scholarly journals WASP integrates substrate topology and cell polarity to guide neutrophil migration

2021 ◽  
Vol 221 (2) ◽  
Author(s):  
Rachel M. Brunetti ◽  
Gabriele Kockelkoren ◽  
Preethi Raghavan ◽  
George R.R. Bell ◽  
Derek Britain ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Neutrophil Migration ◽  
Geometric Features ◽  
Actin Assembly ◽  
Membrane Deformation ◽  
Induced Membrane ◽  
Superresolution Imaging ◽  
Directed Migration ◽  
Membrane Deformations

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP’s front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.

scholarly journals WASP integrates substrate topology and cell polarity to guide neutrophil migration

2021 ◽  
Author(s):  
Rachel M Brunetti ◽  
Gabriele Kockelkoren ◽  
Preethi Raghavan ◽  
George R. R. Bell ◽  
Derek Britain ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Super Resolution ◽  
Leading Edge ◽  
Live Cells ◽  
Immune Disorder ◽  
Induced Membrane ◽  
Mediated Contact ◽  
Membrane Deformations

To control their shape and movement, cells leverage nucleation promoting factors (NPFs) to regulate when and where they polymerize actin. Here we investigate the role of the immune-specific NPF WASP during neutrophil migration. Endogenously-tagged WASP localizes to substrate-induced plasma membrane deformations. Super-resolution imaging of live cells reveals that WASP preferentially enriches to the necks of these substrate-induced membrane invaginations, a distribution that could support substrate pinching. Unlike other curvature-sensitive proteins, WASP only enriches to membrane deformations at the cell front, where it controls Arp2/3 complex recruitment and actin polymerization. Despite relatively normal migration on flat substrates, WASP depletion causes defects in topology sensing and directed migration on textured substrates. WASP therefore both responds to and reinforces cell polarity during migration. Surprisingly, front-biased WASP puncta continue to form in the absence of Cdc42. We propose that WASP integrates substrate topology with cell polarity for 3D guidance by selectively polymerizing actin around substrate-induced membrane deformations at the leading edge. A misregulation of WASP-mediated contact guidance could provide insight into the immune disorder Wiskott-Aldrich syndrome.

The RHO GTPASE CDC42 Regulator CDC42GAP Plays a Critical Role in Neutrophil Migration Via Podosome-Like Formation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 651-651 ◽  
Author(s):  
Marie-Dominique Filippi ◽  
Haiming Xu ◽  
Jason Towe ◽  
Chad E. Harris ◽  
Kathleen Szczur ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Rho Gtpase ◽  
Critical Role ◽  
Neutrophil Migration ◽  
Cell Movement ◽  
Directed Migration ◽  
Mutant Cells

