Cellular Regulation of Macropinocytosis.

Interest in macropinocytosis has risen in recent years owing to its function in tumorigenesis, immune reaction, and viral infection. Cancer cells utilize macropinocytosis to acquire nutrients to support their uncontrolled proliferation and energy consumption. Macropinocytosis, a highly dynamic endocytic and vesicular process, is regulated by a series of cellular signaling pathways. The activation of small GTPases in conjunction with phosphoinositide signaling pivotally regulates the process of macropinocytosis. In this review, we summarize important findings about the regulation of macropinocytosis and provide information to increase our understanding of the regulatory mechanism underlying it.

Architecture of RabL2-associated complexes at the ciliary base: A structural modeling perspective: Deciphering the structural organization of ciliary RabL2 complexes.

Cilia are slender, micrometer-long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so-called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N-terminal domain of CEP43 forms a homo-dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N-terminal domain of CEP43 from the C-terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2-CEP19 associates with the IFT-B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT-B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled-coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.

Targeting guanine nucleotide exchange factors for novel cancer drug discovery.

INTRODUCTION: Guanine nucleotide exchange factors (GEFs) regulate the activation of small GTPases (G proteins) of the Ras superfamily proteins controlling cellular functions. Ras superfamily proteins act as 'molecular switches' that are turned 'ON' by guanine exchange. There are five major groups of Ras family GTPases: Ras, Ran, Rho, Rab and Arf, with a variety of different GEFs regulating their GTP loading. GEFs have been implicated in various diseases including cancer. This makes GEFs attractive targets to modulate signaling networks controlled by small GTPases. AREAS COVERED: In this review, the roles and mechanisms of GEFs in malignancy are outlined. The mechanism of guanine exchange activity by GEFs on a small GTPase is illustrated. Then, some examples of GEFs that are significant in cancer are presented with a discussion on recent progress in therapeutic targeting efforts using a variety of approaches. EXPERT OPINION: Recently, GEFs have emerged as potential therapeutic targets for novel cancer drug development. Targeting small GTPases is challenging; thus, targeting their activation by GEFs is a promising strategy. Most GEF-targeted drugs are still in preclinical development. A deeper biological understanding of the underlying mechanisms of GEF activity and utilizing advanced technology are necessary to enhance drug discovery for GEFs in cancer.

Regulation of the RhoA exchange factor GEF-H1 by pro-fibrotic stimuli through a positive feedback loop involving RhoA, MRTF and Sp1.

RhoA and its effectors, the transcriptional coactivators Myocardin-Related Transcription Factor (MRTF) and Serum Response Factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor-α or transforming growth factor ß1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling and MRTF, indicative of a positive feed-back cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Importantly, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. Since the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.

From disorder comes function: Regulation of small GTPase function by intrinsically disordered lipidated membrane anchor.

The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs).

Programming Spatial Cell Sorting by Engineering Cadherin Intracellular Activity.

The spatial sorting of cells into appropriate tissue compartments is essential for embryogenesis and tissue development. Spatial cell sorting is controlled by the interplay between cell surface affinity and intracellular mechanical properties. However, intracellular signaling that can sufficiently sort cell populations remains unexplored. In this study, we engineered chimeric cadherins by replacing the cadherin intracellular domain with cytoskeletal regulators to test their ability to induce spatial cell sorting. Using a fibroblast-based reconstitution system, we observed that Rac1 and RhoA activity in the cadherin tail induced outward and inward sorting, respectively. In particular, RhoA activity embedded cells toward the inside of E-cadherin-expressing spheroids and tumor spheroids, leading to tissue invagination. Despite the simplicity of chimeric cadherin design, our results indicate that differences in cadherin intracellular activities can determine the direction of spatial cell sorting, even when cell surface affinity is not different, and provide new molecular tools to engineer tissue architectures.

Vesicle trafficking pathways in defence-related cell wall modifications: papillae and encasements.

Filamentous pathogens that cause plant diseases such as powdery mildew, rust, anthracnose, and late blight continue to represent an enormous challenge for farmers worldwide. Interestingly, these pathogens, although phylogenetically distant, initiate pathogenesis in a very similar way by penetrating the cell wall and establishing a feeding structure inside the plant host cell. To prevent pathogen ingress, the host cell responds by forming defence structures known as papillae and encasements that are thought to mediate pre- and post-invasive immunity, respectively. This form of defence is evolutionarily conserved in land plants and is highly effective and durable against a broad selection of non-adapted filamentous pathogens. As most pathogens have evolved strategies to overcome the defences of only a limited range of host plants, the papilla/encasement response could hold the potential to become an optimal transfer of resistance from one plant species to another. In this review I lay out current knowledge of the involvement of membrane trafficking that forms these important defence structures and highlight some of the questions that still need to be resolved.

Crystal structure of NRAS Q61K with a ligand-induced pocket near switch II.

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K.

κB-Ras proteins are fast-exchanging GTPases and function via nucleotide-independent binding of Ral GTPase-activating protein complexes.

