Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization

1999 ◽  
Vol 112 (19) ◽  
pp. 3195-3203 ◽  
Author(s):  
A.A. Noegel ◽  
F. Rivero ◽  
R. Albrecht ◽  
K.P. Janssen ◽  
J. Kohler ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Axenic Medium ◽  
Terminal Domain ◽  
Defect Growth ◽  
Fluid Phase Endocytosis ◽  
Altered Cell ◽  
Mutant Cells ◽  
Proline Rich Region

The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

scholarly journals Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

2004 ◽  
Vol 15 (2) ◽  
pp. 861-869 ◽  
Author(s):  
Yaya Lefkir ◽  
Marilyne Malbouyres ◽  
Daniel Gotthardt ◽  
Adrian Ozinsky ◽  
Sophie Cornillon ◽  
...  
Keyword(s):  
Early Stage ◽  
Fluid Phase ◽  
Small Particles ◽  
Medium Chain ◽  
Murine Macrophages ◽  
Fluid Phase Endocytosis ◽  
Phagocytic Cups ◽  
Golgi Network ◽  
Mutant Cells

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1-cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1-cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1-cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

Synaptojanin family members are implicated in endocytic membrane traffic in yeast

1998 ◽  
Vol 111 (22) ◽  
pp. 3347-3356 ◽  
Author(s):  
B. Singer-Kruger ◽  
Y. Nemoto ◽  
L. Daniell ◽  
S. Ferro-Novick ◽  
P. De Camilli
Keyword(s):  
Synaptic Vesicles ◽  
Direct Evidence ◽  
Family Members ◽  
Fluid Phase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Close Link ◽  
Fluid Phase Endocytosis ◽  
Synaptojanin 1

The synaptojanins represent a subfamily of inositol 5′-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy

1993 ◽  
Vol 105 (3) ◽  
pp. 861-866 ◽  
Author(s):  
L. Aubry ◽  
G. Klein ◽  
J.L. Martiel ◽  
M. Satre
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Lag Phase ◽  
Nutritive Medium ◽  
Chase Period ◽  
Endosomal Acidification ◽  
Fluid Phase Endocytosis ◽  
Endosomal Ph ◽  
Efflux Kinetics ◽  
Secondary Lysosomes

The evolution of endo-lysosomal pH in Dictyostelium discoideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fluorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an extended lag phase lasting for 45–60 min and corresponding to intracellular transit of FITC-dextran cohort. During the chase period, endosomal pH decreased during approximately 20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20–40 min to reach pH 6.0-6.2. It was only at this stage that FITC-dextran was released back into the medium with pseudo first-order kinetics. A vacuolar H(+)-ATPase is involved in endosomal acidification as the acidification process was markedly reduced in mutant strain HGR8, partially defective in vacuolar H(+)-ATPase and in parent type strain AX2 by bafilomycin A1, a selective inhibitor of this enzyme. Our data suggest that endocytic cargo is channeled from endosomes to secondary lysosomes that are actively linked to the plasma membrane via recycling vesicles.

The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement

2000 ◽  
Vol 113 (4) ◽  
pp. 709-719 ◽  
Author(s):  
J.R. Chubb ◽  
A. Wilkins ◽  
G.M. Thomas ◽  
R.H. Insall
Keyword(s):  
Cell Migration ◽  
Significant Proportion ◽  
Cell Movement ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Cell Cortex ◽  
Fluid Phase Endocytosis ◽  
Ras Family ◽  
Actin Protrusions ◽  
Mutant Cells

Endocytosis and cell migration both require transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. We have generated Dictyostelium cells lacking one member of this family, RasS, and the mutant cells are perturbed in endocytosis and cell migration. Mutant amoebae are defective in phagocytosis and fluid-phase endocytosis and are impaired in growth. Conversely, the rasS(-)cells show an enhanced rate of cell migration, moving three times faster than wild-type controls. The mutant cells display an aberrant morphology, are highly polarised, carry many elongated actin protrusions and show a concomitant decrease in formation of pinocytic crowns on the cell surface. These morphological aberrations are paralleled by changes in the actin cytoskeleton, with a significant proportion of the cortical F-actin relocalised to prominent pseudopodia. Rapid migration and endocytosis appear to be mutually incompatible and it is likely that RasS protein is required to maintain the normal balance between these two actin-dependent processes.