Abstract Neutrophils (PMN) are a critical cell in inflammatory processes. In response to environmental stimuli, they activate various signal transduction pathways allowing them to move rapidly to a site of microbial invasion and to perform phagocytosis, cytokine and oxygen substrate release. Rho GTPase proteins, Rac1, Rac2, CDC42 and Rho, are central regulators of cell movement via actin rearrangement. We have demonstrated the specific role of Rac1 and Rac2 in PMN functions (Gu and Filippi et al, Science 2003; Filippi et al. Nat Immuol., 2004) which raises the question of the specificity of the other Rho GTPases. CDC42 primarily regulates the formation of filopodia. CDC42 controls cell polarity and migration in hematopoietic cell lines. Most of previous studies have utilized dominant active or negative mutants which lack specificity and cannot be easily used to define in vivo cell biology. Here, we used mice genetically deficient in the CDC42 negative regulator CDC42 GTPase Activating Protein (GAP) to study the role of CDC42 in neutrophil functions in vitro and in vivo. Heterozygote (CDC42GAP+/−) or homozygote (CDC42GAP−/−) mutant mice displayed normal neutrophil differentiation in vitro and in vivo. PMN deficient in CDC42GAP displayed 2-fold increased in CDC42 activity. In vivo recruitment of PMN in peritoneal cavities after thioglycollate exposure was significantly impaired in CDC42GAP+/− mice compared with wild type (WT) mice (25.5±0.76 x 105 vs 35.7±0.38 x 105, p<0.05). Both CDC42GAP+/− and CDC42GAP−/− PMN demonstrated defective directed migration in vitro in response to fMLP in a Boyden chamber assay compared with WT (248±31 and 199±20 versus 314±29 migrated cells, p<0.05), suggesting that CDC42 plays a critical role in neutrophil migration in vitro and in vivo. To further understand the role of CDC42GAP in neutrophil migration, single-cell analysis by time-lapse videomicroscopy was performed. Surprisingly, CDC42GAP+/− PMN demonstrated higher migration velocity compared with WT cells in response to fMLP, but this increased speed was associated with an abnormal shape. Upon beta-2 integrin ligation, CDC42GAP+/− PMN demonstrated abnormal elongated trailing tail associated with increased tail filopodia. Importantly, the podosome-like structures identified by a vinculin ring surrounding F-actin at the ventral plasma membrane that are present in the leading edge of WT PMN was absent in the mutant cells. CDC42GAP−/− PMN demonstrated more dramatic F-actin impairment upon integrin ligation compared with CDC42GAP+/− and WT cells and remarkably showed complete loss of cell polarity, consistent with the known role of CDC42 in cell polarity. We hypothesize that the lack of podosome formation in mutant cells could account for the increased speed observed in CDC42GAP+/− cells and therefore result in ineffective directed migration in vivo. Altogether, this suggests that regulation of CDC42 activity plays a pivotal role in neutrophil migration likely via integrin-dependent podosome-like formation. This reinforces the importance of turnover of attachment structures during cell movement and suggests a new role for CDC42 in actin-based attachment structure in neutrophils.

Biophysical approaches to protein-induced membrane deformations in trafficking

2008 ◽  
Vol 20 (4) ◽  
pp. 476-482 ◽  
Author(s):  
Pierre Sens ◽  
Ludger Johannes ◽  
Patricia Bassereau
Keyword(s):  
Induced Membrane ◽  
Membrane Deformations

scholarly journals Functional Genomics of Adhesion, Invasion, and Mycelial Formation in Schizosaccharomyces pombe

2009 ◽  
Vol 8 (8) ◽  
pp. 1298-1306 ◽  
Author(s):  
James Dodgson ◽  
Hema Avula ◽  
Kwang-Lae Hoe ◽  
Dong-Uk Kim ◽  
Han-Oh Park ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Polarity ◽  
Nitrogen Limitation ◽  
Molecular Level ◽  
Actin Assembly ◽  
Rich Media ◽  
Second Mode ◽  
Dependent Cell ◽  
Deletion Library ◽  
Mycelial Development

ABSTRACT Investigation into the switch between single-celled and filamentous forms of fungi may provide insights into cell polarity, differentiation, and fungal pathogenicity. At the molecular level, much of this investigation has fallen on two closely related budding yeasts, Candida albicans and Saccharomyces cerevisiae. Recently, the much more distant fission yeast Schizosaccharomyces pombe was shown to form invasive filaments after nitrogen limitation (E. Amoah-Buahin, N. Bone, and J. Armstrong, Eukaryot. Cell 4:1287-1297, 2005) and this genetically tractable organism provides an alternative system for the study of dimorphic growth. Here we describe a second mode of mycelial formation of S. pombe, on rich media. Screening of an S. pombe haploid deletion library identified 12 genes required for mycelial development which encode potential transcription factors, orthologues of S. cerevisiae Sec14p and Tlg2p, and the formin For3, among others. These were further grouped into two phenotypic classes representing different stages of the process. We show that galactose-dependent cell adhesion and actin assembly are both required for mycelial formation and mutants lacking a range of genes controlling cell polarity all produce mycelia but with radically altered morphology.

scholarly journals A protein interaction map for cell polarity development

2001 ◽  
Vol 154 (3) ◽  
pp. 549-576 ◽  
Author(s):  
Becky L. Drees ◽  
Bryan Sundin ◽  
Elizabeth Brazeau ◽  
Juliane P. Caviston ◽  
Guang-Chao Chen ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Large Scale ◽  
Interaction Network ◽  
Open Reading Frames ◽  
Actin Assembly ◽  
Protein Protein Interactions ◽  
Microtubule Organization ◽  
Two Hybrid ◽  
Secretory Apparatus

