F-Actin Cytoskeleton Network Self-Organization Through Competition and Cooperation.

Many fundamental cellular processes such as division, polarization, endocytosis, and motility require the assembly, maintenance, and disassembly of filamentous actin (F-actin) networks at specific locations and times within the cell. The particular function of each network is governed by F-actin organization, size, and density as well as by its dynamics. The distinct characteristics of different F-actin networks are determined through the coordinated actions of specific sets of actin-binding proteins (ABPs). Furthermore, a cell typically assembles and uses multiple F-actin networks simultaneously within a common cytoplasm, so these networks must self-organize from a common pool of shared globular actin (G-actin) monomers and overlapping sets of ABPs. Recent advances in multicolor imaging and analysis of ABPs and their associated F-actin networks in cells, as well as the development of sophisticated in vitro reconstitutions of networks with ensembles of ABPs, have allowed the field to start uncovering the underlying principles by which cells self-organize diverse F-actin networks to execute basic cellular functions.

Cell Motility and Cytokinesis: From Mysteries to Molecular Mechanisms in Five Decades.

This is the story of someone who has been fortunate to work in a field of research where essentially nothing was known at the outset but that blossomed with the discovery of profound insights about two basic biological processes: cell motility and cytokinesis. The field started with no molecules, just a few people, and primitive methods. Over time, technological advances in biophysics, biochemistry, and microscopy allowed the combined efforts of scientists in hundreds of laboratories to explain mysterious processes with molecular mechanisms that can be embodied in mathematical equations and simulated by computers. The success of this field is a tribute to the power of the reductionist strategy for understanding biology.

Regulation of branched versus linear Arp2/3-generated actin filaments.

Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides of pre-existing "mother" filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates "linear" actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N-WASP, and WASH destabilize existing branches, as well as SPIN90-Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.

Actin-capping protein regulates actomyosin contractility to maintain germline architecture in C. elegans.

Actin dynamics play an important role in tissue morphogenesis, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report an in vivo role of the actin-capping protein CAP-1 in the Caenorhabditis elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, and its depletion or overexpression led to severe structural defects in the syncytial germline and oocytes. A 60% reduction in the level of CAP-1 caused a twofold increase in F-actin and non-muscle myosin II activity, and laser incision experiments revealed an increase in rachis contractility. Cytosim simulations pointed to increased myosin as the main driver of increased contractility following loss of actin-capping protein. Double depletion of CAP-1 and myosin or Rho kinase demonstrated that the rachis architecture defects associated with CAP-1 depletion require contractility of the rachis actomyosin corset. Thus, we uncovered a physiological role for actin-capping protein in regulating actomyosin contractility to maintain reproductive tissue architecture.

Aspartyl proteases target host actin nucleator complex protein to limit epithelial innate immunity.

Epithelial-immune cell communication is pivotal to control microbial infections. We show that glycosylphosphatidylinositol-linked aspartyl proteases (Yapsins) of the human opportunistic pathogenic yeast Candida glabrata (Cg) thwart epithelial cell (EC)-neutrophil signalling by targeting the EC protein, Arpc1B (actin nucleator Arp2/3 complex subunit), which leads to actin disassembly and impeded IL-8 secretion by ECs. Further, the diminished IL-8 secretion inhibits neutrophil migration, and protects Cg from the neutrophil-mediated killing. CgYapsin-dependent Arpc1B degradation requires Arginine-142 in Arpc1B, and leads to reduced Arpc1B-p38 MAPK interaction and downregulated p38 signalling. Consistently, Arpc1B or p38 deletion promotes survival of the Cg aspartyl protease-deficient mutant in ECs. Importantly, kidneys of the protease-deficient mutant-infected mice display elevated immune cell infiltration and cytokine secretion, implicating CgYapsins in immune response suppression in vivo. Besides delineating Cg-EC interplay, our results uncover a novel target, Arpc1B, that pathogens attack to constrain the host signalling networks, and link Arpc1B mechanistically with p38 activation.

Cytoskeletal mechanisms regulating attaching/effacing bacteria interactions with host cells: It takes a village to build the pedestal.

