Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

Evolution of the thermostability of actin-depolymerizing factors enhances the adaptation of pollen germination to high temperature.

Double fertilization in many flowering plants (angiosperms) often occurs during the hot summer season, but the mechanisms that enable angiosperms to adapt specifically to high temperatures are largely unknown. The actin cytoskeleton is essential for pollen germination and the polarized growth of pollen tubes, yet how this process responds to high temperatures remains unclear. Here, we reveal that the high thermal stability of 11 Arabidopsis (Arabidopsis thaliana) actin-depolymerizing factors (ADFs) is significantly different: ADFs that specifically accumulate in tip-growing cells (pollen and root hairs) exhibit high thermal stability. Through ancestral protein reconstruction, we found that subclass II ADFs (expressed specifically in pollen) have undergone a dynamic wave-like evolution of the retention, loss, and regeneration of thermostable sites. Additionally, the sites of AtADF7 with high thermal stability are conserved in ADFs specific to angiosperm pollen. Moreover, the high thermal stability of ADFs is required to regulate actin dynamics and turnover at high temperatures to promote pollen germination. Collectively, these findings suggest strategies for the adaptation of sexual reproduction to high temperature in angiosperms at the cell biology level.

Activation of actin-depolymerizing factor by CDPK16-mediated phosphorylation promotes actin turnover in Arabidopsis pollen tubes.

As the stimulus-responsive mediator of actin dynamics, actin-depolymerizing factor (ADF)/cofilin is subject to tight regulation. It is well known that kinase-mediated phosphorylation inactivates ADF/cofilin. Here, however, we found that the activity of Arabidopsis ADF7 is enhanced by CDPK16-mediated phosphorylation. We found that CDPK16 interacts with ADF7 both in vitro and in vivo, and it enhances ADF7-mediated actin depolymerization and severing in vitro in a calcium-dependent manner. Accordingly, the rate of actin turnover is reduced in cdpk16 pollen and the amount of actin filaments increases significantly at the tip of cdpk16 pollen tubes. CDPK16 phosphorylates ADF7 at Serine128 both in vitro and in vivo, and the phospho-mimetic mutant ADF7S128D has enhanced actin-depolymerizing activity compared to ADF7. Strikingly, we found that failure in the phosphorylation of ADF7 at Ser128 impairs its function in promoting actin turnover in vivo, which suggests that this phospho-regulation mechanism is biologically significant. Thus, we reveal that CDPK16-mediated phosphorylation up-regulates ADF7 to promote actin turnover in pollen.

Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.

Disassembly of bundled F-actin and cellular remodeling via an interplay of Mical, cofilin, and F-actin crosslinkers.

Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures-but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined-including that it is driven by actin's polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical's disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical's F-actin disassembly in vitro and in vivo-and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them.

Twist response of actin filaments.

Actin cytoskeleton force generation, sensing, and adaptation are dictated by the bending and twisting mechanics of filaments. Here, we use magnetic tweezers and microfluidics to twist and pull individual actin filaments and evaluate their response to applied loads. Twisted filaments bend and dissipate torsional strain by adopting a supercoiled plectoneme. Pulling prevents plectoneme formation, which causes twisted filaments to sever. Analysis over a range of twisting and pulling forces and direct visualization of filament and single subunit twisting fluctuations yield an actin filament torsional persistence length of ~10 µm, similar to the bending persistence length. Filament severing by cofilin is driven by local twist strain at boundaries between bare and decorated segments and is accelerated by low pN pulling forces. This work explains how contractile forces generated by myosin motors accelerate filament severing by cofilin and establishes a role for filament twisting in the regulation of actin filament stability and assembly dynamics.

Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin.

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin-binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using total internal reflection fluorescence microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- versus ADP-Pi-actin-containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

Slingshot homolog-1 amplifies mitochondrial abnormalities by distinctly impairing health and clearance of mitochondria.

