scholarly journals A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

2005 ◽  
Vol 169 (2) ◽  
pp. 321-329 ◽  
Author(s):  
Hitesh Patel ◽  
Diane L. Barber
Keyword(s):  
Dictyostelium Discoideum ◽  
Intracellular Ph ◽  
Actin Polymerization ◽  
De Novo ◽  
Cell Movement ◽  
Leading Edge ◽  
Ph Homeostasis ◽  
Developmentally Regulated ◽  
Evolutionarily Conserved ◽  
Morphological Polarity

Increased intracellular H+ efflux is speculated to be an evolutionarily conserved mechanism necessary for rapid assembly of cytoskeletal filaments and for morphological polarity during cell motility. In Dictyostelium discoideum, increased intracellular pH through undefined transport mechanisms plays a key role in directed cell movement. We report that a developmentally regulated Na-H exchanger in Dictyostelium discoideum (DdNHE1) localizes to the leading edge of polarized cells and is necessary for intracellular pH homeostasis and for efficient chemotaxis. Starved DdNHE1-null cells (Ddnhe1−) differentiate, and in response to the chemoattractant cAMP they retain directional sensing; however, they cannot attain a polarized morphology, but instead extend mislocalized pseudopodia around the cell and exhibit decreased velocity. Consistent with impaired polarity, in response to chemoattractant, Ddnhe1− cells lack a leading edge localization of F-actin and have significantly attenuated de novo F-actin polymerization but increased abundance of membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). These findings indicate that during chemotaxis DdNHE1 is necessary for establishing the kinetics of actin polymerization and PI(3,4,5)P3 production and for attaining a polarized phenotype.

scholarly journals Dictyostelium Dock180-related RacGEFs Regulate the Actin Cytoskeleton during Cell Motility

2009 ◽  
Vol 20 (2) ◽  
pp. 699-707 ◽  
Author(s):  
Alessia Para ◽  
Miriam Krischke ◽  
Sylvain Merlot ◽  
Zhouxin Shen ◽  
Michael Oberholzer ◽  
...  
Keyword(s):  
Cell Motility ◽  
Protein Function ◽  
Actin Polymerization ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Cortex ◽  
Proteomic Approach ◽  
Evolutionarily Conserved ◽  
Membrane Protrusions

Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P3 production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved.

scholarly journals EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells

1998 ◽  
Vol 111 (2) ◽  
pp. 199-211 ◽  
Author(s):  
A.Y. Chan ◽  
S. Raft ◽  
M. Bailly ◽  
J.B. Wyckoff ◽  
J.E. Segall ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Mammary Adenocarcinoma ◽  
Actin Dynamics ◽  
Actin Nucleation ◽  
Nucleation Sites ◽  
Nucleation Activity ◽  
Pointed End ◽  
Stimulation Of

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/−0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.

scholarly journals Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

2007 ◽  
Vol 179 (7) ◽  
pp. 1539-1553 ◽  
Author(s):  
Rosa Ana Lacalle ◽  
Rosa M. Peregil ◽  
Juan Pablo Albar ◽  
Ernesto Merino ◽  
Carlos Martínez-A ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Polarization ◽  
Type I ◽  
Lipid Kinase ◽  
Erm Proteins ◽  
Chemical Gradients ◽  
C Terminus ◽  
Phosphatidylinositol 4

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.

scholarly journals Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

2004 ◽  
Vol 167 (3) ◽  
pp. 505-518 ◽  
Author(s):  
Atsuo T. Sasaki ◽  
Cheryl Chun ◽  
Kosuke Takeda ◽  
Richard A. Firtel
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Leading Edge ◽  
Ras Signaling ◽  
Phosphatidylinositol 3 Kinase ◽  
Ras Activation ◽  
Directional Movement ◽  
Ras Effectors ◽  
Chemotaxis Receptors ◽  
Intermediate Event

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

Myosin II-dependent cylindrical protrusions induced by quinine inDictyostelium: antagonizing effects of actin polymerization at the leading edge

2001 ◽  
Vol 114 (11) ◽  
pp. 2155-2165
Author(s):  
Kunito Yoshida ◽  
Kei Inouye
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Cell Movement ◽  
Leading Edge ◽  
Contractile Vacuole ◽  
Cell Periphery ◽  
Slowing Down ◽  
On Line ◽  
Cylindrical Protrusion