κB-Ras (NF-κB inhibitor-interacting Ras-like protein) GTPases are small Ras-like GTPases but harbor interesting differences in important sequence motifs. They act in a tumor-suppressive manner as negative regulators of Ral (Ras-like) GTPase and NF-κB signaling, but little is known about their mode of function. Here, we demonstrate that, in contrast to predictions based on primary structure, κB-Ras GTPases possess hydrolytic activity. Combined with low nucleotide affinity, this renders them fast-cycling GTPases that are predominantly GTP-bound in cells. We characterize the impact of κB-Ras mutations occurring in tumors and demonstrate that nucleotide binding affects κB-Ras stability but is not strictly required for RalGAP (Ral GTPase-activating protein) binding. This demonstrates that κB-Ras control of RalGAP/Ral signaling occurs in a nucleotide-binding- and switch-independent fashion.

Molecular and cellular consequences of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD.

Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase.

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

Ras superfamily GTPases and signal transduction in Euglena gracilis.

Biological complexity is challenging to define, but can be considered through one or more features, including overall genome size, number of genes, morphological features, multicellularity, number of life cycle stages and the ability to adapt to different environments. Euglena gracilis meets several of these criteria, with a large genome of ∼38,000 protein coding genes and a considerable ability to survive under many different conditions, some of which can be described as challenging or harsh. Potential molecular exemplars of complexity tying these aspects together are signalling pathways, including GTPases, kinases and ubiquitylation, which increase the functionality of the gene-encoded proteome manyfold. Each of these examples can modulate both protein activity and gene expression. To address the connection between genome size and complexity I have undertaken a brief, and somewhat qualitative, survey of the small ras-like GTPase superfamily of E. gracilis. Unexpectedly, apart from Rab-GTPases which control intracellular transport and organelle identify, the size of the GTPase cohort is modest, and, for example, has not scaled with gene number when compared to the close relatives, trypanosomatids. I suggest that understanding the functions of this protein family will be vital to uncovering the complexity of E. gracilis biology.

Cleft palate and minor metabolic disturbances in a mouse global Arl15 gene knockout.

ARL15, a small GTPase protein, was linked to metabolic traits in association studies. We aimed to test the Arl15 gene as a functional candidate for metabolic traits in the mouse. CRISPR/Cas9 germline knockout (KO) of Arl15 showed that homozygotes were postnatal lethal and exhibited a complete cleft palate (CP). Also, decreased cell migration was observed from Arl15 KO mouse embryonic fibroblasts (MEFs). Metabolic phenotyping of heterozygotes showed that females had reduced fat mass on a chow diet from 14 weeks of age. Mild body composition phenotypes were also observed in heterozygous mice on a high-fat diet (HFD)/low-fat diet (LFD). Females on a HFD showed reduced body weight, gonadal fat depot weight and brown adipose tissue (BAT) weight. In contrast, in the LFD group, females showed increased bone mineral density (BMD), while males showed a trend toward reduced BMD. Clinical biochemistry analysis of plasma on HFD showed transient lower adiponectin at 20 weeks of age in females. Urinary and plasma Mg2+ concentrations were not significantly different. Our phenotyping data showed that Arl15 is essential for craniofacial development. Adult metabolic phenotyping revealed potential roles in brown adipose tissue and bone development.

Pitstop-2 and its novel derivative RVD-127 disrupt global cell dynamics and nuclear pores integrity by direct interaction with small GTPases.

Clathrin-mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop-2 as a potent CME inhibitor, we and others have reported on substantial clathrin-independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop-2, termed RVD-127, to clarify Pitstop-2 diverse effects. Using RVD-127, we were able to trace additional protein targets of Pitstop-2. Besides inhibiting CME, Pitstop-2 and RVD-127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)-like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop-2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop-2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop-2 and RVD-127 open up novel avenues.

Gi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gßγ/PKCα/FARP1/Cdc42 axis.

The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gßγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C ß3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.

Dispensable role of Rac1 and Rac3 after cochlear hair cell specification.

Rac small GTPases play important roles during embryonic development of the inner ear; however, little is known regarding their function in cochlear hair cells (HCs) after specification. Here, we revealed the localization and activation of Racs in cochlear HCs using GFP-tagged Rac plasmids and transgenic mice expressing a Rac1-fluorescence resonance energy transfer (FRET) biosensor. Furthermore, we employed Rac1-knockout (Rac1-KO, Atoh1-Cre;Rac1flox/flox) and Rac1 and Rac3 double KO (Rac1/Rac3-DKO, Atoh1-Cre;Rac1flox/flox;Rac3-/-) mice, under the control of the Atoh1 promoter. However, both Rac1-KO and Rac1/Rac3-DKO mice exhibited normal cochlear HC morphology at 13 weeks of age and normal hearing function at 24 weeks of age. No hearing vulnerability was observed in young adult (6-week-old) Rac1/Rac3-DKO mice even after intense noise exposure. Consistent with prior reports, the results from Atoh1-Cre;tdTomato mice confirmed that the Atoh1 promoter became functional only after embryonic day 14 when the sensory HC precursors exit the cell cycle. Taken together, these findings indicate that although Rac1 and Rac3 contribute to the early development of sensory epithelia in cochleae, as previously shown, they are dispensable for the maturation of cochlear HCs in the postmitotic state or for hearing maintenance following HC maturation. KEY MESSAGES: Mice with Rac1 and Rac3 deletion were generated after HC specification. Knockout mice exhibit normal cochlear hair cell morphology and hearing. Racs are dispensable for hair cells in the postmitotic state after specification. Racs are dispensable for hearing maintenance after HC maturation.

