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The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Large-scale chromosomal translocations are frequent oncogenic drivers in acute myeloid leukemia (AML). These translocations often occur in critical transcriptional/epigenetic regulators and contribute to malignant cell growth through alteration of normal gene expression. Despite this knowledge, the specific gene expression alterations that contribute to the development of leukemia remain incompletely understood. Here, through characterization of transcriptional regulation by the RUNX1-ETO fusion protein, we have identified Ras-association domain family member 2 (RASSF2) as a critical gene that is aberrantly transcriptionally repressed in t(8;21)-associated AML. Re-expression of RASSF2 specifically inhibits t(8;21) AML development in multiple models. Through biochemical and functional studies, we demonstrate RASSF2-mediated functions to be dependent on interaction with Hippo kinases, MST1 and MST2, but independent of canonical Hippo pathway signaling. Using proximity-based biotin labeling we define the RASSF2-proximal proteome in leukemia cells and reveal association with Rac GTPase-related proteins, including an interaction with the guanine nucleotide exchange factor, DOCK2. Importantly, RASSF2 knockdown impairs Rac GTPase activation, and RASSF2 expression is broadly correlated with Rac-mediated signal transduction in AML patients. Together, these data reveal a previously unappreciated mechanistic link between RASSF2, Hippo kinases, and Rac activity with potentially broad functional consequences in leukemia.

Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.

Gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are heterodimers of a common α subunit and unique β subunits. Regulation of their levels, primarily by GnRH, is critical for reproductive function. Several other hormones modulate gonadotropin expression, either independently or by modifying the responsiveness to GnRH. Pituitary adenylate cyclase activating peptide (PACAP) is one such hormone. Four-hour treatment of female mouse primary pituitary cells by either GnRH or PACAP induced FSHβ expression, while 24-h treatment repressed FSHβ. Both PACAP and GnRH caused FSH secretion into the medium. In the gonadotropes, PACAP activates primarily Gαs and increases concentration of cAMP, while GnRH primarily functions via Gαq and increases calcium concentration. Herein, we compared PACAP and GnRH signaling pathways that lead to the induction of FSHβ expression. Interestingly, constitutively active Gαs represses LHβ and induces FSHβ expression, while Gαq induces both β-subunits. We determined that FSHβ induction by PACAP requires functional EPAC, a cAMP sensor protein that serves as a guanine exchange factors for small G proteins that then bridges cAMP signaling to MAPK pathway. We further demonstrate that in addition to the prototypical small G protein Ras, two members of the Rho subfamily, Rac and CDC42 are also necessary for PACAP induction of FSHβ, likely via activation of p38 MAPK that leads to induction of cFOS, a critical transcription factor that is necessary and sufficient for FSHβ induction. Therefore, PACAP-induced cAMP pathway leads to MAPK activation that stimulates cFOS induction, to induce the expression of FSHβ subunit and increase FSH concentration.

During embryogenesis, the germ layers, including the endoderm, undergo convergence and extension movements to narrow and elongate the body plan. In zebrafish, the dorsal migration of endodermal cells during gastrulation is controlled by chemokine signaling, but little is known about how they migrate during segmentation. Here, we show that glypican 4 (Gpc4), a member of the heparin sulfate proteoglycan family, is required for efficient migration of anterior endodermal cells during early segmentation, regulating Rac activation to maintain polarized actin-rich lamellipodia. An endoderm transplantation assay showed that Gpc4 regulates endoderm migration in a non-cell-autonomous fashion. Further analyses revealed that the impaired endoderm migration in gpc4 mutants results from increases in the expression and assembly of fibronectin and laminin, major components of the extracellular matrix (ECM). Notably, we found that matrix metalloproteinase 14 (Mmp14a/b) is required for the control of ECM expression during endoderm migration, with Gpc4 acting through Mmp14a/b to limit ECM expression. Our results suggest that Gpc4 is crucial for generating the environment required for efficient migration of endodermal cells, uncovering a novel function of Gpc4 during development.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.

Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades.