Accumulating toxic protein assemblies, including Aß and tau, and dysfunctional mitochondria are associated with synaptic and neuronal loss in Alzheimer's disease (AD). Such accumulations are thought to be owing to clearance defects in the autophagy-lysosome pathway. Mitochondrial dysfunction is evident in AD brains and animal models at multiple levels, such as mitochondrial genomic mutations, disrupted bioenergetics, deregulated mitochondrial dynamics and impaired clearance of damaged mitochondria (mitophagy). Slingshot homolog-1 (SSH1) is a phosphatase activated by oxidative stress, high intracellular levels of Ca2+ and Aß42 oligomers (Aß42O), known for its function to dephosphorylate/activate cofilin through the N-terminal region. SSH1-mediated cofilin dephosphorylation results in Ab42O-induced severing of F-actin and translocation of cofilin to mitochondria, which promotes mitochondria-mediated apoptosis, synaptic loss and synaptic deficits. On the other hand, SSH1-mediated dephosphorylation/deactivation of the autophagy-cargo receptor p62 (SQSTM1), through its C-terminal region, inhibits p62 autophagy flux. However, the interplay between these two different activities of SSH1 in Aß42O-induced mitochondrial toxicity remains unclear. In this study, we assessed the role of endogenous SSH1 and different regions of SSH1 in regulating mitochondrial health, mitochondrial respiration, clearance of damaged mitochondria and synaptic integrity in vitro and in vivo. Our results indicate that SSH1 suppresses mitochondrial health and respiration through the cofilin-binding N-terminal region, whereas SSH1 impairs mitophagy through a newly identified ~ 100 residue p62-binding domain in the C-terminal region. These results indicate that both N-terminal and C-terminal regions negatively impact mitochondria by distinct and independent modalities to amplify mitochondrial abnormalities, making SSH1 an excellent target to mitigate AD pathogenesis.

Actin turnover protects the cytokinetic contractile ring from structural instability.

In common with other actomyosin contractile cellular machineries, actin turnover is required for normal function of the cytokinetic contractile ring. Cofilin is an actin-binding protein contributing to turnover by severing actin filaments, required for cytokinesis by many organisms. In fission yeast cofilin mutants, contractile rings suffer bridging instabilities in which segments of the ring peel away from the plasma membrane, forming straight bridges whose ends remain attached to the membrane. The origin of bridging instability is unclear. Here, we used molecularly explicit simulations of contractile rings to examine the role of cofilin. Simulations reproduced the experimentally observed cycles of bridging and reassembly during constriction, and the occurrence of bridging in ring segments with low density of the myosin II protein Myo2. The lack of cofilin severing produced ∼2-fold longer filaments and, consequently, ∼2-fold higher ring tensions. Simulations identified bridging as originating in the boosted ring tension, which increased centripetal forces that detached actin from Myo2, which was anchoring actin to the membrane. Thus, cofilin serves a critical role in cytokinesis by providing protection from bridging, the principal structural threat to contractile rings.

Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells.

BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

RABV induces biphasic actin cytoskeletal rearrangement through Rac1 activity modulation.

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.

CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

Functions of cofilin in cell locomotion and invasion.

Recently, a consensus has emerged that cofilin severing activity can generate free actin filament ends that are accessible for F-actin polymerization and depolymerization without changing the rate of G-actin association and dissociation at either filament end. The structural basis of actin filament severing by cofilin is now better understood. These results have been integrated with recently discovered mechanisms for cofilin activation in migrating cells, which led to new models for cofilin function that provide insights into how cofilin regulation determines the temporal and spatial control of cell behaviour.

Forgetting is regulated through Rac activity in Drosophila.

Initially acquired memory dissipates rapidly if not consolidated. Such memory decay is thought to result either from the inherently labile nature of newly acquired memories or from interference by subsequently attained information. Here we report that a small G protein Rac-dependent forgetting mechanism contributes to both passive memory decay and interference-induced forgetting in Drosophila. Inhibition of Rac activity leads to slower decay of early memory, extending it from a few hours to more than one day, and to blockade of interference-induced forgetting. Conversely, elevated Rac activity in mushroom body neurons accelerates memory decay. This forgetting mechanism does not affect memory acquisition and is independent of Rutabaga adenylyl cyclase-mediated memory formation mechanisms. Endogenous Rac activation is evoked on different time scales during gradual memory loss in passive decay and during acute memory removal in reversal learning. We suggest that Rac's role in actin cytoskeleton remodeling may contribute to memory erasure.

Suppression of Cofilin function in the somatosensory cortex alters social contact behavior in the BTBR mouse inbred line.

Sensory differences are a core feature of autism spectrum disorders (ASD) and are predictive of other ASD core symptoms such as social difficulties. However, the neurobiological substrate underlying the functional relationship between sensory and social functioning is poorly understood. Here, we examined whether misregulation of structural plasticity in the somatosensory cortex modulates aberrant social functioning in BTBR mice, a mouse model for autism spectrum disorder-like phenotypes. By locally expressing a dominant-negative form of Cofilin (CofilinS3D; a key regulator of synaptic structure) in the somatosensory cortex, we tested whether somatosensory suppression of Cofilin activity alters social functioning in BTBR mice. Somatosensory Cofilin suppression altered social contact and nest-hide behavior of BTBR mice in a social colony, assessed for seven consecutive days. Subsequent behavioral testing revealed that altered social functioning is related to altered tactile sensory perception; CofilinS3D-treated BTBR mice showed a time-dependent difference in the sensory bedding preference task. These findings show that Cofilin suppression in the somatosensory cortex alters social functioning in BTBR mice and that this is associated with tactile sensory processing, a critical indicator of somatosensory functioning.

