© 2021. Published by The Company of Biologists Ltd | Journal of Cell Science (2021) 134, jcs253534. doi:10.1242/jcs.253534

REVIEW SUBJECT COLLECTION: CELL BIOLOGY AND DISEASE Emerging functions of cytoskeletal in immune diseases Sara Tur-Gracia1 and Narcisa Martinez-Quiles1,2,*

ABSTRACT from the formin family (Courtemanche, 2018) and the Arp2/3 Immune cells are especially dependent on the proper functioning of the complex in particular (Fig. 1) are responsible for overcoming this , and both innate and adaptive responses rely on it. barrier (Plastino and Blanchoin, 2018). Leukocytes need to adhere not only to substrates but also to cells in There is also a third nucleation mechanism that appear to act order to form synapses that pass on instructions or kill infected cells. synergistically with the nucleation factors mentioned above Neutrophils literally squeeze their cell body during blood extravasation (Dominguez, 2016) and that brings together actin monomers by the and efficiently migrate to the inflammatory focus. Moreover, the action of proteins containing several repeats, often located in tandem, – development of immune cells requires the remodeling of their such as the Wiskott Aldrich syndrome (WASP) homology 2 cytoskeleton as it depends on, among other processes, adhesive (WH2) domain (Carlier et al., 2013). contacts and migration. In recent years, the number of reports Apart from WH2 domain-containing proteins, other actin-binding describing cytoskeletal defects that compromise the immune system proteins (ABPs) supply the vast majority of G-actin monomers has increased immensely. Furthermore, a new emerging paradigm during filament elongation. These ABPs include family points toward a role for the cellular actin content as an essential proteins and proteins with a 43-residue repeat sequence known as the β β component of the so-called homeostasis-altering molecular processes -thymosin domain (Renault, 2016). Thymosin 4sequestersactin ‘ ’ that induce the activation of innate immune signaling pathways. Here, monomers and maintains them in a non-polymerizable pool we review the role of critical actin-cytoskeleton-remodeling proteins, (Renault, 2016). An additional level of control for actin including the Arp2/3 complex, cofilin, coronin and WD40-repeat polymerization is exerted by capping proteins that preferentially containing protein 1 (WDR1), in immune pathophysiology, with a bind monomeric actin at the barbed end, preventing elongation special focus on autoimmune and autoinflammatory traits. (Shekhar et al., 2016). Finally, there is another important category of proteins controlling actin dynamics, the actin-severing proteins, KEY WORDS: Arp2/3 complex Cell Biology and Disease Cofilin, which includes cofilins, coronins and WD40-repeat containing Coronin, WD40-repeat containing protein 1, WDR1, Actin-interacting protein 1 (WDR1) (Buracco et al., 2019). protein 1, AIP1 Initial observations of immunopathologies mediated by defective ABPs included the description of immunodeficient patients that Introduction display an ineffective response of the immune system to pathogens The eukaryotic cytoskeleton is composed of actin and and consequently suffered severe life-threatening infections. One of networks, as well as intermediate filaments that contribute to the the first such studies was the characterization of the WAS in the late complexity and flexibility of the system. The actin cytoskeleton 1990s (Derry et al., 1994). However, in many cases, undergoes constant remodeling in order to accommodate the immunodeficiency diseases present concomitant autoimmune and motility that governs physiological functions. inflammatory traits, whose cause remain unclear still many years later. Cycles of actin polymerization and depolymerization contribute Thus, WAS (the encoding WASP) mutations exemplify that a to what is termed actin turnover. Actin is a globular monomer called dysfunctional actin cytoskeleton can not only result in a lack of the G-actin (42 kDa) that possesses three different isoforms, α, β and γ, proper response against pathogens, but also induce an exacerbated according to its electrophoretic mobility (Garrels and Gibson, immune response, causing excessive inflammation partially mediated 1976). These monomers polymerize into either branched or by activation of the NLRP3 inflammasome (Lee et al., 2017), which unbranched filamentous actin (F-actin). The actin filament is a points to a deregulation of the innate immune system (Manthiram polar double-stranded helix, in which the fast-growing (barbed) plus et al., 2017). Consequently, a recent concept is emerging proposing end preferentially incorporates new monomers (ATP-actin), while that actin homeostasis is an essential part of the so-called homeostasis- depolymerization predominates (ADP-actin) at the (pointed) minus altering molecular processes (HAMPs) that activate the innate end (Plastino and Blanchoin, 2018). When actin is assembled, ATP immune response (Liston and Masters, 2017). is rapidly hydrolyzed, generating ADP-inorganic phosphate (Pi)- Soon after, other WASP homologous proteins were described, actin, which slowly releases Pi to generate ADP-actin subunits. The such as neural WASP (N-WASP; also known as WASL) (Miki et al., two ends of F-actin differ in their monomer concentration 1996) and SCAR (suppressor of cAR; Bear et al., 1998), which are requirements, called critical concentration (Hill, 1981). However, currently grouped together as WASP family of proteins (WASPs). the formation of an initial dimer or trimer of actin through Importantly SCAR and N-WASP were established as Arp2/3 nucleation presents a thermodynamic barrier, and nucleation factors complex activators (Machesky and Insall, 1998; Rohatgi et al., 1999), prompting investigations into the possible role of Arp2/3 in immunodeficiencies. 1 Department of Immunology, Ophthalmology and ENT, Complutense University However, although the field has made good progress in School of Medicine, 28040 Madrid, Spain. 2Gregorio Marañón Health Research Institute, 28007 Madrid, Spain. deciphering the role of cytoskeletal proteins in immunodeficiency diseases, the characterization of the molecular basis of autoimmune *Author for correspondence ([email protected]) and autoinflammatory diseases have moved at slower pace. The aim

