Studying the role of Arp2/3 complex in cell migration

The actin cytoskeleton plays important roles in maintaining cell shape and mediating cell motility. Deregulation of actin dynamics occurs during pathological processes such as cancer metastasis, regeneration defects and developmental diseases. One specific form of actin cytoskeleton, the highly dynamic branched actin network, is critical for the formation and regulation of cell cortex and protrusions during cell migration and matrix degradation. The key nucleator for this branched actin network is the seven-protein Arp2/3 complex. Functional studies of Arp2/3 in vivo have been severely hampered by effects on viability observed upon loss of this complex in a variety of organisms. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner. Arp2/3-branched actin is critical for cell morphology and migration. However, perturbations and diseases affecting this network have phenotypes that cannot be fully explained by cell-autonomous effects. Using this stable Arp2/3 knockdown cell line, we also report the non-autonomous effects upon Arp2/3 depletion. We show that the main class of genes with altered expression levels was genes encoding secreted factors including chemokines, growth factors and matrix metaloproteases resembling the senescence associated secretory phenotype (SASP). These factors affect EGF chemotaxis in a non-autonomous way, resolving the recent contradictions about the role of Arp2/3 in chemotaxis. We indicate that these genes are targeted by NF-κB, via a CCM2-MEKK3 pathway that has been implicated in osmotic stress signaling. Thus, perturbations of Arp2/3 have potential non-autonomous effects which should be considered when evaluating diseases affecting the Arp2/3-actin cytoskeleton. Our work has provided strong evidence that Arp2/3 complex is critical for lamellipodia and revealed the roles of Arp2/3-branched actin in cell-matrix interaction. We have also indentified and characterized novel non-cell-autonomous effects of perturbing the branched actin network which has potential clinical relevance.Doctor of Philosoph

RPL23 Links Oncogenic RAS Signaling to p53-Mediated Tumor Suppression

The ribosomal protein (RP)-MDM2 interaction is a p53 response pathway critical for preventing oncogenic c-MYC-induced tumorigenesis. To investigate whether the RP-MDM2-p53 pathway is a broad anti-oncogenic mechanism, we crossed mice bearing an MDM2C305F mutation, which disrupts RPL11 binding to MDM2, with mice expressing an oncogenic HrasG12V transgene. Interestingly, the MDM2C305F mutant mice, which are hypersensitive to c-MYC-induced tumorigenesis, are not hypersensitive to oncogenic HrasG12V-induced tumorigenesis. Unlike c-MYC, which induces expression of RPL11, RAS overexpression leads to an increase in RPL23 mRNA and protein while RPL11 expression remains unchanged. The induction of RPL23 involves both MEK and PI3K signaling pathways and requires mTOR function. Increased expression of RPL23, which maintains binding to MDM2C305F mutant, correlates with increased p53 expression in MDM2C305F cells. Furthermore, RAS overexpression can induce p53 in the absence of p19ARF, and the induction can be abolished by down-regulation of RPL23. Thus, while the RPL11-MDM2-p53 pathway coordinates with the p19ARF-MDM2-p53 pathway against oncogenic c-MYC-induced tumorigenesis, the RPL23-MDM2-p53 pathway coordinates with the p19ARF-MDM2-p53 pathway against oncogenic RAS-induced tumorigenesis

Multi-Grained Named Entity Recognition

This paper presents a novel framework, MGNER, for Multi-Grained Named Entity Recognition where multiple entities or entity mentions in a sentence could be non-overlapping or totally nested. Different from traditional approaches regarding NER as a sequential labeling task and annotate entities consecutively, MGNER detects and recognizes entities on multiple granularities: it is able to recognize named entities without explicitly assuming non-overlapping or totally nested structures. MGNER consists of a Detector that examines all possible word segments and a Classifier that categorizes entities. In addition, contextual information and a self-attention mechanism are utilized throughout the framework to improve the NER performance. Experimental results show that MGNER outperforms current state-of-the-art baselines up to 4.4% in terms of the F1 score among nested/non-overlapping NER tasks.Comment: In ACL 2019 as a long pape

Arp2/3 Is Critical for Lamellipodia and Response to Extracellular Matrix Cues but Is Dispensable for Chemotaxis

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner

Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes

Profilin-1 Serves as a Gatekeeper for Actin Assembly by Arp2/3-Dependent and -Independent Pathways

Cells contain multiple F-actin assembly pathways including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay/competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2−/− cells lack lamellipodia and migrate slower than WT cells, but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2−/− cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP, but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. These observations suggest that antagonism of the Arp2/3 complex by profilin-1 in cells maintains actin homeostasis by balancing Arp2/3 complex-dependent and independent actin assembly pathways

Loss of Arp2/3 induces an NF-κB–dependent, nonautonomous effect on chemotactic signaling

A decrease in Arp2/3 levels results in an NF-κB–dependent increase in the expression of several secreted factors, resulting in nonautonomous effects on chemotaxis.Arp2/3-branched actin is critical for cytoskeletal dynamics and cell migration. However, perturbations and diseases affecting this network have phenotypes that cannot be fully explained by cell-autonomous effects. In this paper, we report nonautonomous effects of Arp2/3 depletion. We show that, upon Arp2/3 depletion, the expression of numerous genes encoding secreted factors, including chemokines, growth factors, and matrix metalloproteases, was increased, a signature resembling the senescence-associated secretory phenotype. These factors affected epidermal growth factor chemotaxis in a nonautonomous way, resolving the recent contradictions about the role of Arp2/3 in chemotaxis. We demonstrate that these genes were activated by nuclear factor κB via a CCM2–MEKK3 pathway that has been implicated in hyperosmotic stress signaling. Consistent with this, Arp2/3-depleted cells showed misregulation of volume control and reduced actin in the submembranous cortex. The defects in osmotic signaling in the Arp2/3-depleted cells can be rescued by hypoosmotic treatment. Thus, perturbations of Arp2/3 have nonautonomous effects that should be considered when evaluating experimental manipulations and diseases affecting the Arp2/3-actin cytoskeleton