The actin cytoskeleton is a key cellular structure subverted by pathogens to infect and survive in or on host cells. Several pathogenic strains of Escherichia coli, such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), developed a unique mechanism to remodel the actin cytoskeleton that involves the assembly of actin filament-rich pedestals beneath the bacterial attachment sites. Actin pedestal assembly is driven by bacterial effectors injected into the host cells, and this structure is important for EPEC and EHEC colonization. While the interplay between bacterial effectors and the actin polymerization machinery of host cells is well-understood, how other mechanisms of actin filament remodelling regulate pedestal assembly and bacterial attachment are poorly investigated. This review discusses the gaps in our understanding of the complexity of the actin cytoskeletal remodelling during EPEC and EHEC infection. We describe possible roles of actin depolymerizing, crosslinking and motor proteins in pedestal dynamics, and bacterial interactions with the host cells. We also discuss the biological significance of pedestal assembly for bacterial infection.

Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor.

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and ß subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Polymerization force-regulated actin filament-Arp2/3 complex interaction dominates self-adaptive cell migrations.

Cells migrate by adapting their leading-edge behaviors to heterogeneous extracellular microenvironments (ECMs) during cancer invasions and immune responses. Yet it remains poorly understood how such complicated dynamic behaviors emerge from millisecond-scale assembling activities of protein molecules, which are hard to probe experimentally. To address this gap, we establish a spatiotemporal "resistance-adaptive propulsion" theory based on the interactions between Arp2/3 complexes and polymerizing actin filaments and a multiscale dynamic modeling system spanning from molecular proteins to the cell. We quantitatively find that cells can accurately self-adapt propulsive forces to overcome heterogeneous ECMs via a resistance-triggered positive feedback mechanism, dominated by polymerization-induced actin filament bending and the bending-regulated actin-Arp2/3 binding. However, for high resistance regions, resistance triggers a negative feedback, hindering branched filament assembly, which adapts cellular morphologies to circumnavigate the obstacles. Strikingly, the synergy of the two opposite feedbacks not only empowers the cell with both powerful and flexible migratory capabilities to deal with complex ECMs but also enables efficient utilization of intracellular proteins by the cell. In addition, we identify that the nature of cell migration velocity depending on ECM history stems from the inherent temporal hysteresis of cytoskeleton remodeling. We also show that directional cell migration is dictated by the competition between the local stiffness of ECMs and the local polymerizing rate of actin network caused by chemotactic cues. Our results reveal that it is the polymerization force-regulated actin filament-Arp2/3 complex binding interaction that dominates self-adaptive cell migrations in complex ECMs, and we provide a predictive theory and a spatiotemporal multiscale modeling system at the protein level.

CK-666 protects against ferroptosis and renal ischemia-reperfusion injury through a microfilament-independent mechanism.

Ferroptosis is a type of regulated cell death driven by iron-dependent accumulation of lipid peroxidation, exhibiting unique morphological changes. While actin microfilaments are crucial for various cellular processes, including morphogenesis, motility, endocytosis, and cell death, their role in ferroptosis remains unclear. Here, our study reveals that actin microfilaments undergo remodeling and disassembly during ferroptosis. Interestingly, inhibitors that target actin microfilament remodeling do not affect cell sensitivity to ferroptosis, with the exception of CK-666 and its structural analogue CK-636. Mechanistically, CK-666 attenuates ferroptosis independently of its canonical function in inhibiting the Arp2/3 complex. Further investigation revealed that CK-666 modulates the ferroptotic transcriptome, prevents lipid degradation, and diminishes lipid peroxidation. In addition, CK-666 does not impact the labile iron pool within cells, nor does the inhibition of FSP1 impact its anti-ferroptosis activity. Notably, the results of DPPH assay and liposome leakage assay suggest that CK-666 mitigates ferroptosis by directly eliminating lipid peroxidation. Importantly, CK-666 significantly ameliorated renal ischemia-reperfusion injury and ferroptosis in renal tissue, underscoring its potential therapeutic impact.

Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae.

Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site.