S.T.-G., 0000-0003-2262-0430; N.M.-Q., 0000-0002-0366-6591 of this Review is to emphasize the role of actin regulatory proteins Journal of Cell Science

1 REVIEW Journal of Cell Science (2021) 134, jcs253534. doi:10.1242/jcs.253534

P Actin preexisting filament (Mullins et al., 1998). Activation of the Arp2/3 P WDR1 complex is triggered by actin nucleation-promoting factors (NPFs) Cofilin (Blanchoin et al., 2000). Binding of an NPF to the Arp2/3 complex at the side of an actin filament renders the ARP2 and ARP3 subunits into an active conformation, which is equivalent to an actin dimer. Other subunits, such as ARPC2 and ARPC4, have been implicated in mediating the interaction of Arp2/3 with the side of the mother Coronin filament (Humphries et al., 2002). There are two types of NPFs; type I comprises WASPs, WAVEs and others factors that have been more recently described, including the WASP and SCAR homologue (WASH) complex, Arp2/3 complex WASP homolog associated with actin, membranes and (WHAMM), and junction-mediating regulatory protein (JMY) (Campellone and Welch, 2010). Type II NPFs comprise cortactin and its immune homolog hematopoietic cell- specific protein 1 (HS1; also known as HCLS1) (Castro-Ochoa et al., 2019). Briefly, the Arp2/3 complex is activated by a verprolin- Fig. 1. Coordinated action of coronin, cofilin and WDR1 results in cofilin-acidic (VCA) domain found in type I NPFs (Campellone severing of actin filaments. The Arp2/3 complex promotes the and Welch, 2010), or by an acidic conserved motif (DDW) found polymerization of actin by means of incorporating a branch to a preexisting actin filament. In contrast, polymerized actin can be fragmented by the action of in type II NPFs (Weed et al., 2000), with each of these promoting severing proteins, such as coronin, cofilin and WDR1. Binding of coronin might the ramification of actin. change the actin filament twist slightly, favoring the binding of cofilin nearby, and the subsequent binding of WDR1 promotes efficient severing of the Arp2/3-mediated immunopathology filament. It is proposed that WDR1 displaces one or two cofilin molecules ArpC3-deficient mice are embryonically lethal, indicating that certain (yellow). Free cofilin is phosphorylated, whereas actin-bound cofilin is subunits are indispensable for development (Yae et al., 2006). unphosphorylated. Interestingly, three of the proteins shown display WD40- However, mutations in ARPC1B are not lethal owing to the fact that repeat (WDR) domains (depicted as a seven-bladed daisy); the ARPC1 ARPC1A subunit of Arp2/3, coronin and WDR1. The C-terminal coiled-coil region of is ubiquitously expressed. In fact the description of the first coronin is depicted as a helix. immunodeficiency that is based on improper functioning of the Arp2/3 complex allowed to comprehend the specific pattern of expression of isoforms 1A and 1B of the ARPC subunit (Kuijpers not only in immunodeficiencies, but also in autoimmune and et al., 2017). Here, proteomic analysis performed in neutrophils, T autoinflammatory diseases that have received less attention. cells and platelets indicated that the Arp2/3 complex in hematopoietic Therefore, we focus here on cytoskeletal proteins, such as the cells is composed of a specific array of subunits, ARPC1B, ARPC2, Arp2/3 complex and coronins, whose mutations are well established ARPC3, ARPC4, ARPC5 and ARPC5B, whereas ARPC1A is not as the cause of immunodeficiencies. In addition, we highlight recent present (Kuijpers et al., 2017). Conversely, non-hematopoietic cells findings that have linked cofilins to autoimmune diseases, as well as preferentially express ARPC1A, although they also express ARPC1B present the role of WDR1 in autoinflammatory diseases. (Kuijpers et al., 2017). Both isoforms share 90% similarity at the protein level and have regulatory roles that contribute to the stability Actin nucleators – the Arp2/3 complex of the Arp2/3 complex; however, the capacity to assemble stable The actin-related protein 2/3 (Arp2/3) complex comprises seven branched actin networks under rapid demand is significantly higher subunits; two of these are similar to G-actin and are called actin- for ARPC1B (Abella et al., 2016). related proteins ARP2 (44 kDa), and ARP3 (47 kDa), encoded by the The first ARPC1B pathogenic variant was found in a seven-year- ACTR2 and ACTR3 , respectively (Machesky et al., 1994). The old male patient born from consanguineous parents, resulting in other subunits are the actin-related protein complex (ARPC) proteins, combined immunodeficiency, immune dysregulation and bleeding ARPC1 (also named p41 in vertebrates, with an A and B isoform), complications (Table S1) (Kuijpers et al., 2017). Soon after, three ARPC2 (p34), ARPC3 (p21), ARPC4 (p20) and ARPC5 (also new patients were reported from two families with pathogenic known as p16, with an A and B isoform) (Pizarro-Cerda et al., 2017). variants in ARPC1B, who did not present combined Interestingly, ARPC1 is a 40 kDa protein that is structurally immunodeficiency, as the number of leukocytes was even slightly similar to a WD40-repeat (WDR) domain (Robinson et al., 2001). increased with respect to healthy controls. However, platelet defects WDR domains are abundant, with ∼360 described to date (Schapira were prominent, and the patients presented additional autoimmune et al., 2017). Although WDR domains can have five to eight blades, and autoinflammatory manifestations (Table S1) (Kahr et al., 2017). a canonical WDR domain comprises seven blades, with each Patient one has a homozygous frameshift mutation (p.V91Wfs*30) comprising 40 to 60 (aa) residues that possess a that leads to the loss of ARPC1B, resulting in the total absence of conserved glycine-histidine and tryptophan-aspartate (WD) motif, Arp2/3 in hematopoietic cells. This patient presented with and the seven blades adopt a β-propeller structure (Schapira et al., microthrombocytopenia (reduced number of platelets and of small 2017). The array of subunits that conform the complex may vary size than healthy individuals) and defective platelet spreading that depending on the type of structure upon which actin has to be was characterized by their having limited lamellipodia but prominent nucleated and, for instance, hybrid complexes containing filopodia, confirming the essential role of the Arp2/3 complex in but lacking ARPC1 have been described at focal adhesions in HeLa mediating lamellipodia formation during cell spreading (Kahr et al., cells (Chorev et al., 2014). In accordance with the dendritic 2017). nucleation model (Mullins et al., 1998), the Arp2/3 complex is able In contrast, the other two patients, who were brothers, shared a to add a characteristic 70° angle branch (Fig. 1) to the side of a p.A105V missense mutation in ARPC1B that yields a residual Journal of Cell Science