New methods are needed to reduce dynamic blebbing which inhibits cell attachment and survival during passaging of pluripotent stem cells. We tested the hypothesis that activation of the P2X7 receptor by extracellular ATP during passaging initiates dynamic blebbing. The P2X7 receptor was present in human embryonic stem cells (hESC), but not in differentiating cells. Extracellular ATP concentrations were 14× higher in medium during passaging. Addition of ATP to culture medium prolonged dynamic blebbing and inhibited attachment. Inhibition of P2X7 by specific drugs or by siRNA significantly reduced dynamic blebbing and improved cell attachment. When cells were incubated in calcium chelators (EGTA or BAPTA), blebbing decreased and attachment improved. Calcium influx was observed using Fura-4 when ATP was added to culture medium and inhibited in the presence of the P2X7 inhibitor. Over-expressing activated Rac in hESC reduced blebbing and promoted cell attachment, while a Rac inhibitor prolonged blebbing and reduced attachment. These data identify a pathway involving P2X7 that initiates and prolongs dynamic blebbing during hESC passaging. This pathway provides new insight into factors that increase dynamic blebbing and identifies new targets, such as P2X7, that can be used to improve the culture of cells with therapeutic potential.

BackgroundThere have been a number of candidate gene association studies of cancer cachexia-related traits, but no genome-wide association study (GWAS) has been published to date. Cachexia presents in patients with a number of complex traits, including both cancer and COPD. The objective of the current investigation was to search for a shared genetic aetiology for change in body mass index (ΔBMI) among cancer and COPD by using GWAS data in the Framingham Heart Study.MethodsA linear mixed effects model accounting for age, sex, and change in smoking status was used to calculate ΔBMI in participants over 40 years of age with three consecutive BMI time points (n = 4162). Four GWAS of ΔBMI using generalized estimating equations were performed among 1085 participants with a cancer diagnosis, 204 with gastrointestinal (GI) cancer, 112 with lung cancer, and 237 with COPD to test for association with 418 365 single-nucleotide polymorphisms (SNPs).ResultsTwo SNPs reached a level of genome-wide significance (P

Hirschsprung disease (HSCR) is a congenital disorder caused by the defective function of the embryonic enteric neural crest. The impaired migration of embryonic enteric neural crest plays an important role in the pathogenesis of this disease. Recent studies showed that the ARP2/3 complex and RAC isoforms had effects on actin cytoskeleton remodelling, which contributes to migration. Moreover, some regulatory relationships were identified between ARP2/3 complex and RAC isoforms. Although microRNAs (miRNAs) have been known to modulate target gene expression on the post-transcriptional level, little is known about the regulation among miRNAs, ARP2/3 complex and RAC isoforms. Here, we report that down-regulation of ARP2 and ARP3, two main subunits of ARP2/3 complex, suppressed migration and proliferation in 293T and SH-SY5Y cell lines via the inhibition of RAC1 and RAC2. Meanwhile, as the target genes, ARP2 and ARP3 are reduced by increased miR-24-1* and let-7a*, respectively, in 70 HSCR samples as compared with 74 normal controls. Co-immunoprecipitation showed that aberrant reduction in ARP2 and ARP3 could weaken the function of ARP2/3 complex. Our study demonstrates that the miR-24-1*/let-7a*-ARP2/3 complex-RAC isoforms pathway may represent a novel pathogenic mechanism for HSCR.

Neutrophils and other amoeboid cells chemotax by steering their front ends towards chemoattractant. Although Ras, Rac, Cdc42 and RhoA small GTPases all regulate chemotaxis, it has been unclear how they spatiotemporally control polarization and steering. Using fluorescence biosensors in neutrophil-like PLB-985 cells and photorelease of chemoattractant, we show that local Cdc42 signals, but not those of Rac, RhoA or Ras, precede cell turning during chemotaxis. Furthermore, pre-existing local Cdc42 signals in morphologically unpolarized cells predict the future direction of movement on uniform stimulation. Moreover, inhibition of actin polymerization uncovers recurring local Cdc42 activity pulses, suggesting that Cdc42 has the excitable characteristic of the compass activity proposed in models of chemotaxis. Globally, Cdc42 antagonizes RhoA, and maintains a steep spatial activity gradient during migration, whereas Ras and Rac form shallow gradients. Thus, chemotactic steering and de novo polarization are both directed by locally excitable Cdc42 signals.