The role of phosphodiesterase in aggregation of Dictyostelium discoideum

1978 ◽  
Vol 31 (1) ◽  
pp. 233-243
Author(s):  
M. Darmon ◽  
J. Barra ◽  
P. Brachet
Keyword(s):  
Dictyostelium Discoideum ◽  
Direct Contact ◽  
Signal To Noise Ratio ◽  
Signal To Noise ◽  
Extracellular Medium ◽  
Camp Phosphodiesterase ◽  
Cellular Slime Mould ◽  
Cellular Slime ◽  
Mutant Cells

The role of cAMP phosphodiesterase in the cAMP-mediated aggregation of the cellular slime mould Dictyostelium discoideum was investigated with a morphogenetic mutant defective in phosphodiesterase production. Mutant cells become capable of aggregating normally when incubated in the presence of exogenous phosphodiesterase isolated from Idictyostelium or rat brain. Direct contact between enzyme and the cell membrane is not required for this phenotypic suppression. The aggregateless character of this strain presumably results from an over-accumulation of cAMP in the extracellular medium since aggregation can be induced in the absence of added phosphodiesterase under conditions facilitating diffusion of the nucleotide. This suggests that phosphodiesterase is not involved in the generation or recognition of cAMP signals, but that the enzyme is essential in the control of the cAMP signal-to-noise ratio.

Stimulation of P(i) transport in opossum kidney cells by hyposmotic media

1993 ◽  
Vol 264 (4) ◽  
pp. F585-F592
Author(s):  
M. Loghman-Adham ◽  
G. T. Motock
Keyword(s):  
Fluid Phase ◽  
Kinetic Studies ◽  
Uptake Kinetic ◽  
Opossum Kidney ◽  
Pi Transport ◽  
Opossum Kidney Cells ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake ◽  
Stimulation Of

Exposure of various cells to hyposmotic media (Hypo) results in a rapid inhibition of both receptor-mediated and fluid-phase endocytosis. We used this maneuver to investigate the role of endocytosis in regulation of Pi transport in opossum kidney (OK) cells. Following exposure to Hypo, Na(+)-dependent Pi uptake increased rapidly, reaching a maximum within 5 min, and remained elevated up to 30 min. This was associated with a simultaneous reduction of horseradish peroxidase uptake. Kinetic studies showed increased apparent Vmax for Pi (9.38 +/- 0.93 vs. 13.08 +/- 1.04 nmol.mg-1.5 min-1 for control and Hypo, respectively; P < 0.05, n = 6) with no change in apparent Km. The effect was specific for Pi with no change in the Na(+)-dependent or -independent uptake of L-proline, L-glutamine, or methyl-alpha-D-glucopyranoside. Stimulation of Pi transport persisted when control and Hypo had identical ionic compositions. Stimulation of Pi transport was rapidly reversed when cells were returned to an isosmotic medium. Preincubation with Hypo at 4 degrees C had no effect on Pi transport. Addition of cycloheximide or actinomycin D did not prevent the increased Pi uptake after exposure to Hypo. The effect also persisted after protein kinase C downregulation. Stimulation of Pi transport by Hypo is consistent with reduced endocytic retrieval of Na(+)-Pi cotransporters from brush-border membrane (BBM), resulting in an increase in their number on the BBM.

scholarly journals Endocytosis of soluble immune complexes leads to their clearance by FcγRIIIB but induces neutrophil extracellular traps via FcγRIIA in vivo

Blood ◽  
2012 ◽  
Vol 120 (22) ◽  
pp. 4421-4431 ◽  
Author(s):  
Kan Chen ◽  
Hiroshi Nishi ◽  
Richard Travers ◽  
Naotake Tsuboi ◽  
Kimberly Martinod ◽  
...  
Keyword(s):  
Immune Complexes ◽  
Fluid Phase ◽  
Neutrophil Extracellular Traps ◽  
Humoral Factors ◽  
Extracellular Traps ◽  
Fluid Phase Endocytosis ◽  
Soluble Immune Complexes ◽  
The Individual