2 REVIEW Journal of Cell Science (2021) 134, jcs253534. doi:10.1242/jcs.253534 amount of ARPC1B and a normal number of platelets (Table S1). Additionally, five out of the six patients presented Nevertheless, platelets share features such as aberrant filopodia in thrombocytopenia (Brigida et al., 2018). the three patients, as shown by spreading studies carried out (Kahr The most recent description of ARPC1B-deficient patients with et al., 2017). homozygous mutations c.491_495delinsCCTGCCC (the first Around the same time, a different study described two sibling mutation described for ARPC1B; Kuijpers et al., 2017) or sons of congenital parents with an ARPC1B homozygous p.V208 c.897_910delCGAGCGCTTCCAGA includes a reduced number frameshift variant who displayed susceptibility to infections and and malfunctioning of CD8+ cytotoxic T cells (CTLs) (Randzavola mild vessel inflammation but no major T cell alterations (Table S1) et al., 2019). Interestingly, western blot analysis of lysates from the (Somech et al., 2017). The authors developed a zebrafish knockout patient with the first mutation confirmed the complete loss of (KO) model to help understand the role of ARPC1B in the ARPC1B as previously described, but also detected an upregulation development of thrombocytes and T cells. The zebrafish mutants of ARPC1A compared with lysates from a healthy control showed altered development of both thrombocytes and T cells. (Randzavola et al., 2019). The major function of CTLs is to kill Importantly, the defects could be rescued by the wild-type (WT) virus-infected cells, and they use the T cell receptor (TCR) to human ARPC1B ortholog, but not by the variant found in the recognize viral peptides that are presented by the major patients, corroborating its pathogenicity (Somech et al., 2017). histocompatibility complex class I (MHC-I) molecules. This No major T cell defects where reported in previous studies of interaction results in the formation of a lytic IS and the ARPC1B-deficient patients, until T lymphocytes were investigated degranulation of cytotoxic vesicles. Both processes are partially in detail and six additional patients carrying different homozygous dependent on Arp2/3-mediated F-actin polymerization, and in fact, mutations were found to have mild T cell lymphopenia (reduced ARPC2 and ARPC3 colocalize with F-actin in the periphery of the number of lymphocytes) (Table S1) (Brigida et al., 2018). lytic synapse (Butler and Cooper, 2009). ARPC1B-deficient cells Interestingly, the patients have reduced, but detectable levels of fail to assemble proper immunological synapses with a decrease in ARPC1B in T-, B- and natural killer (NK) cells, as well as in F-actin and the absence of invaginations at the contact area, which monocytes, in contrast to the previously described p.V208fs-mutant remains flat, or emit long filopodia (Fig. 2) (Randzavola et al., patient with a complete loss of ARPC1B (Somech et al., 2017), who 2019). was included in this study as a control (Brigida et al., 2018). The study by Randzavola et al. (2019) investigated cell migration Remarkably, the patients present mosaicism with a variable over ICAM-1-coated surfaces, both with WT and ARPC1B- proportion of cells expressing normal levels of ARPC1B deficient CTLs. WT CTLs have thick lamellipodia at the leading compared to healthy controls, which contributes to the phenotype age, whereas ARPC1B-deficient CTLs fail to form lamellipodia and complexity upon Arp2/3 deficiency. Experiments mimicking instead produce long filopodia-like projections that were unable to immune synapse (IS) formation by allowing the cells to spread reorganize upon TCR activation (Randzavola et al., 2019). Here, the over glass covered with intercellular adhesion molecule 1 (ICAM-1) activation of formins could have resulted in the long filopodia-like and anti-CD3 antibody, show that T blasts had defects in generating projections instead of lamellipodia in the absence of Arp2/3 radial lamellipodia, which prevents the proper assembly of the IS. subunits. Therefore, the lack of properly functioning CTLs could