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

scholarly journals Effects of BIG1 and KANK1 on cell polarity and directed migration during wound healing

2011 ◽  
Vol 25 (S1) ◽  
Author(s):  
CHUN‐CHUN LI ◽  
Jean‐Cheng Kuo ◽  
Ryoiti Kiyama ◽  
Joel Moss ◽  
Martha Vaughan
Keyword(s):  
Wound Healing ◽  
Cell Polarity ◽  
Directed Migration

Rho GTPase Cdc42 Maintains Neutrophil Polarity during Directed Migration by Regulating CD11b Integrin Signals.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 238-238
Author(s):  
Kathleen Szczur ◽  
Yi Zheng ◽  
Marie-Dominique Filippi
Keyword(s):  
Cell Polarity ◽  
Rho Gtpase ◽  
Critical Role ◽  
Leading Edge ◽  
Negative Regulator ◽  
Blocking Antibody ◽  
Directed Migration ◽  
Cellular Defense ◽  
Membrane Protrusions ◽  
Actin Protrusions

Abstract Neutrophil (PMN) migration to sites of infection is the first line of cellular defense. Among others, a key event of cell migration is the maintenance of a polarized morphology characterized by a single protrusive leading edge of F-actin and a contractile uropod devoid of F-actin protrusions. Using mice genetically deficient in the Cdc42 negative regulator Cdc42 GTPase Activating Protein, we previously demonstrated that Cdc42 activity suppresses membrane protrusions at the uropod of the cells to maintain stable polarity during directed migration (Szczur et al, Blood 2006). However, the underlying molecular mechanism of Cdc42-mediated neutrophil polarity remains to be understood. Here, using mice with a conditional Cdc42 (flox) allele, we showed by video microscopy that Cdc42−/− PMNs exhibited multiple membrane extensions in various directions and failed to maintain cell polarity and directionality towards formyl-methionyl-leucyl-phenylalanin (fMLP) gradient compared to wild type (WT) cells. Consistent with this observation, Cdc42−/− PMNs exhibited increased lamellipodia protrusions of F-actin all around the cells compared to WT PMNs, in response to fMLP stimulation and fibrinogen ligation, confirming that Cdc42 maintains stable polarity by preventing abnormal membrane protrusions outside the leading edge. To understand how Cdc42 orchestrates neutrophil polarity at a mechanistic level, we explored the possibility of a role for integrins in this process since Cdc42 appears to regulate neutrophil polarity in a manner, at least in part, dependent on integrin ligation (Szczur et al, Blood 2006). Expression of the neutrophil integrin, CD11b/CD18, on resting or fMLP-stimulated PMNs was similar between the genotypes. Stimulation of WT PMNs with fMLP and ligation to fibrinogen induced a polarized distribution of CD11b into clusters mostly concentrated at the uropod of the cells. Remarkably, the numbers of CD11b clusters of Cdc42−/− PMNs were significantly decreased compared to WT cells. Furthermore, inhibition of CD11b clustering in WT PMNs, using anti-CD11b blocking antibody, significantly increased membrane protrusions associated with loss of stable polarity during directed migration, similarly to Cdc42-deficiency. Enforcing CD11b clustering by CD11b cross-linking in Cdc42−/− PMNs partially rescued cell polarity and F-actin distribution concentrated only at the leading edge of the cells to WT levels. These results strongly suggest that CD11b clustering is regulated by Cdc42 activity and contributes to suppress F-actin protrusions at the uropod of neutrophils during migration. The uropod distribution of CD11b suggests that CD11b may recruit contractile proteins, such as the myosin regulator myosin light chain (MLC), to antagonize membrane protrusions. To test this hypothesis, we analyzed the distribution of phosphorylated MLC (p-MLC). Upon stimulation, p-MLC strongly translocated to the uropod of WT cells. In contrast, p-MLC remained diffuse and non polarized in the cytoplasm of Cdc42−/− cells. Blocking CD11b function in WT cells abrogated the polarized distribution of p-MLC mimicking Cdc42−/− PMNs. Enforcing CD11b clustering in Cdc42−/− PMNs rescued p-MLC signals concentrated at the uropod of the cells. Altogether, this study suggests that Cdc42 activity maintains neutrophil polarity during directed migration by regulating CD11b clustering/distribution and subsequent outside/in signals to suppress lateral membrane protrusions. This study uncovers a critical role for CD11b in maintaining neutrophil polarity during migration.