Abstract Soluble immune complexes (ICs) are abundant in autoimmune diseases, yet neutrophil responses to these soluble humoral factors remain uncharacterized. Moreover, the individual role of the uniquely human FcγRIIA and glycophosphatidylinositol (GPI)–linked FcγRIIIB in IC-mediated inflammation is still debated. Here we exploited mice and cell lines expressing these human neutrophil FcγRs to demonstrate that FcγRIIIB alone, in the absence of its known signaling partners FcγRIIA and the integrin Mac-1, internalizes soluble ICs through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis. FcγRIIA also uses this pathway. As shown by intravital microscopy, FcγRIIA but not FcγRIIIB-mediated neutrophil interactions with extravascular soluble ICs results in the formation of neutrophil extracellular traps (NETs) in tissues. Unexpectedly, in wild-type mice, IC-induced NETosis does not rely on the NADPH oxidase, myeloperoxidase, or neutrophil elastase. In the context of soluble ICs present primarily within vessels, FcγRIIIB-mediated neutrophil recruitment requires Mac-1 and is associated with the removal of intravascular IC deposits. Collectively, our studies assign a new role for FcγRIIIB in the removal of soluble ICs within the vasculature that may serve to maintain homeostasis, whereas FcγRIIA engagement of tissue soluble ICs generates NETs, a proinflammatory process linked to autoimmunity.

scholarly journals Overproduction of discoidin I by a temperature-sensitive motility mutant of Dictyostelium discoideum.

1984 ◽  
Vol 4 (6) ◽  
pp. 1035-1041 ◽  
Author(s):  
S Biswas ◽  
S C Kayman ◽  
M Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Axenic Medium ◽  
Two Dimensional Gel Electrophoresis ◽  
Motility Mutant ◽  
And Migration ◽  
Mutant Cells

Dictyostelium discoideum MC2 is a temperature-sensitive motility mutant of AX3. Mutant cells are incapable of growth, phagocytosis, and migration under restrictive conditions (Kayman et al., J. Cell Biol. 92:705-711, 1982). We show here that at the restrictive temperature MC2 cells grown axenically or on bacteria synthesized excessive quantities of the lectin discoidin I. By two-dimensional gel electrophoresis and peptide mapping, the proteins overproduced by MC2 cells were indistinguishable from discoidin I synthesized at lower levels in AX3 cells. At least two of the three species of discoidin I were overproduced. This protein family constituted 9% of the total protein in cells that were incubated overnight at 27 degrees C in axenic medium. Although MC2 cells are defective in nutrient uptake under restrictive conditions, the overproduction of discoidin I did not appear to be part of a pleiotropic response to starvation. We propose that transcription of the coordinately regulated discoidin I genes is altered in mutant cells. This alteration may be related to the motility defects manifested by MC2.

scholarly journals Structure-based inhibitors reveal roles for the clathrin terminal domain and its W-box binding site in CME

2020 ◽  
Author(s):  
Zhiming Chen ◽  
Rosa Mino ◽  
Marcel Mettlen ◽  
Peter Michaely ◽  
Madhura Bhave ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fluid Phase ◽  
Structure Design ◽  
Coated Pits ◽  
Bulk Fluid ◽  
Lysosomal Trafficking ◽  
Terminal Domain ◽  
Binding Surface ◽  
Fluid Phase Endocytosis ◽  
Late Stages

AbstractClathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain (TD), which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the TD with or without the distal leg specifically inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. We designed small membrane-penetrating peptides that mimic the four known binding sites on the TD. A peptide, Wbox2, designed to mimic to the W-box motif binding surface on TD binds to SNX9 and AP2, and potently and acutely inhibits CME, while not perturbing AP1-dependent lysosomal trafficking from the Golgi or bulk, fluid phase endocytosis.SummaryChen et al define the role the N-terminal domain (TD) of clathrin heavy chain in early and late stages of clathrin-mediated endocytosis, and guided by its structure, design a membrane-penetrating peptide, Wbox2, that acutely and potently inhibits CME.

scholarly journals The conserved Pkh–Ypk kinase cascade is required for endocytosis in yeast

2002 ◽  
Vol 156 (2) ◽  
pp. 241-248 ◽  
Author(s):  
Amy K.A. deHart ◽  
Joshua D. Schnell ◽  
Damian A. Allen ◽  
Linda Hicke
Keyword(s):  
Fluid Phase ◽  
Receptor Internalization ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Extracellular Signals ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery ◽  
Kinase Cascade ◽  
Signaling Receptors ◽  
Mutant Cells

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast α-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base–regulated serine–threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum– and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in α-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in α-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

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