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Journal of Cell Science Aug 2022Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin...
Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP.
Topics: Actin Cytoskeleton; Actin-Related Protein 2-3 Complex; Actins; Cell Movement; Pseudopodia; Wiskott-Aldrich Syndrome Protein Family
PubMed: 35971979
DOI: 10.1242/jcs.260364 -
European Journal of Cell Biology Jun 2023The actin cytoskeleton impacts practically every function of a eukaryotic cell. Historically, the best-characterized cytoskeletal activities are in cell morphogenesis,...
The actin cytoskeleton impacts practically every function of a eukaryotic cell. Historically, the best-characterized cytoskeletal activities are in cell morphogenesis, motility, and division. The structural and dynamic properties of the actin cytoskeleton are also crucial for establishing, maintaining, and changing the organization of membrane-bound organelles and other intracellular structures. Such activities are important in nearly all animal cells and tissues, although distinct anatomical regions and physiological systems rely on different regulatory factors. Recent work indicates that the Arp2/3 complex, a broadly expressed actin nucleator, drives actin assembly during several intracellular stress response pathways. These newly described Arp2/3-mediated cytoskeletal rearrangements are coordinated by members of the Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation-promoting factors. Thus, the Arp2/3 complex and WASP-family proteins are emerging as crucial players in cytoplasmic and nuclear activities including autophagy, apoptosis, chromatin dynamics, and DNA repair. Characterizations of the functions of the actin assembly machinery in such stress response mechanisms are advancing our understanding of both normal and pathogenic processes, and hold great promise for providing insights into organismal development and interventions for disease.
Topics: Animals; Wiskott-Aldrich Syndrome Protein Family; Actins; Actin-Related Protein 2-3 Complex; Actin Cytoskeleton; Cytoskeleton; Wiskott-Aldrich Syndrome Protein; Actin-Related Protein 3
PubMed: 36907023
DOI: 10.1016/j.ejcb.2023.151301 -
Frontiers in Cellular and Infection... 2016The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. The disease is characterized by bacterial invasion of intestinal... (Review)
Review
The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. The disease is characterized by bacterial invasion of intestinal cells, dissemination within the colonic epithelium through direct spread from cell to cell, and massive inflammation of the intestinal mucosa. Here, we review the mechanisms supporting S. flexneri dissemination. The dissemination process primarily relies on actin assembly at the bacterial pole, which propels the pathogen throughout the cytosol of primary infected cells. Polar actin assembly is supported by polar expression of the bacterial autotransporter family member IcsA, which recruits the N-WASP/ARP2/3 actin assembly machinery. As motile bacteria encounter cell-cell contacts, they form plasma membrane protrusions that project into adjacent cells. In addition to the ARP2/3-dependent actin assembly machinery, protrusion formation relies on formins and myosins. The resolution of protrusions into vacuoles occurs through the collapse of the protrusion neck, leading to the formation of an intermediate membrane-bound compartment termed vacuole-like protrusions (VLPs). VLP formation requires tyrosine kinase and phosphoinositide signaling in protrusions, which relies on the integrity of the bacterial type 3 secretion system (T3SS). The T3SS is also required for escaping double membrane vacuoles through the activity of the T3SS translocases IpaB and IpaC, and the effector proteins VirA and IcsB. Numerous factors supporting envelope biogenesis contribute to IcsA exposure and maintenance at the bacterial pole, including LPS synthesis, membrane proteases, and periplasmic chaperones. Although less characterized, the assembly and function of the T3SS in the context of bacterial dissemination also relies on factors supporting envelope biogenesis. Finally, the dissemination process requires the adaptation of the pathogen to various cellular compartments through transcriptional and post-transcriptional mechanisms.
Topics: Actin-Related Protein 2-3 Complex; Actins; Antigens, Bacterial; Bacterial Proteins; Biological Transport; Cell Surface Extensions; DNA-Binding Proteins; Dysentery, Bacillary; Epithelial Cells; Humans; Intestinal Mucosa; Shigella flexneri; Transcription Factors; Type III Secretion Systems; Vacuoles; Virulence Factors; Wiskott-Aldrich Syndrome Protein, Neuronal
PubMed: 27014639
DOI: 10.3389/fcimb.2016.00029 -
Nature Medicine Jan 2019In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in...
In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-β. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.