Metabolic Abnormalities in G6PC3 Deficient Human Neutrophils Result in Severe Functional Defects

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1024-1024
Author(s):  
Christopher M. McKinney ◽  
Michael A. Ellison ◽  
Natalie Briones ◽  
Angelina Baroffio ◽  
Julie A. Reisz ◽  
...  
Keyword(s):  
Mouse Models ◽  
Bactericidal Activity ◽  
Superoxide Anion ◽  
Tca Cycle ◽  
Surface Expression ◽  
Actin Assembly ◽  
Human Patient ◽  
Superoxide Anion Production ◽  
Directed Migration ◽  
Anion Production

Abstract Introduction Severe congenital neutropenia, type 4 results from a mutation in the G6PC3 gene. This gene encodes the enzyme glucose-6-phosphatase (G6Pase) which is ubiquitously expressed in endoplasmic reticulum and is responsible for the final step of gluconeogenesis and glycogenolysis. G6pc3-/- mouse models have demonstrated decreased neutrophil (PMN) function including impaired chemotaxis and superoxide anion production and increased apoptosis. Metabolically, g6pc3-/-mice have impaired intracellular glucose transport and reduced levels of ATP and lactic acid. We present the most comprehensive functional and metabolic analysis to date on a patient with a novel compound heterozygous mutation in the G6PC3 gene, c.325G>A and c.758G>A, validating mouse model findings in a human. Methods PMNs were isolated from heparinized whole blood by dextran sedimentation and hypotonic lysis of red cells. Chemotactic assays were performed under agarose techniques with zymosan activated serum (ZAS), fMLF, and buffer control. Expression of CD18 and F-actin assembly were analyzed after incubation with buffer, PMA, and fMLF using standard flow cytometric techniques. Bactericidal activity was measured using variable ratios of S Aureus:PMNs in the presence of 10% normal human serum. Superoxide anion production was measured with SOD-inhibitable chemiluminescence with Diogenes reagent. Chemiluminescence was employed to measure caspase expression. Metabolomics and tracing experiments were performed incubating cells with U-13C-glucose prior to UHPLC/MS analysis. Results Cell motility was significantly impaired in human G6Pase deficient (patient) PMNs. Non-directed migration was reduced by over 50% compared to controls. Directed migration of patient PMNs was only 35% and 23% of control when stimulated with ZAS and 0.1 µM fMLF respectively. Surface expression of CD18 in patient cells did not increase in response to either stimulus but more than doubled in normal PMNs. F-actin was present at significantly higher levels in unstimulated patient PMNs and did not increase in the presence of 200 ng/mL PMA or 1 µM fMLF. Bactericidal activity was normal at a bacteria:PMN ratio of 1:1 but abnormal at ratios > 5:1 with over a 20% increase in viable bacteria at 120 minutes for patient PMNs. Superoxide anion production was markedly impaired with over 70% reduction compared to control when stimulated with either 200 ng/mL PMA or 1 µM fMLF. Caspase expression was not significantly different between patient and control PMNs, but the patient was on filgrastim which has antiapoptotic effects. Tracing experiments with 13C6-glucose showed decreased flux through glycolysis (significant reductions in labeled isotopologues for G6P and lactate) and marked inhibition of the NADPH-generating pentose phosphate pathway (PPP) in patient PMNs stimulated with 1µM fMLF. Glutaminolysis appears to be increased with increased glutamine consumption in patient PMNs, decreased glucogenic amino acids, and increased TCA cycle intermediates. Discussion Metabolic reprogramming is a recently appreciated hallmark of immune cell activation. Decreases in G6P affect multiple metabolic pathways including glycolysis, PPP and TCA cycle. Deficiency of NADPH-dependent superoxide anion production likely results from impairment in the NADPH-generating PPP. Interestingly the bactericidal defect is not as severe as would be predicted given the degree of impairment in superoxide anion production. The most striking functional defect is with PMN motility. Defects in the glycolytic/PPP likely contribute to this phenotype. ATP-dependent actin assembly is critical for cell migration. Our data demonstrate that in human patient PMNs, nearly all actin is in the filamentous form and does not increase further in response to chemoattractants. This could result in impaired hydrolysis of the F-actin filament and reassembly at the leading edge. We also see reduction in surface expression of CD11b/CD18 which is vital for PMN adhesion and migration. In conclusion, metabolic defects resulting from G6PC3 deficiency contribute to global PMN dysfunction and absolute neutropenia. Patients may be at increased risk for infections in spite of correction of neutropenia with filgrastim. These novel findings in human patient cells elucidate further the pathogenesis of this disease which has largely been dependent on data from mouse models. Disclosures No relevant conflicts of interest to declare.