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching., Competing Interests: Declaration of interests L.L. is a member of the advisory board for Cell., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The molecular pathogenesis of acute myeloid leukemia (AML) was dramatically clarified over the latest two decades. Several important molecular markers were discovered in patients with AML that have helped to improve the risk stratification. However, developing new treatment strategies for relapsed/refractory acute myeloid leukemia (AML) is crucial due to its poor prognosis., Procedure: To overcome this difficulty, we performed an assay for transposase-accessible chromatin with sequencing (ATAC-seq) in 10 AML patients with various gene alterations. ATAC-seq is based on direct in vitro sequencing adaptor transposition into native chromatin, and is a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq analysis revealed increased accessibility of the DOCK1 gene in patients with AML harboring poor prognostic factors. Following the ATAC-seq results, quantitative reverse transcription polymerase chain reaction was used to measure DOCK1 gene expression levels in 369 pediatric patients with de novo AML., Results: High DOCK1 expression was detected in 132 (37%) patients. The overall survival (OS) and event-free survival (EFS) among patients with high DOCK1 expression were significantly worse than those patients with low DOCK1 expression (3-year EFS: 34% vs. 60%, p < .001 and 3-year OS: 60% vs. 80%, p < .001). To investigate the significance of high DOCK1 gene expression, we transduced DOCK1 into MOLM14 cells, and revealed that cytarabine in combination with DOCK1 inhibitor reduced the viability of these leukemic cells., Conclusions: Our results indicate that a DOCK1 inhibitor might reinforce the effects of cytarabine and other anti-cancer agents in patients with AML with high DOCK1 expression., (© 2024 Wiley Periodicals LLC.)

During development, axons must integrate directional information encoded by multiple guidance cues and their receptors. Axon guidance receptors, such as UNC-40 (DCC) and SAX-3 (Robo), can function individually or combinatorially with other guidance receptors to regulate downstream effectors. However, little is known about the molecular mechanisms that mediate combinatorial guidance receptor signaling. Here, we show that UNC-40, SAX-3 and the SYD-1 RhoGAP-like protein function interdependently to regulate the MIG-2 (Rac) GTPase in the HSN axon of C. elegans. We find that SYD-1 mediates an UNC-6 (netrin) independent UNC-40 activity to promote ventral axon guidance. Genetic analysis suggests that SYD-1 function in axon guidance requires both UNC-40 and SAX-3 activity. Moreover, the cytoplasmic domains of UNC-40 and SAX-3 bind to SYD-1 and SYD-1 binds to and negatively regulates the MIG-2 (Rac) GTPase. We also find that the function of SYD-1 in axon guidance is mediated by its phylogenetically conserved C isoform, indicating that the role of SYD-1 in guidance is distinct from its previously described roles in synaptogenesis and axonal specification. Our observations reveal a molecular mechanism that can allow two guidance receptors to function interdependently to regulate a common downstream effector, providing a potential means for the integration of guidance signals.

BackgroundFcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis. It is well-known that Fcγ receptor (FcγR) crosslinking induces the tyrosine phosphorylation of CBL which is associated with FcγR mediated phagocytosis, however how signaling molecules coordinate to desensitize these receptors is unclear. An investigation of the mechanisms involved in receptor desensitization will provide new insight into potential mechanisms by which signaling molecules may downregulate tyrosine phosphorylation dependent signaling events to terminate important signaling processes.ResultsUsing the U937IF cell line, we observed that FcγR1 crosslinking induces the tyrosine phosphorylation of CBL, which is maximal at 5 min. followed by a kinetic pattern of dephosphorylation. An investigation of the mechanisms involved in receptor desensitization revealed that pretreatment of U937IF or J774 cells with PMA followed by Fcγ receptor crosslinking results in the reduced tyrosine phosphorylation of CBL and the abrogation of downstream signals, such as CBL-CRKL binding, Rac-GTP activation and the phagocytic response. Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response. We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation. The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S).ConclusionsOur results suggest a functional model by which PKC interacts with SHP1 to affect the phosphorylation state of CBL, the activation state of Rac and the negative regulation of ITAM signaling i.e. Fcγ receptor mediated phagocytosis. These findings suggest a mechanism for Fcγ receptor desensitization by which a serine-threonine kinase e.g. PKC downregulates tyrosine phosphorylation dependent signaling events via the reduced tyrosine phosphorylation of the complex adapter protein, CBL.

