scholarly journals HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease

Science ◽  
2020 ◽  
Vol 369 (6500) ◽  
pp. 202-207 ◽  
Author(s):  
Sarah A. Cook ◽  
William A. Comrie ◽  
M. Cecilia Poli ◽  
Morgan Similuk ◽  
Andrew J. Oler ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Synapse Formation ◽  
Immune Cell ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Akt Phosphorylation ◽  
Actin Networks ◽  
Evolutionarily Conserved ◽  
Regulatory Complex ◽  
Immune Cell Migration

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)–dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

scholarly journals Genetic immunodeficiency and autoimmune disease reveal distinct roles of Hem1 in the WAVE2 and mTORC2 complexes

2019 ◽  
Author(s):  
William A. Comrie ◽  
M. Cecilia Poli ◽  
Sarah A. Cook ◽  
Morgan Similuk ◽  
Andrew J. Oler ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Synapse Formation ◽  
Immune Cell ◽  
Loss Of Function ◽  
Cortical Actin ◽  
Akt Phosphorylation ◽  
Regulatory Complex

AbstractImmunodeficiency often coincides with immune hyperresponsiveness such as autoimmunity, lymphoproliferation, or atopy, but the molecular basis of this paradox is typically unknown. We describe four families with immunodeficiency coupled with atopy, lymphoproliferation, cytokine overproduction, hemophagocytic lymphohistocytosis, and autoimmunity. We discovered loss-of-function variants in the gene NCKAP1L, encoding the hematopoietic-specific Hem1 protein. Three mutations cause Hem1 protein and WAVE regulatory complex (WRC) loss, thereby disrupting actin polymerization, synapse formation, and immune cell migration. Another mutant, M371V encodes a stable Hem1 protein but abrogates binding of the Arf1 GTPase and identifies Arf1 as a critical Hem1 regulator. All mutations reduce the cortical actin barrier to cytokine release explaining immune hyperresponsiveness. Finally, Hem1 loss blocked mTORC2-dependent AKT phosphorylation, T cell proliferation, and effector cytokine production during T cell activation. Thus, our data show that Hem1 independently governs two key regulatory complexes, the WRC and mTORC2, and how Hem1 loss causes a combined immunodeficiency and immune hyperresponsiveness disease.One sentence summaryHem1 loss of function mutations cause a congenital immunodysregulatory disease and reveal its role regulating WAVE2 and mTORC2 signaling.

scholarly journals Small GTP-binding Protein TC10 Differentially Regulates Two Distinct Populations of Filamentous Actin in 3T3L1 Adipocytes

2002 ◽  
Vol 13 (7) ◽  
pp. 2334-2346 ◽  
Author(s):  
Makoto Kanzaki ◽  
Robert T. Watson ◽  
June Chunqiu Hou ◽  
Mark Stamnes ◽  
Alan R. Saltiel ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Actin Polymerization ◽  
Coat Proteins ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Amino Terminal ◽  
Gtp Binding ◽  
Subcellular Compartmentalization ◽  
Constitutively Active

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (ΔN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/ΔVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

scholarly journals Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

2010 ◽  
Vol 21 (20) ◽  
pp. 3529-3539 ◽  
Author(s):  
Tim Ting Chiu ◽  
Nish Patel ◽  
Alisa E. Shaw ◽  
James R. Bamburg ◽  
Amira Klip
Keyword(s):  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Muscle Cells ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Glut4 Translocation ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Rac Gtpase ◽  
Skeletal Myoblasts

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

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 Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno

2020 ◽  
Vol 21 (7) ◽  
pp. 2457 ◽  
Author(s):  
Vikash Singh ◽  
Anthony C. Davidson ◽  
Peter J. Hume ◽  
Vassilis Koronakis
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Nucleotide Exchange Factor ◽  
Regulatory Complex

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

Mechanisms Underlying Oxytocin-Induced Excitation of Supraoptic Neurons: Prostaglandin Mediation of Actin Polymerization

2006 ◽  
Vol 95 (6) ◽  
pp. 3933-3947 ◽  
Author(s):  
Yu-Feng Wang ◽  
Glenn I. Hatton
Keyword(s):  
Actin Polymerization ◽  
Cyclooxygenase 2 ◽  
Filamentous Actin ◽  
Excitatory Effect ◽  
Actin Networks ◽  
Oxytocin Receptors ◽  
Activating Agent ◽  
Supraoptic Neurons ◽  
Pm 10 ◽  
Actin Expression