Target cell Fig. 2. Deficiencies in ARP1B and CORO1A result CTL/NK cell in immunological synapse defects. Invaginations are formed at the contact area between the cytotoxic synapse of a wild-type cytotoxic T cell (CTL) and a target cell, for example a virus-infected cell. In contrast, aberrant ARPC1B induces a flat surface or even prominent filopodia; this prevents lysis of the target cell, which requires proper signaling for polymerizing actin, thus contributing to the viral susceptibility found in ARPC1B-deficient patients. Similarly, coronin 1A (CORO1A)-deficient patients display viral susceptibility that can be partially explained by defects of NK cells in assembling the cytolytic synapse, which shows increased F-actin content that impedes exocytosis of the lytic granules. Cytotoxic/cytolytic immunological synapse

Wild-type ARPC1B deficiency CORO1A deficiency Journal of Cell Science

3 REVIEW Journal of Cell Science (2021) 134, jcs253534. doi:10.1242/jcs.253534 contribute to the susceptibility to viral infections in patients with 2017). In addition, autoantibodies against the three autoantigens were mutations in ARPC1B (Table S1) (Randzavola et al., 2019). detected at higher titers in patients with MALT than in patients with In conclusion, studies of immunodeficient patients with pSS (Cui et al., 2017). One potential carcinogenic function of cofilin mutations in Arp2/3 have demonstrated an essential role for is through controlling invadopodia dynamics together with cortactin ARPC1B during cell spreading of T cells, neutrophils and (Magalhaes et al., 2011), and, therefore, as previously described for platelets, as well as for CTL IS assembly. Furthermore, they have other lymphomas, we speculate this role might contribute to the shown that long filopodia are formed when lamellipodia are blocked development of MALT. due to improper functioning or total absence of Arp2/3. As a Remarkably, in Alzheimer’s disease, equimolar amounts of consequence, the affected patients present a spectrum of diseases, cofilin–actin give rise to anomalous rods (bundles of filaments including combined immunodeficiency, thrombocytopenia, which composed of cofilin–actin in a 1:1 complex), which are a can be accompanied with autoimmune and autoinflammatory pathognomonic biomarker (Bamburg and Bernstein, 2016). manifestations (Table S1). Another type of rod-like structures, called nemaline bodies, are found in patients suffering from , a genetically Actin-severing proteins – cofilin, WDR1 and coronin heterogeneous disease characterized by muscle weakness (Sewry Cofilin et al., 2019). While the majority of cases have pathogenic variants of By severing the actin meshwork, actin depolymerizing factor nebulin (NEB) and skeletal α-actin (ACTA1) (Table S1), other genes (ADF), also known as cofilin, contributes to actin turnover (Fig. 1). implicated in the disease are T1 (TNNT1), β- Cofilin is a small 19 kDa protein that exists in two isoforms, cofilin (TPM2)andα-tropomyosin (TPM3) (Agrawal et al., 2007). 1 and cofilin 2. Cofilin 1 is ubiquitously expressed, with the Interestingly, in rare instances, pathogenic cofilin 2 (CFL2) gene exception of muscle cells, while cofilin 2 is mainly expressed in variants are present in patients with nemaline myopathy (Table S1). muscles (Ono et al., 1994). The actin-severing activity of cofilin is The first case reported a homozygous p.A35T CFL2 missense inhibited by of residue 3 by LIM kinases change found in two siblings (Agrawal et al., 2007). The patients (LIMK1 and LIMK2; LIM is an acronym of the three gene products presented at birth with hypotonia, followed by delayed achievement Lin-11, Isl-1 and Mec-3), and this inhibitory effect is reversed by of motor milestones, numerous falls and incapacity to run. The dephosphorylation mediated by the slingshot phosphatases mutation is located immediately following a highly conserved (Mizuno, 2013). nuclear localization signal, and might cause misfolding and The net outcome of cofilin action depends on various factors, degradation of the protein (Agrawal et al., 2007). It would including the ratio between cofilin and actin filaments. Depending certainly be interesting to determine whether there are similarities on that ratio, cofilin can bind cooperatively to the side of ADP- between the rods in Alzheimer’s patients and the nemaline bodies bound F-actin filaments (Carlier et al., 1999). In addition, the found in nemaline myopathy patients with mutant CFL2. binding of cofilin to F-actin shortens the pitch of the helix, creating specific filaments domains defined as ‘cofilactin’ (McGough et al., WDR1 1997). However, to sever actin filaments, cofilin has to act in WDR1 is a 60–65 kDa protein that consists mainly of two seven conjunction with other proteins, such as WDR1 and coronins bladed β-propellers (Ono, 2018); it was initially described as a yeast (discussed below; Fig. 1). The combined action of cofilin, WDR1 ABP and named actin-interacting protein 1 (AIP1) (Adler et al., and coronins generates actin fragments and thus promotes filament 1999). As mentioned above, WDR1 functions in conjunction with disassembly. However, they can nevertheless promote barbed-end cofilin and coronin in severing of actin filaments (Fig. 1). Since in extension of new filaments owing to the pool of monomeric actin vitro cofilin is not very efficient in severing, it is though that the that is generated by F-actin disassembly (Shekhar et al., 2016). coordinated action of both cofilin and WDR1 is especially important when a rapid actin turnover is required (Elam et al., Cofilin-mediated immunopathology 2013). The binding of WDR1 to actin and severing of actin Patients with primary Sjogren’s syndrome (pSS) display filaments occurs preferentially at the edges of those regions that are autoantibodies against cofilin 1 in rare cases, as reported by a sole bound by cofilin and bare of any ABPs (Fig. 1), thereby promoting study (Cui et al., 2017). pSS is an autoimmune disease characterized the fracture of filaments at their cofilin-bound edges (boundary- by damage of the salivary and lacrimal glands, resulting in dry eye and fracture model) (Rodal et al., 1999). dry mouth, and additional exocrine glands and other tissues might also Human WDR1 is regulated by Src kinase-mediated tyrosine be involved in the disease. Pathological studies indicate progressive phosphorylation at residue 238, which promotes its interaction with lymphocytic infiltration of the affected gland by T, B and NK cells. In cofilin-bound F-actin, while it is dephosphorylated by eyes absent advanced cases, persistent activation of B cells is linked to the homolog 3 (EYA3), a dual phosphatase containing an additional development of mucosal-associated lymphoid tissue lymphoma transcriptional domain (Mentel et al., 2018). (MALT) (Martín-Nares and Hernández-Molina, 2019). Autoimmune diseases are characterized by immune deregulation and the presence of Mutant WDR1 – a driver of autoinflammation, immunodeficiency autoantibodies or autoreactive T cells against our own cellular or both? components (mainly proteins, which are then considered as Ablation of the Wdr1 locus in mice is embryonically lethal at the autoantigens). In ∼70% of pSS cases, autoantibodies reactive with peri-implantation stage (Xiao et al., 2017). Murine hypomorphic Ro52 are present. Ro52 is a subtype of the Ro/SSA autoantigen, a mutations cause macrothrombocytopenia (a significantly reduced RNA–protein complex composed of Ro60 and Ro52 (Wolska et al., number of platelets displaying anomalous augmented size) and 2016). autoinflammation that is characterized by neutrophilia, massive In an attempt to find biomarkers capable of predicting malignancy, neutrophilic migration and infiltration into inflamed tissues, such as a proteomic analysis identified four and studied three overexpressed the skin (Kile et al., 2007). Importantly, the use of the RAG 1/2- proteins in the parotid gland of pSS patients, namely, cofilin 1, deficient mice indicated that the manifestations are T and B cell