E-cadherin is a major homophilic cell-cell adhesion molecule that inhibits motility of individual cells on matrix. However, its contribution to migration of cells through cell-rich tissues is less clear. We developed an in vivo sensor of mechanical tension across E-cadherin molecules, which we combined with cell-type-specific RNAi, photoactivatable Rac, and morphodynamic profiling, to interrogate how E-cadherin contributes to collective migration of cells between other cells. Using the Drosophila ovary as a model, we found that adhesion between border cells and their substrate, the nurse cells, functions in a positive feedback loop with Rac and actin assembly to stabilize forward-directed protrusion and directionally persistent movement. Adhesion between individual border cells communicates direction from the lead cell to the followers. Adhesion between motile cells and polar cells holds the cluster together and polarizes each individual cell. Thus, E-cadherin is an integral component of the guidance mechanisms that orchestrate collective chemotaxis in vivo.

Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

Hippocampus-dependent learning and memory relies on synaptic plasticity as well as network adaptations provided by the addition of adult-born neurons. We have previously shown that activity-induced intracellular signaling through the Rho family small GTPase Rac1 is necessary in forebrain projection neurons for normal synaptic plasticity in vivo, and here we show that selective loss of neuronal Rac1 also impairs the learning-evoked increase in neurogenesis in the adult mouse hippocampus. Earlier work has indicated that experience elevates the abundance of adult-born neurons in the hippocampus primarily by enhancing the survival of neurons produced just before the learning event. Loss of Rac1 in mature projection neurons did reduce learning-evoked neurogenesis but, contrary to our expectations, these effects were not mediated by altering the survival of young neurons in the hippocampus. Instead, loss of neuronal Rac1 activation selectively impaired a learning-evoked increase in the proliferation and accumulation of neural precursors generated during the learning event itself. This indicates that experience-induced alterations in neurogenesis can be mechanistically resolved into two effects: (1) the well documented but Rac1-independent signaling cascade that enhances the survival of young postmitotic neurons; and (2) a previously unrecognized Rac1-dependent signaling cascade that stimulates the proliferative production and retention of new neurons generated during learning itself.

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1(+/-) and Atg7-deficient mammary epithelial cells (MECs) produced 'competent' apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment. Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance.

Recent genetic-based studies have implicated a number of immune-related genes in the pathogenesis of inflammatory bowel disease (IBD). Our recent genetic studies showed that RAC2 is associated with human IBD; however, its role in disease pathogenesis is unclear. Given Rac2's importance in various fundamental immune cell processes, we investigated whether a defect in Rac2 may impair host immune responses in the intestine and promote disease in the context of an infection-based (Citrobacter rodentium) model of colitis. In response to infection, Rac2(-/-) mice showed i) worsened clinical symptoms (days 13-18), ii) increased crypt hyperplasia at days 11 and 22 (a time when crypt hyperplasia was largely resolved in wild-type mice; WT), and iii) marked mononuclear cell infiltration characterized by higher numbers of T (CD3(+)) cells (day 22), compared to WT-infected mice. Moreover, splenocytes harvested from infected Rac2(-/-) mice and stimulated in vitro with C. rodentium lysate produced considerably higher levels of interferon-γ and interleukin-17A. The augmented responses observed in Rac2(-/-) mice did not appear to stem from Rac2's role in NADPH oxidase-driven reactive oxygen species production as no differences in crypt hyperplasia, nor inflammation, were observed in infected NOX2(-/-) mice compared to WT. Collectively, our findings demonstrate that Rac2(-/-) mice develop more severe disease when subjected to a C. rodentium-induced model of infectious colitis, and suggest that impaired Rac2 function may promote the development of IBD in humans.

Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts.

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin alpha subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans.

P21 activated kinase (PAK), PAK interacting exchange factor (PIX), and G protein coupled receptor kinase interactor (GIT) compose a highly conserved signaling module controlling cell migrations, immune system signaling, and the formation of the mammalian nervous system. Traditionally, this signaling module is thought to facilitate the function of RAC and CDC-42 GTPases by allowing for the recruitment of a GTPase effector (PAK), a GTPase activator (PIX), and a scaffolding protein (GIT) as a regulated signaling unit to specific subcellular locations. Instead, we report here that this signaling module functions independently of RAC/CDC-42 GTPases in vivo to control the cell shape and migration of the distal tip cells (DTCs) during morphogenesis of the Caenorhabditis elegans gonad. In addition, this RAC/CDC-42-independent PAK pathway functions in parallel to a classical GTPase/PAK pathway to control the guidance aspect of DTC migration. Among the C. elegans PAKs, only PAK-1 functions in the GIT/PIX/PAK pathway independently of RAC/CDC42 GTPases, while both PAK-1 and MAX-2 are redundantly utilized in the GTPase/PAK pathway. Both RAC/CDC42-dependent and -independent PAK pathways function with the integrin receptors, suggesting that signaling through integrins can control the morphology, movement, and guidance of DTC through discrete pathways. Collectively, our results define a new signaling capacity for the GIT/PIX/PAK module that is likely to be conserved in vertebrates and demonstrate that PAK family members, which are redundantly utilized as GTPase effectors, can act non-redundantly in pathways independent of these GTPases.

Cell-cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and beta(1)-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.

Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes), scaffolded by hematopoietic protein 1 (Hem-1), that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE)2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a) dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b) substantially weakens Rac activation and phosphatidylinositol-(3,4,5)-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5)-tris-phosphate)/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c) prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling.

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling.

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

Background: The effect of DOCK1 gene on the biological behavior of endometrial carcinoma cells and its related pathway has not been reported., Methods: The immunohistochemical method and western blot were utilized to analyze DOCK1 protein expression in endometrial tissues and cells, respectively. CCK-8, BrdU, transwell and flow cytometry were performed to analyze the effect of DOCK1 expression changes on the viability, proliferation, invasion, migration and apoptosis of endometrial cancer cells, respectively. The effects of DOCK1 gene on Bcl-2, MMP9, Ezrin, E-cadherin and c-RAF/ERK1/2 signaling pathway were evaluated by western blot. The xenograft models were constructed to analyze the effect of DOCK1 in vivo., Results: DOCK1 expression was increased in endometrial cancer tissues and cells compared with those in normal adjacent tissues and cells. DOCK1 knockout could inhibit the malignant biological behavior of endometrial cancer cells, while DOCK1 overexpression played the opposite effect. The expression of E-cadherin was upregulated and those of MMP9, Ezrin, Bcl-2, p-c-RAF (S338) and p-ERK1/2 (T202/Y204) were downregulated after DOCK1 knockout, while DOCK1 overexpression played the opposite effect. Additionally, Raf inhibitor LY3009120 reversed the function of DOCK1 on malignant biological behavior. In vivo experiment results showed that the growth and weight of transplanted tumors in nude mice were inhibited after DOCK1 knockout. The changes of E-cadherin, MMP9, Ezrin and Bcl-2 expressions in the transplanted tumors were consistent with those in vitro., Conclusion: DOCK1 could enhance the malignant biological behavior of endometrial cancer cells, which might be through c-RAF/ERK1/2 signaling pathways in vitro and in vivo., (© 2024. The Author(s).)