Mechanism of actin filament branch formation by Arp2/3 complex revealed by a high-resolution cryo-EM structureof the branch junction.

We reconstructed the structure of actin filament branch junctions formed by fission yeast Arp2/3 complex at 3.5 Å resolution from images collected by electron cryo-microscopy. During specimen preparation, all of the actin subunits and Arp3 hydrolyzed their bound adenosine triphosphate (ATP) and dissociated the γ-phosphate, but Arp2 retained the γ-phosphate. Binding tightly to the side of the mother filament and nucleating the daughter filament growing as a branch requires Arp2/3 complex to undergo a dramatic conformational change where two blocks of structure rotate relative to each other about 25° to align Arp2 and Arp3 as the first two subunits in the branch. During branch formation, Arp2/3 complex acquires more than 8,000 Å2 of new buried surface, accounting for the stability of the branch. Inactive Arp2/3 complex binds only transiently to the side of an actin filament, because its conformation allows only a subset of the interactions found in the branch junction.

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.

Molecular dynamics simulations support a multistep pathway for activation of branched actin filament nucleation by Arp2/3 complex.

Actin-related protein 2/3 complex (Arp2/3 complex) catalyzes the nucleation of branched actin filaments that push against membranes in processes like cellular motility and endocytosis. During activation by WASP proteins, the complex must bind WASP and engage the side of a pre-existing (mother) filament before a branched filament is nucleated. Recent high-resolution structures of activated Arp2/3 complex revealed two major sets of activating conformational changes. How these activating conformational changes are triggered by interactions of Arp2/3 complex with actin filaments and WASP remains unclear. Here we use a recent high-resolution structure of Arp2/3 complex at a branch junction to design all-atom molecular dynamics simulations that elucidate the pathway between the active and inactive states. We ran a total of ∼4.6 microseconds of both unbiased and steered all-atom molecular dynamics simulations starting from three different binding states, including Arp2/3 complex within a branch junction, bound only to a mother filament, and alone in solution. These simulations indicate that the contacts with the mother filament are mostly insensitive to the massive rigid body motion that moves Arp2 and Arp3 into a short pitch helical (filament-like) arrangement, suggesting actin filaments alone do not stimulate the short pitch conformational change. In contrast, contacts with the mother filament stabilize subunit flattening in Arp3, an intrasubunit change that converts Arp3 from a conformation that mimics an actin monomer to one that mimics a filamentous actin subunit. Our results support a multistep activation pathway that has important implications for understanding how WASP-mediated activation allows Arp2/3 complex to assemble force-producing actin networks.

The Rab GTPase-binding protein EHBP1L1 and its interactors CD2AP/CIN85 negatively regulate the length of primary cilia via actin remodeling.

Primary cilia are organelles consisting of axonemal microtubules and plasma membranes, and they protrude from the cell surface to the extracellular region and function in signal sensing and transduction. The integrity of cilia, including the length and structure, is associated with signaling functions; however, factors involved in regulating the integrity of cilia have not been fully elucidated. Here, we showed that the Rab GTPase-binding protein EHBP1L1 and its newly identified interactors CD2AP and CIN85, known as adaptor proteins of actin regulators, are involved in ciliary length control. Immunofluorescence microscopy showed that EHBP1L1 and CD2AP/CIN85 are localized to the ciliary sheath. EHBP1L1 depletion caused mislocalization of CD2AP/CIN85, suggesting that CD2AP/CIN85 localization to the ciliary sheath is dependent on EHBP1L1. Additionally, we determined that EHBP1L1- and CD2AP/CIN85-depleted cells had elongated cilia. The aberrantly elongated cilia phenotype and the ciliary localization defect of CD2AP/CIN85 in EHBP1L1-depleted cells were rescued by the expression of WT EHBP1L1, although this was not observed in the CD2AP/CIN85-binding-deficient mutant, indicating that the EHBP1L1-CD2AP/CIN85 interaction is crucial for controlling ciliary length. Furthermore, EHBP1L1- and CD2AP/CIN85-depleted cells exhibited actin nucleation and branching defects around the ciliary base. Taken together, our data demonstrate that the EHBP1L1-CD2AP/CIN85 axis negatively regulates ciliary length via actin network remodeling around the basal body.

Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell-cell junctions.

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

Lamellipodia-like actin networks in cells lacking WAVE regulatory complex.

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP.

The stabilization of Arp2/3 complex generated actin filaments.

The Arp2/3 complex, which generates both branched but also linear actin filaments via activation of SPIN90, is evolutionarily conserved in eukaryotes. Several factors regulate the stability of filaments generated by the Arp2/3 complex to maintain the dynamics and architecture of actin networks. In this review, we summarise recent studies on the molecular mechanisms governing the tuning of Arp2/3 complex nucleated actin filaments, which includes investigations using microfluidics and single-molecule imaging to reveal the mechanosensitivity, dissociation and regeneration of actin branches. We also discuss the high-resolution cryo-EM structure of cortactin bound to actin branches, as well as the differences and similarities between the stability of Arp2/3 complex nucleated branches and linear filaments. These new studies provide a clearer picture of the stabilisation of Arp2/3 nucleated filaments at the molecular level. We also identified gaps in our understanding of how different factors collectively contribute to the stabilisation of Arp2/3 complex-generated actin networks.

Plant KASH proteins SINE1 and SINE2 have synergistic and antagonistic interactions with actin-branching and actin-bundling factors.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of outer nuclear membrane KASH proteins, interacting in the nuclear envelope lumen with inner nuclear membrane SUN proteins and connecting the nucleus and cytoskeleton. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light-dark transitions and abscisic acid (ABA), which requires F-actin reorganization. SINE2 influences actin depolymerization and SINE1 actin repolymerization. The actin-related protein 2/3 (ARP2/3) complex, an actin nucleator, and the plant actin-bundling and -stabilizing factor SCAB1 are involved in stomatal aperture control. Here, we have tested the genetic interaction of SINE1 and SINE2 with SCAB1 and the ARP2/3 complex. We show that SINE1 and the ARP2/3 complex function in the same pathway during ABA-induced stomatal closure, while SINE2 and the ARP2/3 complex play opposing roles. The actin repolymerization defect observed in sine1-1 is partially rescued in scab1-2 sine1-1, while SINE2 is epistatic to SCAB1. In addition, SINE1 and ARP2/3 act synergistically in lateral root development. The absence of SINE2 renders trichome development independent of the ARP2/3 complex. Together, these data reveal complex and differential interactions of the two KASH proteins with the actin-remodeling apparatus and add evidence to the proposed differential role of SINE1 and SINE2 in actin dynamics.

Disulfidptosis: a novel cell death modality induced by actin cytoskeleton collapse and a promising target for cancer therapeutics.

Disulfidptosis is a novel discovered form of programmed cell death (PCD) that diverges from apoptosis, necroptosis, ferroptosis, and cuproptosis, stemming from disulfide stress-induced cytoskeletal collapse. In cancer cells exhibiting heightened expression of the solute carrier family 7 member 11 (SLC7A11), excessive cystine importation and reduction will deplete nicotinamide adenine dinucleotide phosphate (NADPH) under glucose deprivation, followed by an increase in intracellular disulfide stress and aberrant disulfide bond formation within actin networks, ultimately culminating in cytoskeletal collapse and disulfidptosis. Disulfidptosis involves crucial physiological processes in eukaryotic cells, such as cystine and glucose uptake, NADPH metabolism, and actin dynamics. The Rac1-WRC pathway-mediated actin polymerization is also implicated in this cell death due to its contribution to disulfide bond formation. However, the precise mechanisms underlying disulfidptosis and its role in tumors are not well understood. This is probably due to the multifaceted functionalities of SLC7A11 within cells and the complexities of the downstream pathways driving disulfidptosis. This review describes the critical roles of SLC7A11 in cells and summarizes recent research advancements in the potential pathways of disulfidptosis. Moreover, the less-studied aspects of this newly discovered cell death process are highlighted to stimulate further investigations in this field.

SIK2 orchestrates actin-dependent host response upon Salmonella infection.

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.