α-enolase, and Rho GDP-dissociation inhibitor 2 (RGI2) (Cui et al., independent. Instead, the major cell subtypes with malfunctions Journal of Cell Science

4 REVIEW Journal of Cell Science (2021) 134, jcs253534. doi:10.1242/jcs.253534 include megakaryocytes, which show defects in their morphology, recurrent infections, stomatitis and neutropenia (abnormally reduced maturation and shedding into platelets, as well as neutrophils, which number of neutrophils), as well as both abnormal neutrophil random display abnormally distributed F-actin content, implying WDR1 is (chemokinesis) and directed (chemotaxis) migration. Patients may required for the function of both cell types (Kile et al., 2007). also present frequent severe skin and mucosal ulcerations. Some of However, the first clear indication of the molecular mechanism that the neutrophilic defects include an abnormal herniation of the nuclear explains how aberrant actin found in mutant WDR1 mouse induces lobes after spreading on glass, reduced number of granules and a four- autoinflammation came by analyzing possible cytokine mediators fold increase in F-actin content, altogether indicating that the (Kile et al., 2007). Indeed, using the above mouse model (Kile et al., cytoskeleton of neutrophils is altered (Fig. 3). In this study, among 2007), it was shown that autoinflammation was mediated by IL-18, others (Table S1), a homozygous missense mutation c.76G.A, which is mainly produced by monocytes through activation of the p.D26N was found in WDR1, a position that is highly conserved pyrin inflammasome (Kim et al., 2015) (Fig. 1). Inflammasomes are among species and is located on blade 1 of the first β-propeller; In molecular platforms that preferentially activate caspase-1, which in fact, all five identified mutations fell within structurally invariant β- turn cleaves and contributes to the secretion of highly inflammatory strand regions of the blades, possibly causing relevant structural cytokines, such as IL-1β and IL-18. Mutations in inflammasome changes (Kuhns et al., 2016). components cause autoinflammatory diseases (Alehashemi and Mirroring the described IL-18 overproduction in mice with Wdr1 Goldbach-Mansky, 2020; Martinez-Quiles and Goldbach-Mansky, mutations (Kile et al., 2007), two siblings with high serum levels of 2018). IL-18 presented with key autoinflammatory markers (Standing et al., The study of patients from three families affected by the previously 2017). In addition, the patients displayed associated recognized so-called ‘lazy leukocyte syndrome’ (Miller et al., 1971) immunodeficiency and thrombocytopenia. Therefore, the term shed light onto WDR1-mediated inflammation in humans (Kuhns PFIT (periodic fever, immunodeficiency and thrombocytopenia) et al., 2016). The syndrome is characterized by life-threatening was coined to describe the main traits of the disease presented by the