Cisplatin-based chemotherapy is considered the primary treatment option for patients with advanced bladder cancer (BCa). However, the objective response rate to chemotherapy is often unsatisfactory, leading to a poor 5-year survival rate. Furthermore, current strategies for evaluating chemotherapy response and prognosis are limited and inefficient. In this study, we aimed to address these challenges by establishing a chemotherapy response type gene (CRTG) signature consisting of 9 genes and verified the prognostic value of this signature using TCGA and GEO BCa cohorts. The risk scores based on the CRTG signature were found to be associated with advanced clinicopathological status and demonstrated favorable predictive power for chemotherapy response in the TCGA cohort. Meanwhile, tumors with high risk scores exhibited a tendency toward a "cold tumor" phenotype. These tumors showed a low abundance of T cells, CD8+ T cells and cytotoxic lymphocytes, along with a high abundance of cancer-associated fibroblasts. Moreover, they displayed higher mRNA levels of these immune checkpoints: CD200, CD276, CD44, NRP1, PDCD1LG2 (PD-L2), and TNFSF9. Furthermore, we developed a nomogram that integrated the CRTG signature with clinicopathologic risk factors. This nomogram proved to be a more effective tool for predicting the prognosis of BCa patients. Additionally, we identified Rac family small GTPase 3 (RAC3) as a biomarker in our model. RAC3 was found to be overexpressed in chemoresistant BCa tissues and enhance the chemotherapeutic resistance of BCa cells in vitro and in vivo by regulating the PAK1-ERK1/2 pathway. In conclusion, our study presents a novel CRTG model for predicting chemotherapy response and prognosis in BCa. We also highlight the potential of combining chemotherapy with immunotherapy as a promising strategy for chemoresistant BCa and that RAC3 might be a latent target for therapeutic intervention., (© 2023 Wiley Periodicals LLC.)

Background and Purpose: Bladder tumors are among the most prevalent malignancies in the urinary system, and RAC3 has been linked to various types of cancer. This article seeks to explore the potential of RAC3 as both an early diagnostic marker for bladder tumors and a novel therapeutic target., Methods/patients: The expression of RAC3 in bladder tissue was detected using immunohistochemical staining. Additionally, the protein expression of RAC3 was measured and quantified through enzyme-linked immunosorbent assay (ELISA). Subsequently, the correlation between the expression level of RAC3 and bladder tumors was investigated through multifactorial analysis and survival analysis., Results: Our findings revealed that RAC3 expression was upregulated in bladder tumor tissues. Moreover, we observed higher levels of RAC3 expression in the serum and urine of patients with bladder tumors compared to those with non-bladder tumors. Additionally, we identified a significant positive correlation between RAC3 expression levels and the stage, degree of differentiation, and infiltration of bladder tumors. Importantly, high RAC3 expression emerged as an influential factor in the poor prognosis of bladder tumors, as patients with high RAC3 expression exhibited a lower overall survival rate than those with low RAC3 expression., Conclusion: Based on our results, RAC3 shows promise as both a marker for early diagnosis of bladder tumors and a potential therapeutic target., (© 2023. The Author(s).)

Background: Paclitaxel, a tubulin-binding agent, is a Food and Drug Administration-approved first-line drug for the treatment of non-small cell lung cancer (NSCLC), for both squamous and non-squamous cell lung carcinoma, with paclitaxel/carboplatin + bevacizumab a common chemotherapy regimen for stage IV non-squamous NSCLC; however, primary or acquired resistance to paclitaxel is gradually increasing, leading to treatment failure., Methods: Our results show that Ras-related C3 botulinum toxin substrate 3 (RAC3) is overexpressed in cultured paclitaxel-resistant cells and that RAC3 expression levels are negatively correlated with sensitivity of lung adenocarcinoma cells to paclitaxel. Pulsatilla saponin D could inhibit RAC3 expression, and we hypothesize that it may block paclitaxel resistance. Further, we found that treatment with paclitaxel combined with Pulsatilla saponin D, can overcome lung adenocarcinoma cell resistance to paclitaxel alone in cell culture and mouse xenograft models., (© 2024. The Author(s).)

Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a "molecular switch" for the induction of cell migration and appears to contribute to the invasive property of tumors.

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.