scholarly journals PakD, a Putative p21-Activated Protein Kinase in Dictyostelium discoideum, Regulates Actin

2013 ◽  
Vol 13 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Miguel Garcia ◽  
Sibnath Ray ◽  
Isaiah Brown ◽  
Jon Irom ◽  
Derrick Brazill
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Dictyostelium Discoideum ◽  
Cell Function ◽  
Actin Dynamics ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Actin Organization ◽  
Cellular Processes

ABSTRACT Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum . We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD − cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD − cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD − cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD − cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.

scholarly journals The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum

2010 ◽  
Vol 9 (10) ◽  
pp. 1557-1565 ◽  
Author(s):  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Signal Transduction Pathway ◽  
Protein Accumulation ◽  
Proliferation Inhibition ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Green Fluorescent

ABSTRACT AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA − cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA − cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.

scholarly journals A Retinoblastoma Orthologue Is Required for the Sensing of a Chalone in Dictyostelium discoideum

2014 ◽  
Vol 13 (3) ◽  
pp. 376-382 ◽  
Author(s):  
Deenadayalan Bakthavatsalam ◽  
Michael J. V. White ◽  
Sarah E. Herlihy ◽  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Accumulation Rate ◽  
Protein Accumulation ◽  
Wild Type ◽  
Spore Viability ◽  
Content Type ◽  
Multicellular Development ◽  
Normal Mass ◽  
Inhibit Cell Proliferation

ABSTRACT Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA − cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA − cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA − cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA − cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA − cells. Similar to aprA − cells, rblA − cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium , suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.

scholarly journals Actin Cytoskeleton Regulates Calcium Dynamics and NFAT Nuclear Duration

2004 ◽  
Vol 24 (4) ◽  
pp. 1628-1639 ◽  
Author(s):  
Fabiola V. Rivas ◽  
James P. O'Keefe ◽  
Maria-Luisa Alegre ◽  
Thomas F. Gajewski
Keyword(s):  
T Cell ◽  
Actin Cytoskeleton ◽  
T Cell Activation ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Cell Function ◽  
Immunological Synapse ◽  
Interleukin 2 ◽  
Actin Dynamics ◽  
Activated T Cells

ABSTRACT T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca2+ ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca2+-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.

FGF signalling is required for differentiation-induced cytoskeletal reorganisation and formation of actin-based processes by podocytes

2001 ◽  
Vol 114 (18) ◽  
pp. 3359-3366 ◽  
Author(s):  
Gary Davidson ◽  
Rosanna Dono ◽  
Rolf Zeller
Keyword(s):  
Cell Differentiation ◽  
Actin Cytoskeleton ◽  
Mutant Mouse ◽  
Wild Type Mouse ◽  
Wild Type ◽  
General Role ◽  
Cellular Processes ◽  
Cells In Culture ◽  
Fgf Signalling

To examine the potential role of fibroblast growth factor (FGF) signalling during cell differentiation, we used conditionally immortalised podocyte cells isolated from kidneys of Fgf2 mutant and wild-type mice. Wild-type mouse podocyte cells upregulate FGF2 expression when differentiating in culture, as do maturing podocytes in vivo. Differentiating wild-type mouse podocyte cells undergo an epithelial to mesenchymal-like transition, reorganise their actin cytoskeleton and extend actin-based cellular processes; all of these activities are similar to the activity of podocytes in vivo. Molecular analysis of Fgf2 mutant mouse podocyte cells reveals a general disruption of FGF signalling as expression of Fgf7 and Fgf10 are also downregulated. These FGF mutant mouse podocyte cells in culture fail to activate mesenchymal markers and their post-mitotic differentiation is blocked. Furthermore, mutant mouse podocyte cells in culture fail to reorganise their actin cytoskeleton and form actin-based cellular processes. These studies show that FGF signalling is required by cultured podocytes to undergo the epithelial to mesenchymal-like changes necessary for terminal differentiation. Together with other studies, these results point to a general role for FGF signalling in regulating cell differentiation and formation of actin-based cellular processes during morphogenesis.

scholarly journals CbfA, the C-Module DNA-Binding Factor, Plays an Essential Role in the Initiation of Dictyostelium discoideum Development

2004 ◽  
Vol 3 (5) ◽  
pp. 1349-1358 ◽  
Author(s):  
Thomas Winckler ◽  
Negin Iranfar ◽  
Peter Beck ◽  
Ingo Jennes ◽  
Oliver Siol ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Density ◽  
Dictyostelium Discoideum ◽  
Ectopic Expression ◽  
Development Program ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Wild Type ◽  
Gene Encoding ◽  
Multicellular Development

ABSTRACT We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.

scholarly journals DprA-Dependent Exit from the Competent State Regulates Multifaceted Streptococcus pneumoniae Virulence

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Jingjun Lin ◽  
Gee W. Lau
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Cell Separation ◽  
Protein A ◽  
Wild Type ◽  
Content Type ◽  
Cellular Processes ◽  
Virulence Attenuation ◽  
Specific Induction ◽  
Competence System