scholarly journals Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion.

1993 ◽  
Vol 120 (3) ◽  
pp. 785-798 ◽  
Author(s):  
S P Colgan ◽  
C A Parkos ◽  
C Delp ◽  
M A Arnaout ◽  
J L Madara
Keyword(s):  
Protein Synthesis ◽  
Epithelial Cells ◽  
Qualitative Difference ◽  
Neutrophil Migration ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Ifn Gamma ◽  
Directed Migration ◽  
Disease States ◽  
Epithelial Barriers

Neutrophil, or polymorphonuclear leukocyte (PMN), migration across intestinal epithelial barriers, such as occurs in many disease states, appears to result in modifications of epithelial barrier and ion transport functions (Nash, S., J. Stafford, and J. L. Madara. 1987. J. Clin. Invest. 80:1104-1113; Madara, J. L., C. A. Parkos, S. P. Colgan, R. J. MacLeod, S. Nash, J. B. Matthews, C. Delp, and W. I. Lencer. 1992. J. Clin. Invest. 89:1938-1944). Here we investigate the effects of epithelial exposure to IFN-gamma on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was initially assessed in the apical-to-basolateral direction, since previous studies indicate general qualitative similarities between PMN migration in the apical-to-basolateral and in the basolateral-to-apical directions. In the apical-to-basolateral direction, epithelial exposure to IFN-gamma markedly upregulated transepithelial migration of PMN in a dose- and time-dependent fashion as measured by both electrical and myeloperoxidase assays. This IFN-gamma-elicited effect on transmigration was specifically due to a IFN-gamma effect on epithelial cells and was not secondary to IFN-gamma effects on epithelial tight junction permeability. Moreover, this IFN-gamma effect was dependent on epithelial protein synthesis, and involved a pathway in which CD11b/18, but not ICAM-1 or CD11a/18, appeared to play a crucial role in PMN-epithelial adhesion. IFN-gamma also substantially modified PMN transepithelial migration in the natural, basolateral-to-apical direction. The IFN-gamma effect on naturally directed transmigration was also specifically due to an IFN-gamma effect on epithelial cells, showed comparable time and dose dependency to that of oppositely directed migration, was CD11b/18 dependent, and required epithelial protein synthesis. Additionally, however, important qualitative differences existed in how IFN-gamma affected transmigration in the two directions. In contrast to apical-to-basolateral directed migration, IFN-gamma markedly downregulated transepithelial migration of PMN in the natural direction. This downregulation of PMN migration in the natural direction, however, was not due to failure of PMN to move across filters and into monolayers. Indeed, IFN-gamma exposure to epithelia increased the number of PMN which had moved into the basolateral space of the epithelium in naturally directed transmigration. These results represent the first detailed report of influences on PMN transepithelial migration by a cytokine, define conditions under which a qualitative difference in PMN transepithelial migration exists, and suggest that migration of PMN across epithelia in the natural direction may involve multiple steps which can be differentially regulated by cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Sign in / Sign up

Export Citation Format

Share Document