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc− cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general.Movies available on-line

scholarly journals Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

2011 ◽  
Vol 193 (5) ◽  
pp. 831-839 ◽  
Author(s):  
Michael Carnell ◽  
Tobias Zech ◽  
Simon D. Calaminus ◽  
Seiji Ura ◽  
Monica Hagedorn ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Dictyostelium Discoideum ◽  
Adenosine Triphosphatase ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Coated Vesicles ◽  
Filamentous Actin ◽  
Evolutionarily Conserved ◽  
Related Process

WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

scholarly journals Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

1997 ◽  
Vol 139 (5) ◽  
pp. 1243-1253 ◽  
Author(s):  
R.J. Eddy ◽  
J. Han ◽  
J.S. Condeelis
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Actin Assembly ◽  
Directed Movement ◽  
Capping Protein ◽  
End Capping

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

scholarly journals Association of Mouse Actin-binding Protein 1 (mAbp1/SH3P7), an Src Kinase Target, with Dynamic Regions of the Cortical Actin Cytoskeleton in Response to Rac1 Activation

2000 ◽  
Vol 11 (1) ◽  
pp. 393-412 ◽  
Author(s):  
Michael M. Kessels ◽  
Åsa E. Y. Engqvist-Goldstein ◽  
David G. Drubin
Keyword(s):  
Actin Cytoskeleton ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Membrane Dynamics ◽  
Cellular Growth ◽  
Actin Binding ◽  
Cortical Actin ◽  
Binding Domains

Yeast Abp1p is a cortical actin cytoskeleton protein implicated in cytoskeletal regulation, endocytosis, and cAMP-signaling. We have identified a gene encoding a mouse homologue of Abp1p, and it is identical to SH3P7, a protein shown recently to be a target of Src tyrosine kinases. Yeast and mouse Abp1p display the same domain structure including an N-terminal actin-depolymerizing factor homology domain and a C-terminal Src homology 3 domain. Using two independent actin-binding domains, mAbp1 binds to actin filaments with a 1:5 saturation stoichiometry. In stationary cells, mAbp1 colocalizes with cortical F-actin in fibroblast protrusions that represent sites of cellular growth. mAbp1 appears at the actin-rich leading edge of migrating cells. Growth factors cause mAbp1 to rapidly accumulate in lamellipodia. This response can be mimicked by expression of dominant-positive Rac1. mAbp1 recruitment appears to be dependent on de novo actin polymerization and occurs specifically at sites enriched for the Arp2/3 complex. mAbp1 is a newly identified cytoskeletal protein in mice and may serve as a signal-responsive link between the dynamic cortical actin cytoskeleton and regions of membrane dynamics.

scholarly journals Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

2004 ◽  
Vol 166 (5) ◽  
pp. 697-708 ◽  
Author(s):  
Ghassan Mouneimne ◽  
Lilian Soon ◽  
Vera DesMarais ◽  
Mazen Sidani ◽  
Xiaoyan Song ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Actin Polymerization ◽  
Carcinoma Cell ◽  
Cell Movement ◽  
Leading Edge ◽  
Carcinoma Cells ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase ◽  
Blocking Antibodies ◽  
Interfering Rna

The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

scholarly journals Intracellular pH in Dictyostelium discoideum: a 31P nuclear magnetic resonance study.

1985 ◽  
Vol 101 (3) ◽  
pp. 778-784 ◽  
Author(s):  
J E Jentoft ◽  
C D Town
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Dictyostelium Discoideum ◽  
Intracellular Ph ◽  
Nuclear Magnetic Resonance Study ◽  
Published Data ◽  
Cellular Slime Mold ◽  
Ph Homeostasis ◽  
Cytosolic Ph ◽  
Nuclear Magnetic

We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum. We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils. Cells continued to develop normally in this set-up. Spectra acquired under these conditions typically show two peaks in the inorganic phosphate region corresponding to pH values of 7.16 +/- 0.03 and 6.48 +/- 0.02. These peaks are believed to represent the mitochondrial and cytosolic compartments respectively, based on a comparison of these values with published data and the collapse of the two compartments upon addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. Dictyostelium cells show a remarkable degree of intracellular pH homeostasis. Both mitochondrial and cytosolic pH remained unchanged as extracellular pH was varied from 4.3 to 8.1. There was also no apparent change in the pH of either compartment after up to 13.5 hours' development in suspension.

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