ABSTRACT Streptococcus pneumoniae (pneumococcus) causes multiple infectious diseases. The pneumococcal competence system facilitates genetic transformation, spreads antibiotic resistance, and contributes to virulence. DNA-processing protein A (DprA) regulates the exit of pneumococcus from the competent state. Previously, we have shown that DprA is important in both bacteremia and pneumonia infections. Here, we examined the mechanisms of virulence attenuation in a ΔdprA mutant. Compared to the parental wild-type D39, the ΔdprA mutant enters the competent state when exposed to lower concentrations of the competence-stimulating peptide CSP1. The ΔdprA mutant overexpresses ComM, which delays cell separation after division. Additionally, the ΔdprA mutant overexpresses allolytic factors LytA, CbpD, and CibAB and is more susceptible to detergent-triggered lysis. Disabling of the competent-state-specific induction of ComM and allolytic factors compensated for the virulence loss in the ΔdprA mutant, suggesting that overexpression of these factors contributes to virulence attenuation. Finally, the ΔdprA mutant fails to downregulate the expression of multiple competence-regulated genes, leading to the excessive energy consumption. Collectively, these results indicate that an inability to properly exit the competent state disrupts multiple cellular processes that cause virulence attenuation in the ΔdprA mutant.

scholarly journals Protein Kinase B Gene Homologue pkbR1 Performs One of Its Roles at First Finger Stage of Dictyostelium

2011 ◽  
Vol 10 (4) ◽  
pp. 512-520 ◽  
Author(s):  
Hiroshi Ochiai ◽  
Kosuke Takeda ◽  
Masashi Fukuzawa ◽  
Atsushi Kato ◽  
Shigeharu Takiya ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Stalk Cell ◽  
Null Mutant ◽  
Wild Type ◽  
Cell Region ◽  
Content Type ◽  
Mutant Forms ◽  
Second Wave ◽  
Gene Homologue

ABSTRACT Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB , which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene ( pkaC ) only in the prestalk region of the pkbR1 null mutant.

scholarly journals Cross Talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A Pathways in Cryptococcus neoformans

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Maureen J. Donlin ◽  
Rajendra Upadhya ◽  
Kimberly J. Gerik ◽  
Woei Lam ◽  
Laura G. VanArendonk ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Cryptococcus Neoformans ◽  
Signaling Pathway ◽  
Cell Wall Integrity ◽  
Wild Type ◽  
Content Type ◽  
Kinase A

ABSTRACTCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely,PKC1,BCK1,MKK2, andMPK1results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions ofBCK1,MKK2, andMPK1compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.IMPORTANCECryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

scholarly journals Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

2015 ◽  
Vol 26 (6) ◽  
pp. 1084-1097 ◽  
Author(s):  
Wei-Chi Lin ◽  
Liang-Chen Wang ◽  
Te-Ling Pang ◽  
Mei-Yu Chen
Keyword(s):  
Cell Migration ◽  
Dictyostelium Discoideum ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Protein Domain ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Protein G ◽  
Wild Type ◽  
Actin Binding Protein

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG− cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG− cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG− cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.

scholarly journals MoTea4-Mediated Polarized Growth Is Essential for Proper Asexual Development and Pathogenesis in Magnaporthe oryzae

2010 ◽  
Vol 9 (7) ◽  
pp. 1029-1038 ◽  
Author(s):  
Rajesh N. Patkar ◽  
Angayarkanni Suresh ◽  
Naweed I. Naqvi
Keyword(s):  
Actin Cytoskeleton ◽  
Magnaporthe Oryzae ◽  
Fluorescent Protein ◽  
Function Analysis ◽  
Coiled Coil ◽  
Polarized Growth ◽  
Wild Type ◽  
Cortical Actin ◽  
Content Type ◽  
Aerial Hyphae

ABSTRACT Polarized growth is essential for cellular development and function and requires coordinated organization of the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly and bipolar growth in fission yeast and directional mycelial growth in Aspergillus. Here, we characterize Tea4 in the rice blast fungus Magnaporthe oryzae (MoTea4). Similar to its orthologs, MoTea4-green fluorescent protein (MoTea4-GFP) showed punctate distribution confined to growth zones, particularly in the mycelial tips, aerial hyphae, conidiophores, conidia, and infection structures (appressoria) in Magnaporthe. MoTea4 was dispensable for vegetative growth in Magnaporthe. However, loss of MoTea4 led to a zigzag morphology in the aerial hyphae and a huge reduction in conidiation. The majority of the tea4Δ conidia were two celled, as opposed to the tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coil domains of MoTea4 are necessary for proper conidiation in Magnaporthe. The tea4Δ conidia failed to produce proper appressoria and consequently failed to infect the host plants. The tea4Δ conidia and germ tubes showed disorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to the wild-type conidia, the tea4Δ conidia showed aberrant germination, poor cytoplasmic streaming, and persistent accumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment of germinating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorial development in Magnaporthe. We show that MoTea4 plays an important role in organizing the F-actin cytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and pathogenic development in Magnaporthe.

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