Mutant WDR1

WASPs/N-Wasp ↑ F-actin content WASPs HS1/Cortactin Mutant WASPs Pyrin/NLRP3 WAVE Arp2/3 Caspase 1 Mutant ARPC1B

IL1-β/IL-18 Lamellipodia Filopodium Inflammasome

Uropod

Nucleus

Leading edge

fMLP Integrins CXCL1 SDF-1α Extracellular matrix

Fig. 3. Overview of cytoskeletal signaling pathways in leukocytes that are relevant for immunodeficiencies and autoinflammatory diseases. After sensing chemokine factor fMLP or chemokine (CXC1, SDF-1α) gradients, leukocytes polarize and display a leading edge and a rear end (uropod). Activation of the Arp2/3 complex by the Wiskott–Aldrich syndrome protein family (WASPs) promotes actin branching as found in lamellipodia, whereas filopodia are formed by unbranched actin filaments. Mutations in the proteins of the depicted pathways give rise to primary immunodeficiencies. For instance, ARPC1B mutation results in an altered function of the Arp2/3 complex function, and the cell tends to assemble filopodia instead of lamellipodia, thus producing combined immunodeficiency, thrombocytopenia, together with autoimmune and autoinflammatory manifestations. A novel aspect of the factors illustrated here is their role in inducing concomitant inflammation. Indeed, mutant WDR1 can induce both immunodeficiency and inflammation. WDR1 participates in actin severing; as consequence of its abnormal functioning, cells accumulate cytoplasmic actin clusters that activate the pyrin inflammasome, and the nucleus acquires a typical lobulated shape (depicted in blue). Apart from causing the immunodeficiency Wiskott–Aldrich syndrome (WAS) it is named after, mutant WASP has also been linked to aberrant activation of the NLRP3 inflammasome, giving rise to autoinflammatory conditions. Journal of Cell Science