PI3K couples long-term synaptic potentiation with cofilin recruitment and actin polymerization in dendritic spines via its regulatory subunit p85α.

Long-term synaptic plasticity is typically associated with morphological changes in synaptic connections. However, the molecular mechanisms coupling functional and structural aspects of synaptic plasticity are still poorly defined. The catalytic activity of type I phosphoinositide-3-kinase (PI3K) is required for specific forms of synaptic plasticity, such as NMDA receptor-dependent long-term potentiation (LTP) and mGluR-dependent long-term depression (LTD). On the other hand, PI3K signaling has been linked to neuronal growth and synapse formation. Consequently, PI3Ks are promising candidates to coordinate changes in synaptic strength with structural remodeling of synapses. To investigate this issue, we targeted individual regulatory subunits of type I PI3Ks in hippocampal neurons and employed a combination of electrophysiological, biochemical and imaging techniques to assess their role in synaptic plasticity. We found that a particular regulatory isoform, p85α, is selectively required for LTP. This specificity is based on its BH domain, which engages the small GTPases Rac1 and Cdc42, critical regulators of the actin cytoskeleton. Moreover, cofilin, a key regulator of actin dynamics that accumulates in dendritic spines after LTP induction, failed to do so in the absence of p85α or when its BH domain was overexpressed as a dominant negative construct. Finally, in agreement with this convergence on actin regulatory mechanisms, the presence of p85α in the PI3K complex determined the extent of actin polymerization in dendritic spines during LTP. Therefore, this study reveals a molecular mechanism linking structural and functional synaptic plasticity through the coordinate action of PI3K catalytic activity and a specific isoform of the regulatory subunits.

Cdc42 activation is necessary for heterosynaptic cooperation and competition.

Synapses change their weights in response to neuronal activity and in turn, neuronal networks alter their response properties and ultimately allow the brain to store information as memories. As for memories, not all events are maintained over time. Maintenance of synaptic plasticity depends on the interplay between functional changes at synapses and the synthesis of plasticity-related proteins that are involved in stabilizing the initial functional changes. Different forms of synaptic plasticity coexist in time and across the neuronal dendritic area. Thus, homosynaptic plasticity refers to activity-dependent synaptic modifications that are input-specific, whereas heterosynaptic plasticity relates to changes in non-activated synapses. Heterosynaptic forms of plasticity, such as synaptic cooperation and competition allow neurons to integrate events that occur separated by relatively large time windows, up to one hour. Here, we show that activation of Cdc42, a Rho GTPase that regulates actin cytoskeleton dynamics, is necessary for the maintenance of long-term potentiation (LTP) in a time-dependent manner. Inhibiting Cdc42 activation does not alter the time-course of LTP induction and its initial expression but blocks its late maintenance. We show that Cdc42 activation is involved in the phosphorylation of cofilin, a protein involved in modulating actin filaments and that weak and strong synaptic activation leads to similar levels on cofilin phosphorylation, despite different levels of LTP expression. We show that Cdc42 activation is required for synapses to interact by cooperation or competition, supporting the hypothesis that modulation of the actin cytoskeleton provides an activity-dependent and time-restricted permissive state of synapses allowing synaptic plasticity to occur. We found that under competition, the sequence in which synapses are activated determines the degree of LTP destabilization, demonstrating that competition is an active destabilization process. Taken together, we show that modulation of actin cytoskeleton by Cdc42 activation is necessary for the expression of homosynaptic and heterosynaptic forms of plasticity. Determining the temporal and spatial rules that determine whether synapses cooperate or compete will allow us to understand how memories are associated.

Single-molecule analysis of actin filament debranching by cofilin and GMF.

Eukaryotic cells contain branched actin networks that are essential for endocytosis, motility, and other key cellular processes. These networks, which are formed by filamentous actin and the Arp2/3 complex, must subsequently be debranched to allow network remodeling and to recycle the Arp2/3 complex. Debranching appears to be catalyzed by two different members of the actin depolymerizing factor homology protein family: cofilin and glial maturation factor (GMF). However, their mechanisms of debranching are only partially understood. Here, we used single-molecule fluorescence imaging of Arp2/3 complex and actin filaments under physiological ionic conditions to observe debranching by GMF and cofilin. We demonstrate that cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch-junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin-actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament-Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.

Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.

The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes implicated in both the complete demolition and fine size control of microtubule-based arrays are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here, we use atomic force microscopy (AFM) to image depolymerizing arrays at single-microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-scale cytoskeletal structures.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.