Topics: Anaplastic Lymphoma Kinase; Animals; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytoskeletal Proteins; Down-Regulation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Guanosine Triphosphate; Humans; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Lymphoma, T-Cell; MAP Kinase Signaling System; Mice; Protein Binding; STAT3 Transcription Factor; T-Lymphocytes; Tumor Suppressor Proteins; Wiskott-Aldrich Syndrome Protein; cdc42 GTP-Binding Protein
PubMed: 30510251
DOI: 10.1038/s41591-018-0262-9 -
Therapeutic Advances in Rare Disease 2021Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency X-linked genetic disorder. It is often featured with a clinical triad of thrombocytopenia with low mean...
UNLABELLED
Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency X-linked genetic disorder. It is often featured with a clinical triad of thrombocytopenia with low mean platelet volume, eczematoid dermatitis and recurrent infections. The clinical manifestation of WAS, depending on the underlying variant, shows wide heterogeneity. We present a case of a 10-month-old boy who came in with a history of recurrent fever, skin lesions since birth and episodes of bloody diarrhoea. He had severe anaemia and thrombocytopenia (with normal mean platelet volume). Genetic analysis revealed the patient to be hemizygous for a pathogenic WAS gene splice variant (NM_000377.2:c.360+1G>A). He was managed with supportive treatment and regular follow up, but died 4 months later. As it is a rare genetic disease, the diagnosis of WAS can easily be missed, especially in settings with scarce healthcare resources that do not have easy access to genetic testing. Thus, a high index of suspicion is needed when a male child presents with recurrent infections and bleeding tendencies.
PLAIN LANGUAGE SUMMARY
Management challenges of a rare genetic disorder in a resource-limited country: a case report of Wiskott-Aldrich syndrome in TanzaniaWiskott-Aldrich syndrome (WAS) is a rare inherited disease that mainly affects boys. Patients will typically present with low levels of a single line of little particles of cells that clot the blood called platelets, whole-body skin rashes and recurrent infections. Nevertheless, the clinical presentation can vary between individuals. We present a case of a 10-month-old boy who came in with a history of recurrent fever, skin rash since birth and episodes of bloody diarrhoea. He had very low levels of red blood cells and platelets. Genetic analysis confirmed the patient to have WAS. He was managed with supportive treatment, followed up on a regular clinic but unfortunately died 4 months later. Being a rare genetic disease, the diagnosis of WAS can easily be missed, especially in regions with scarce healthcare resources that do not have easy access to genetic testing. Thus, doctors should suspect WAS in boys presenting with recurrent infections and bleeding problems.
PubMed: 37181115
DOI: 10.1177/26330040211009905 -
Cell Reports Apr 2024Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic...
Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that β1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of β1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase β1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.
Topics: Humans; Integrin beta1; ras GTPase-Activating Proteins; Neoplasm Metastasis; Cell Line, Tumor; Serum Response Factor; Male; Female; Prostatic Neoplasms; Animals; Trans-Activators; Cell Adhesion; Wiskott-Aldrich Syndrome Protein, Neuronal; Breast Neoplasms; Mice; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; cdc42 GTP-Binding Protein
PubMed: 38536816
DOI: 10.1016/j.celrep.2024.113989 -
Orphanet Journal of Rare Diseases Dec 2022Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency disorder caused by abnormal expression of the WAS protein (WASp) due to mutations in the WAS gene, and...
BACKGROUND
Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency disorder caused by abnormal expression of the WAS protein (WASp) due to mutations in the WAS gene, and is generally characterized by microthrombocytopenia, eczema, recurrent infections, and high susceptibility to autoimmune complications and hematological malignancies.
RESULTS
Herein, we identified a novel WAS mutation (c.158 T > C) using next-generation sequencing in a Chinese pedigree with WAS. The expression of WASp in the patients and their families was detected by flow cytometry and western blot analysis. To explore the exon-splicing effect of intron mutations and the correlation between the genotype and clinical phenotype, four groups of wild-type (WT), exon mutant, intron mutant, and combined mutant recombinant plasmids were transfected into COS-7 cells in vitro. The proband showed dramatically decreased WASp expression, while the female carriers showed a slightly lower level of WASp. The expression of products in the mutant and WT recombinant plasmids was detected by real-time fluorescence quantitative polymerase chain reaction (PCR), which showed a significant reduction in the combined mutant group than in the WT, exon mutant, and intron mutant groups. The length of the expression products in the four groups showed no differences, each containing 360 base pairs. Sequence analysis confirmed that the c.158 T > C mutation appeared in the exon mutant and combined mutant groups, whereas the intron variant c.273 + 14C > T caused no other sequence changes.