5 REVIEW Journal of Cell Science (2021) 134, jcs253534. doi:10.1242/jcs.253534 patients that was not initially recognized as ‘lazy leukocyte actin branch (Fig. 1), replacing the complex and modifying the syndrome’ (Fig. 3; Table S1). The study found two missense angle of the branch (i.e. increasing the angle between the mother and mutations in WDR1, and investigations focused on monocyte-derived daughter filaments), thereby possibly contributing to remodeling of dendritic cells (DCs), which possess augmented levels of the branched actin and its turnover at lamellipodia, as demonstrated polymerized F-actin and larger podosomes than control cells by coronin 1B-labeling studies (Cai et al., 2008). This action (Standing et al., 2017). WDR1 contributes to the formation of counteracts the opposite stabilizing effects exerted by F-actin-bound podosomes, adhesion structures that play a part in protease-dependent cortactin (Cai et al., 2008), a type II NPF that activates Arp2/3 3D migration of DCs (Cougoule et al., 2018). DCs migrate from the (Weaver et al., 2001). infection or inflammatory focus to the regional lymph nodes to Furthermore, for efficient filament disassembly, coronin, cofilin initiate adaptive immune responses (including production of and WDR1 have been shown to act together (Brieher et al., 2006; antibodies, and expansion and activation of cytotoxic T cells); Kueh et al., 2008) (Fig. 1). Seminal biochemical studies established therefore, any defect that affects their migration will strongly impact that coronin and WDR1 are required for actin severing by cofilin immune defense against pathogens that would contribute to the under physiological conditions (i.e. high concentrations of underlying immunodeficiency. polymerizable G actin), and that a lesser amount of cofilin was Finally, the study of six patients from three families with biallelic needed to depolymerize actin if both proteins were present (Brieher WDR1 mutations confirms the previously reported WDR1-associated et al., 2006). Later in vitro studies have shown that coronin, cofilin disease hallmarks, such as the typical neutrophil nuclear herniation and WDR1 bind to the actin filament sequentially (Jansen et al., and their migratory defects. Interestingly, WDR1 mutant lymphocytes 2015). Indeed, the binding of coronin 1B to in vitro preformed actin display striking cytoskeletal alterations, including excess of actin arcs, filaments accelerates the otherwise slow binding of cofilin 1 (Elam actin spikes and filopodia, all structures that mainly rely on formin- et al., 2013). This suggests that coronin could slightly alter the mediated actin nucleation (Courtemanche, 2018). Whether this filament twist, favoring the binding of cofilin in the proximity. Here, nucleation mechanism is upregulated to counteract the lack of a fully the binding of both coronin and cofilin in the absence of WDR1 functioning Arp2/3-mediated route remains unclear. would have an overall filament-stabilizing effect. However, when Taken together, one of the main WDR1-deficiency hallmarks is WDR1 is present, rapid and efficient severing of the filament takes aberrant herniation of the nuclear lobes of neutrophils, together with place (Jansen et al., 2015). Based on previous findings (Elam et al., reduced number of granules, and increase in F-actin content, and 2013), the authors proposed that WDR1 might displace one or more both immunodeficiency and autoinflammation can arise as a cofilin molecules from a patch, generating a local discontinuity that consequence of WDR1 malfunctioning. destabilizes the filaments (Jansen et al., 2015). Wdr1-deficient neutrophils from zebrafish embryos have Coronin increased death rates accompanied by nuclear disorganization Here, we only highlight the basic aspects of coronin that are (Bowes et al., 2019). These cells have cytoplasmic clusters of F- important for understanding how it functions in conjunction with actin, and interestingly, cofilin is found unphosphorylated and other ABPs and contributes to immune disease. For a detailed bound to F-actin in these cells, which corroborates the functional discussion of coronin, we refer the reader to recent reviews (Chánez- interdependence of WDR1 and cofilin. Unexpectedly, depletion of Paredes et al., 2019; Mori et al., 2018). coronin 1A restored these defects and even allowed the cells to The first description of coronin was in the amoeba Dictyostelium migrate in vivo (Bowes et al., 2019). Therefore, the spatiotemporal as an ABP associated with crown-shaped cell surface projections recruitment and activation of coronin, cofilin and WDR1 constitutes that regulate chemotaxis (de Hostos et al., 1991), and it was later a fine-tuned mechanism that controls actin dynamics. shown to have a WDR domain (Suzuki et al., 1995). Since these initial studies, coronins have been shown to be involved in Coronin-mediated immunopathology phagocytosis, including during Mycobacterium persistence in The description of a p.E26K coronin 1A mutation in the peripheral macrophage phagosomes (Jayachandran et al., 2007), actin-tail T-cell-deficient (Ptcd) murine strain (Shiow et al., 2008), prompted induction during Listeria infection (David et al., 1998), as well as in these investigators to explore whether pathogenic variants of human cytokinesis (de Hostos et al., 1993), among other cellular motility coronin 1A gene (CORO1A, MIM 605000) might be involved in processes. human immunodeficiency (Shiow et al., 2008). In the p.E26K There are seven mammalian genes encoding for the 50 to 70 kDa coronin mutant Ptcd mouse, the F-actin-binding capacity of coronin coronin proteins, which group into three classes. Classes I and II is preserved, but it has an increased affinity for the Arp2/3 complex possess one typical WDR domain, whereas class III coronins and therefore is more efficient in inhibiting the Arp2/3 complex than possesses two. Apart from the WDR domain, some coronins also WT coronin, as determined by actin polymerization assays have a coiled-coil region at their C-terminus, which is required for performed in vitro (Shiow et al., 2008). Therefore, pE26K coronin oligomerization (type I and II), or an acidic domain (type III). The 1A can be categorized as a gain-of-function mutant of coronin most-studied coronins are 1A and 1B (Chánez-Paredes et al., 2019). (Shiow et al., 2008). Coronin 1B (class I) has a highly charged patch of residues at the Accordingly, analysis of human CORO1A by Sanger sequencing surface of the β-propeller that binds to F-actin. Coronin 1B is led to the description of a novel immunodeficiency caused by a ubiquitously expressed, whereas coronin 1A is predominantly deletion of two nucleotides in exon 3 (c.248 249delCT) that has expressed in immune cells, as deduced from the phenotype of been found in a 13-month-old girl who inherited the mutation from coronin 1A-deficient patients (see below). It is possible that her father (Shiow et al., 2008). The deletion is located within the coronins exert various functions in regulating actin turnover, such WDR domain and results in a frameshift mutation that incorporates as participating in lamellipodia dynamics. In fact, class I and II a premature stop codon (p.Pro83ArgfsX10). Additionally, the coronins bind to Arp2/3 and inhibit its activation (Humphries et al., patient presented a deletion of the other allele. Both mutations 2002). The underlying molecular mechanism was revealed by together resulted in the total absence of coronin 1A, as assessed showing that coronin 1B induces the dissociation of Arp2/3 from the using in vitro-derived patient B cell lines [i.e. Epstein Barr virus Journal of Cell Science

