismentioning

confidence: 88%

“…S5). Interestingly, although vegetative scar-null cells are still able to move (Pollitt et al, 2006) multinucleate scar-null cells are stationary and did not significantly move over several hours. SCAR is thus more important for multinucleate cell motility and is essential for traction-mediated cytofission.…”

Section: Scar Is Also Essential For Traction-mediated Cytofissionmentioning

confidence: 99%

“…During interphase, Ax3 cells only have weak SCAR-complex localisation (Pollitt et al, 2006) and HSPC300-GFP only transiently localised to the small pseudopodia that vegetative Dictyostelium cells make (data not shown). However, during cytokinesis, the SCAR complex became strongly localised to the large lamellipodial protrusions of each daughter cell, just ahead of a broad band of actin and ARPC4 and was excluded from the furrow ( Fig.…”

Section: Enhanced Migration and Activation Of Scar At Cytokinesismentioning

confidence: 99%

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SCAR/WAVE is activated at mitosis and drives myosin-independent cytokinesis

King

Veltman

Georgiou

et al. 2010

Journal of Cell Science

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SummaryCell division requires the tight coordination of multiple cytoskeletal pathways. The best understood of these involves myosin-IIdependent constriction around the cell equator, but both Dictyostelium and mammalian cells also use a parallel, adhesion-dependent mechanism to generate furrows. We show that the actin nucleation factor SCAR/WAVE is strongly activated during Dictyostelium cytokinesis. This activation localises to large polar protrusions, driving separation of the daughter cells. This continues for 10 minutes after division before the daughter cells revert to normal random motility, indicating that this is a tightly regulated process. We demonstrate that SCAR activity is essential to drive myosin-II-independent cytokinesis, and stabilises the furrow, ensuring symmetrical division. SCAR is also responsible for the generation of MiDASes, mitosis-specific actin-rich adhesions. Loss of SCAR in both Dictyostelium and Drosophila leads to a similar mitotic phenotype, with severe mitotic blebbing, indicating conserved functionality. We also find that the microtubule end-binding protein EB1 is required to restrict SCAR localisation and direct migration. EB1-null cells also exhibit decreased adhesion during mitosis. Our data reveal a spindle-directed signalling pathway that regulates SCAR activity, migration and adhesion at mitosis.

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Abi Mutants in Dictyostelium Reveal Specific Roles for the SCAR/WAVE Complex in Cytokinesis

Pollitt

Insall

2008

Current Biology

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Actin polymerization drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signaling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins: PIR121, Nap1, Abi2,and HSPC300 (Figure S1A available online) [1-3]. However,there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation, as originally proposed by Edenet al. [1]. It also is unclear whether complex members exclusively regulate SCAR/WAVEs or if they have additional targets [4-6]. Here, we analyze the roles of the unique Dictyostelium Abi. We find that abiA null mutants show less severe defects in motility than do scar null cells, indicating--unexpectedly--that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants and is seen only when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways.

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Cell motility and SCAR localisation in axenically growing Dictyostelium cells

Cited by 21 publications

References 35 publications

Two distinct functions for PI3-kinases in macropinocytosis

Two distinct functions for PI3-kinases in macropinocytosis

SCAR/WAVE is activated at mitosis and drives myosin-independent cytokinesis

Abi Mutants in Dictyostelium Reveal Specific Roles for the SCAR/WAVE Complex in Cytokinesis

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