CONCLUSION
This study confirmed that the intron mutation did not affect the splicing of exons and excluded the influence of the double mutations at the transcription level on the severe clinical manifestations in the cousin. This in vitro study provided new insights into the pathogenesis of intronic mutations in WAS.
Topics: Humans; Female; Wiskott-Aldrich Syndrome; Pedigree; East Asian People; Mutation; RNA Splicing
PubMed: 36550574
DOI: 10.1186/s13023-022-02589-y -
The Journal of Allergy and Clinical... Sep 2019Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and...
BACKGROUND
Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients.
OBJECTIVE
We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction.
METHODS
We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis.
RESULTS
We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up.
CONCLUSIONS
Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.
Topics: Adolescent; Adult; Blood Platelets; Child; Child, Preschool; Female; Genetic Therapy; Hematopoietic Stem Cell Transplantation; Humans; Infant; Lentivirus; Male; Microscopy, Electron, Transmission; Phenotype; Platelet Activation; Platelet Count; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein
PubMed: 30926529
DOI: 10.1016/j.jaci.2019.03.012 -
Biology of Blood and Marrow... Mar 2018Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by a triad of immunodeficiency, eczema, and thrombocytopenia. Currently, hematopoietic stem cell... (Clinical Trial)
Clinical Trial
Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by a triad of immunodeficiency, eczema, and thrombocytopenia. Currently, hematopoietic stem cell transplantation (HSCT) is the most reliable curative treatment with excellent results for patients with HLA-matched family or unrelated donors. However, even after fully myeloablative preparative regimens, mixed donor chimerism is a potential concern. We performed a retrospective chart review of 12 children who underwent allogeneic HSCT for WAS to report our experience. The median age at transplant was 10.5 months (range, 3 to 39). The median nucleated cell dose from the marrow was 4.55 × 10/kg (range, .3 to 7.9). The median times to neutrophil and platelet engraftment were 19 days (range, 13 to 27) and 18.5 days (range, 12 to 31), respectively. The rate of overall survival was 92% with median follow-up of 67 months (range, 3 to 146). Two patients developed grade IV acute graft-versus-host disease, and 1 died on day +99. Five of 12 patient's (42%) had mixed donor chimerism (range, 12% to 85%) at day +180. None of the pretransplant patient parameters was predictive of mixed chimerism. Nonetheless, of these 5 patients, 2 had normalization of the platelet count despite the mixed chimerism, 2 had full donor chimerism after receiving a second transplant with the same donor, and 1 remains transfusion dependent awaiting a second transplant. Hence, even with a significant rate of mixed chimerism, HSCT provides substantial benefit to WAS patients, with excellent overall survival.
Topics: Adolescent; Adult; Allografts; Child; Child, Preschool; Disease-Free Survival; Female; Follow-Up Studies; Hematopoietic Stem Cell Transplantation; Humans; Male; Retrospective Studies; Risk Factors; Survival Rate; Transplantation Conditioning; Wiskott-Aldrich Syndrome
PubMed: 29196075
DOI: 10.1016/j.bbmt.2017.11.019 -
Small GTPases Nov 2019The Arf and Rho subfamilies of small GTPases are nucleotide-dependent molecular switches that act as master regulators of vesicular trafficking and the actin... (Review)
Review
The Arf and Rho subfamilies of small GTPases are nucleotide-dependent molecular switches that act as master regulators of vesicular trafficking and the actin cytoskeleton organization. Small GTPases control cell processes with high fidelity by acting through distinct repertoires of binding partners called effectors. While we understand a great deal about how these GTPases act individually, relatively little is known about how they cooperate, especially in the control of effectors. This review highlights how Arf GTPases collaborate with Rac1 to regulate actin cytoskeleton dynamics at the membrane via recruiting and activating the Wave Regulatory Complex (WRC), a Rho effector that underpins lamellipodia formation and macropinocytosis. This provides insight into Arf regulation of the actin cytoskeleton, while putting the spotlight on small GTPase cooperation with emerging evidence of its importance in fundamental cell biology and interactions with pathogenic bacteria.
Topics: ADP-Ribosylation Factors; Actin Cytoskeleton; Animals; Escherichia coli; Humans; Salmonella; Wiskott-Aldrich Syndrome Protein Family; rho GTP-Binding Proteins
PubMed: 28524754
DOI: 10.1080/21541248.2017.1329691