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(EBV)-immortalized B lymphoid cell lines] (Shiow et al., 2008). cytoskeleton by pathogens, such as those occurring by blocking of The patient presented lymphopenia, but the size of the thymus was the RhoA GTPase and, additionally, it is activated by aberrant cellular normal. Among other infections, she suffered severe varicella actin due to WDR1 malfunction (among other pathways), thereby infection after vaccination with a live attenuated vaccine. The causing autoinflammation (Kim et al., 2015). patient received the diagnosis of T− B+ NK+ severe combined With this in mind, it is possible to understand why perturbations of immunodeficiency (SCID) and was subjected to allogeneic bone the homeostasis of the actin cytoskeleton that are caused by marrow transplantation (Shiow et al., 2008). malfunctioning or deletion of ABPs produce not only a lack of More recently, three siblings have been described who presented immune response as a result of a ‘physical impairment’ of the cell, for with CD4 lymphopenia, normal size thymus and early EBV- instance immunodeficiency caused by Arp2/3, WDR1 and coronin, associated B-cell lymphoproliferative syndrome (Moshous et al., but also an exacerbation or decontextualized response, such as 2013). Whole exome sequence (WES) identified a homozygous autoinflammation caused by aberrant Arp2/3, WDR1 and coronin, or missense mutation p.V134M in CORO1A, also located within the autoimmunity caused by anomalous cofilin. This is best illustrated by WDR domain, leading to residual protein expression that is focusing on the consequences of the expression of WDR1 pathogenic responsible for a hypomorphic defect (Moshous et al., 2013). variants. For example, lack of appropriate actin remodeling by Furthermore, a different study performed WES in two siblings deregulated severing in the absence of functional WDR1 results in an with mucocutaneous-immunodeficiency syndrome from a non- impairment of neutrophil adhesion and migration, as predicted. consanguineous family revealed two CORO1A pathogenic variants: However, the observed increase in inflammatory markers linking the previously described c.248_249delCT (p.P83RfsX10) (Shiow malfunctioning of WDR1 to inflammasome activation was not et al., 2008), and a novel one c.1077delC (p.Q360RfsX44) expected. The question thus remains of how an increase in F-actin is (NM_007074.3). The phenotypic manifestations include absent sensed and which pathways intervene to active the innate immune naïve (CD4+ CD45RA+) T and memory (CD19+ CD27+) B cells, a response, including activation of the pyrin inflammasome. low number of NK cells, as well as an abnormally increased number As mentioned above, it is worth keeping in mind that various of double negative (CD4− CD8−) ϒδ T-cells. Both patients display ABPs bear WDR domains, such as ARPC1, WDR1 and the class I profound viral susceptibility (see Table S1), and, in one of the and II coronins. WDR domains possess mechanical characteristics siblings, telomere shortening was detected, but its relevance is as intuitively implied by the β-propeller structure they conform. unclear (Stray-Pedersen et al., 2014). Therefore, it is possible that β-propellers induce changes in F-actin The susceptibility to viral infections displayed by patients with that contribute to its remodeling. Here, the helical conformation of altered expression levels of coronin 1A might be partially explained by the filament regulates the binding of ABPs, while binding of some a malfunctioning of NK cells. In order to achieve a polarized secretion ABPs, in turn, regulates the helicity of the filament (Jegou and of the granule content at the NK–target-cell interface, known as the Romet-Lemonne, 2020), such as cofilin. However, in order to sever cytolytic synapse, some degree of F-actin deconstruction mediated by the filament, cofilin requires the concerted action of two additional coronin 1A action is required (Fig. 2) (Mace and Orange, 2014). This proteins with WDR domains, coronin and WDR1 (Fig. 1). In this has been proposed based on the increased amount of F-actin found at context, coronin appears to facilitate the binding of cofilin, possibly the NK IS in a coronin-deficient patient who presented compound marking the specific points for cofilin binding, which then defines heterozygous mutations with c.1077delC, in combination with the the site of action of WDR1. previously described c.248_249delCT mutation (Fig. 2) (Shiow et al., As discussed here, in particular the function of neutrophils and 2008). Profound T cell lymphopenia was detected, but the NK cell platelets, is disrupted in various immune diseases that are caused by number was within normal range, implying that coronin 1A is aberrant ABPs. Both cell types possess a high content of G-actin, necessary for NK functioning but not for development (Shiow et al., determined to be in the order of hundreds of micromolar, whereas 2008). However, in order to understand the viral susceptibility of for example HeLa cells have only ∼10% of that amount (Plastino CORO1A-deficient patients, it would be necessary to establish whether and Blanchoin, 2018). In theory, this high G-actin content would CTLs exhibit comparable cytotoxic synapse functional defects. Given allow for a rapid and massive grow of actin filaments required for that mast cell degranulation by exocytosis is orchestrated by the motility underlying immune functions (Plastino and Blanchoin, formation of an actin fence mediated by formin mDia1 (Klein et al., 2018). Certainly, many challenges await us in the fascinating 2019), and that exocytosis takes place at the cytolytic or cytotoxic multidisciplinary cytoskeleton research field. synapse (Phatarpekar and Billadeau, 2020), it would be interesting to investigate whether this represents a general molecular mechanism by which mDia1 assembles an actin barrier to stall degranulation, whereas Competing interests The authors declare no competing or financial interests. coronin 1A would participate in disassembling the fence during exocytosis. Funding Our work in this area was supported by the European Regional Development Fund Concluding remarks and the Spanish Ministry of Science, Innovation and Universities (Fondo Europeo de – The actin cytoskeleton is typically considered as a structure that Desarrollo Regional FEDER/Ministerio de Ciencia, Innovación y Universidades Agencia Estatal de Investigación), grant SAF2017-82967-R to N.M.-Q. (See https:// remodels in order to accommodate cellular functions, www.ciencia.gob.es/stfls/MICINN/AEI/imagenes/MICINN_Gob_AEI_1.jpg). underestimating that it is manipulated by numerous pathogens, not only to invade cells but also to block the cellular counter-response. Supplementary information Therefore, the innate immunity detects pathogen-induced Supplementary information available online at modifications of the actin cytoskeleton as pathogenic clues. https://jcs.biologists.org/lookup/doi/10.1242/jcs.253534.supplemental Consequently, actin is considered an essential part of homeostasis- altering molecular processes (HAMPs) that activate the innate References Abella, J. V. G., Galloni, C., Pernier, J., Barry, D. J., Kjaer, S., Carlier, M.-F. and immune response (Liston and Masters, 2017). In this sense, the Way, M. (2016). Isoform diversity in the Arp2/3 complex determines actin filament pyrin inflammasome is specialized in detecting a manipulation of the dynamics. Nat. 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