DictyosteliumPAKc Is Required for Proper Chemotaxis
45
Citation
58
Reference
10
Related Paper
Citation Trend
Abstract:
We have identified a new Dictyostelium p21-activated protein kinase, PAKc, that we demonstrate to be required for proper chemotaxis. PAKc contains a Rac-GTPase binding (CRIB) and autoinhibitory domain, a PAK-related kinase domain, an N-terminal phosphatidylinositol binding domain, and a C-terminal extension related to the Gbetagamma binding domain of Saccharomyces cerevisiae Ste20, the latter two domains being required for PAKc transient localization to the plasma membrane. In response to chemoattractant stimulation, PAKc kinase activity is rapidly and transiently activated, with activity levels peaking at approximately 10 s. pakc null cells exhibit a loss of polarity and produce multiple lateral pseudopodia when placed in a chemoattractant gradient. PAKc preferentially binds the Dictyostelium Rac protein RacB, and point mutations in the conserved CRIB that abrogate this binding result in misregulated kinase activation and chemotaxis defects. We also demonstrate that a null mutation lacking the PAK family member myosin I heavy chain kinase (MIHCK) shows mild chemotaxis defects, including the formation of lateral pseudopodia. A null strain lacking both PAKc and the PAK family member MIHCK exhibits severe loss of cell movement, suggesting that PAKc and MIHCK may cooperate to regulate a common chemotaxis pathway.Keywords:
Pseudopodia
ABSTRACT Mutants lacking the MAP kinase DdERK2 show reduced chemotactic responses to folate and cAMP. Analysis of cAMP chemotaxis shows that Dderk2- cells are defective in chemotaxis to high concentrations of cAMP. This defect is due to an inability to repolarize in the continued presence of high concentrations of cAMP. Under these conditions, the speed of movement of mutant cells remains low. Instead of generating a leading pseudopod, mutant cells generate transient crown-like structures over multiple regions of the cell surface. These structures differ from pseudopods in that they contain myosin II as well as F actin and coronin. These studies identify a role for MAP kinases in coordinating the formation of cell projections generated in response to chemoattractants.
Cite
Citations (41)
ABSTRACT Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: MyΔ824-941, MyΔ943-1464, MyΔ943-1194 and MyΔ1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, except for MyΔ943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggest that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow.
Cleavage furrow
Cleavage (geology)
Cite
Citations (21)
Cite
Citations (15)
Model Organism
Mycetozoa
Cite
Citations (33)
Neutrophil chemotaxis is a critical component in innate immunity. Recently, using a small-molecule functional screening, we identified NADPHoxidase- dependent Reactive Oxygen Species (ROS) as key regulators of neutrophil chemotactic migration. Neutrophils depleted of ROS form more frequent multiple pseudopodia and lost their directionality as they migrate up a chemoattractant concentration gradient. Here, we further studied the role of ROS in neutrophil chemotaxis and found that multiple pseudopodia formation induced by NADPH inhibitor diphenyleneiodonium chloride (DPI) was more prominent in relatively shallow chemoattractant gradient. It was reported that, in shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. Directional sensing is mediated by maintaining the most accurate existing pseudopod, and destroying pseudopods facing the wrong direction by actin depolymerization. We propose that NADPH-mediated ROS production may be critical for disruption of misoriented pseudopods in chemotaxing neutrophils. Thus, inhibition of ROS production will lead to formation of multiple pseudopodia.
Pseudopodia
Cite
Citations (44)
Conventional myosin plays a key role in the cytoskeletal reorganization necessary for cytokinesis, migration, and morphological changes associated with development in nonmuscle cells. We have made a fusion between the green fluorescent protein (GFP) and the Dictyostelium discoideum myosin heavy chain (GFP-myosin). The unique Dictyostelium system allows us to test the GFP-tagged myosin for activity both in vivo and in vitro. Expression of GFP-myosin rescues all myosin null cell defects. Additionally, GFP-myosin purified from these cells exhibits the same ATPase activities and in vitro motility as wild-type myosin. GFP-myosin is concentrated in the cleavage furrow during cytokinesis and in the posterior cortex of migrating cells. Surprisingly, GFP-myosin concentration increases transiently in the tips of retracting pseudopods. Contrary to previous thinking, this suggests that conventional myosin may play an important role in the dynamics of pseudopods as well as filopodia, lamellipodia, and other cellular protrusions.
Pseudopodia
Cleavage furrow
Filopodia
Lamellipodium
Cite
Citations (204)
When starved, Dictyostelium cells respond to extracellular signals, polarize, and move with strong persistence into aggregation centers. Actin and actin-associated proteins play key roles in regulating both the morphology and directed movements of cells during chemotactic aggregation. Recently, we identified an ortholog of Abp1 in Dictyostelium (Dabp1). The first actin binding protein identified in yeast, Abp1 functions in actin-based endocytosis in yeast and in receptor-mediated endocytosis in mammalian cells. To explore the functions for Abp1 in Dictyostelium, we examined the phenotypes of cells that overexpressed the Dabp1 protein and cells that eliminated Dabp1 expression. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild-type cells had a single leading pseudopodium and a single uropodium, whereas more than 27% of Dabp1 null cells projected multiple pseuodpodia. Similarly, approximately 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium.
Pseudopodia
Cite
Citations (9)
Mycetozoa
Cite
Citations (2)
Chemotaxis is one of the most fascinating processes in cell biology. Shallow gradients of chemoattractant direct the movement of cells, and an intricate network of signalling pathways somehow instructs the movement apparatus to induce pseudopods in the direction of these gradients. Exciting new experiments have approached chemotaxis from the perspective of the extending pseudopod. These recent studies have revealed that, in the absence of external cues, cells use endogenous signals for the highly ordered extension of pseudopods, which appear mainly as alternating right and left splits. In addition, chemoattractants activate other signalling molecules that induce a positional bias of this basal system, such that the extending pseudopods are oriented towards the gradient. In this Commentary, I review the findings of these recent experiments, which together provide a new view of cell movement and chemotaxis.
Pseudopodia
Cite
Citations (56)
We have cloned and completely sequenced a gene encoding the heavy chain of Dictyostelium myosin I. Like the myosin I molecules from Acanthamoeba, the Dictyostelium myosin I heavy chain is composed of a globular head domain fused to a 45-kDa glycine-, proline-, and alanine-rich carboxyl-terminal domain, rather than the coiled-coil rod domain of conventional myosins. Comparisons of the Dictyostelium myosin I heavy-chain amino acid sequence with those of the Acanthamoeba myosins I reveal that they are highly similar throughout, including the unconventional carboxyl-terminal domains. The Dictyostelium myosin I gene is expressed in growing cells as a 3600-nucleotide mRNA. Measurements of the steady-state level of this mRNA at different times during starvation-induced aggregation and development are consistent with a role for myosin I in chemotaxis and aggregation. Generation of Dictyostelium cells lacking myosin I by gene disruption and/or antisense RNA production should provide a way to test directly the role of this nonfilamentous myosin in cell motility. These experiments will be simplified by the fact that Southern blot analyses of Dictyostelium genomic DNA are consistent with there being a single myosin I heavy-chain gene.
Cite
Citations (93)