In nonneuronal tissues, activation of oxytocin receptors (OTRs), like other Gαq/11 type G-protein-coupled receptors (Gαq/11/GPCRs), increase prostaglandin (PG) expression. This is not known for the OTRs expressed by central OT neurons. We examined mechanisms underlying OT's effects on supraoptic nucleus (SON) OT and vasopressin (VP) neurons in hypothalamic slices from lactating rats. OT application (10 pM, 10 min) significantly increased firing rates of OT and VP neurons, both of which expressed OTRs. Indomethacin, an inhibitor of PG synthetases, blocked these increases. OTR (but not a V1 receptor) antagonist blocked OT effects without blocking the excitatory effect of PGE2. Tetanus toxin blocked OT effects on fast synaptic inputs and firing activity of SON neurons but not OT-evoked depolarization, suggesting involvement of both pre- and postsynaptic neurons. Indomethacin also blocked the excitatory effects of phenylephrine, another Gαq/11/GPCR activating agent but not those of PGE2, a non-Gαq/11/GPCR activating agent in the SON. OT or phenylephrine, but not glutamate or KCl, enhanced cyclooxygenase 2 expression at cytosolic loci in SON neurons and nearby astrocytes, as revealed by immunocytochemistry. This OT effect was not blocked by TTX. Western blot analyses showed that OT significantly increased cyclooxygenase 2 but not actin expression. OT promoted the formation of filamentous actin (F-actin) networks at membrane subcortical areas of both OT and VP neurons. Indomethacin blocked enhancement of F-actin networks by OT but not by PGE2. These results indicate that PGs serve as a common mediator of Gαq/11/GPCR-activating agents in neuronal function.

scholarly journals Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization

2003 ◽  
Vol 371 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Takehito URUNO ◽  
Peijun ZHANG ◽  
Jiali LIU ◽  
Jian-Jiang HAO ◽  
Xi ZHAN
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Primary Component ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Specific Gene ◽  
Actin Assembly ◽  
Related Protein ◽  
Filamentous Actin ◽  
Cortical Actin

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (Kd) of 880nM. Although HS1 is a modest F-actin-binding protein with a Kd of 400nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott–Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

scholarly journals Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

2006 ◽  
Vol 175 (6) ◽  
pp. 957-969 ◽  
Author(s):  
Thomas M. Huckaba ◽  
Thomas Lipkin ◽  
Liza A. Pon
Keyword(s):  
Actin Polymerization ◽  
Budding Yeast ◽  
Type Ii ◽  
Retrograde Flow ◽  
Cortical Actin ◽  
Actin Networks ◽  
Actin Cable ◽  
Intracellular Movement ◽  
Actin Cables ◽  
Type Ii Myosin

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.

scholarly journals Chlamydomonas reinhardtii formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly

2016 ◽  
Author(s):  
Jenna R. Christensen ◽  
Evan W. Craig ◽  
Michael J. Glista ◽  
David M. Mueller ◽  
Yujie Li ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Apical Cell ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Tubule Formation ◽  
Actin Networks ◽  
Cellular Processes ◽  
The Right

ABSTRACTThe regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. PRF1 further favors FOR1-mediated actin assembly by potently inhibiting Arp2/3 complex-mediated actin assembly. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.SUMMARY STATEMENTThe Chlamydomonas reinhardtii formin FOR1 initiates rapid assembly of fertilization tubule actin filaments from monomers associated with the actin-assembly inhibitor profilin PRF1.

scholarly journals First person – Nathan Roy

2020 ◽  
Vol 133 (17) ◽  
pp. jcs253047
Keyword(s):  
Actin Polymerization ◽  
Immune Cell ◽  
Early Career ◽  
First Person ◽  
Upstream Migration ◽  
Postdoctoral Fellow ◽  
Early Career Researchers ◽  
Pathological Conditions ◽  
Immune Cell Migration ◽  
Selection Of

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Nathan Roy is first author on ‘LFA-1 signals to promote actin polymerization and upstream migration in T cells’, published in JCS. Nathan is a postdoctoral fellow in the lab of Janis Burkhardt at the Children's Hospital of Philadelphia, Philadelphia, PA, investigating signaling events that drive immune cell migration in both normal and pathological conditions.

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