The protein kinases of Dictyostelia and their incorporation into a signalome.

Protein kinases are major regulators of cellular processes, but the roles of most kinases remain unresolved. Dictyostelid social amoebas have been useful in identifying functions for 30% of its kinases in cell migration, cytokinesis, vesicle trafficking, gene regulation and other processes but their upstream regulators and downstream effectors are mostly unknown. Comparative genomics can assist to distinguish between genes involved in deeply conserved core processes and those involved in species-specific innovations, while co-expression of genes as evident from comparative transcriptomics can provide cues to the protein complement of regulatory networks. Genomes and developmental and cell-type specific transcriptomes are available for species that span the 0.5 billion years of evolution of Dictyostelia from their unicellular ancestors. In this work we analysed conservation and change in the abundance, functional domain architecture and developmental regulation of protein kinases across the 4 major taxon groups of Dictyostelia. All data are summarized in annotated phylogenetic trees of the kinase subtypes and accompanied by functional information of all kinases that were experimentally studied. We detected 393 different protein kinase domains across the five studied genomes, of which 212 were fully conserved. Conservation was highest (71%) in the previously defined AGC, CAMK, CK1, CMCG, STE and TKL groups and lowest (26%) in the "other" group of typical protein kinases. This was mostly due to species-specific single gene amplification of "other" kinases. Apart from the AFK and α-kinases, the atypical protein kinases, such as the PIKK and histidine kinases were also almost fully conserved. The phylogeny-wide developmental and cell-type specific expression profiles of the protein kinase genes were combined with profiles from the same transcriptomic experiments for the families of G-protein coupled receptors, small GTPases and their GEFs and GAPs, the transcription factors and for all genes that upon lesion generate a developmental defect. This dataset was subjected to hierarchical clustering to identify clusters of co-expressed genes that potentially act together in a signalling network. The work provides a valuable resource that allows researchers to identify protein kinases and other regulatory proteins that are likely to act as intermediates in a network of interest.

Neuronal Exosomes as a New Signaling System.

Number of studies devoted to investigation of neuronal exosomes increases significantly each year. Potential of exosomes as diagnostic markers of neurodegenerative diseases has been examined thoroughly and similar protocols were used to search for the markers of other psychiatric disorders. Biogenesis of exosomes in various types of cells has been studied, physiological role of exosomes has been actively investigated, and many features of their signaling cascades have been clarified. The accumulated data indicate important role of the exosome signaling in interneuronal communication. Do we have enough grounds to recognize exosomes as new non-canonical neurotransmitters in the brain? In this review we discuss this issue and present a concept on the possible role of brain exosomes as a new signaling system to the scientific community.

Rab10 regulates the sorting of internalised TrkB for retrograde axonal transport.

Neurons process real-time information from axon terminals to coordinate gene expression, growth, and plasticity. Inputs from distal axons are encoded as a stream of endocytic organelles, termed signalling endosomes, targeted to the soma. Formation of these organelles depends on target-derived molecules, such as brain-derived neurotrophic factor (BDNF), which is recognised by TrkB receptors on the plasma membrane, endocytosed, and transported to the cell body along the microtubules network. Notwithstanding its physiological and neuropathological importance, the mechanism controlling the sorting of TrkB to signalling endosomes is currently unknown. In this work, we use primary mouse neurons to uncover the small GTPase Rab10 as critical for TrkB sorting and propagation of BDNF signalling from axon terminals to the soma. Our data demonstrate that Rab10 defines a novel membrane compartment that is rapidly mobilised towards the axon terminal upon BDNF stimulation, enabling the axon to fine-tune retrograde signalling depending on BDNF availability at the synapse. These results help clarifying the neuroprotective phenotype recently associated to Rab10 polymorphisms in Alzheimer's disease and provide a new therapeutic target to halt neurodegeneration.

Integrin receptor trafficking in health and disease.

Integrins are a family of 24 different heterodimers that are indispensable for multicellular life. Cell polarity, adhesion and migration are controlled by integrins delivered to the cell surface which in turn is regulated by the exo- and endocytic trafficking of integrins. The deep integration between trafficking and cell signaling determines the spatial and temporal output from any biochemical cue. Integrin trafficking plays a key role in development and many pathological conditions, especially cancer. Several novel regulators of integrin traffic have been discovered in recent times, including a novel class of integrin carrying vesicles, the intracellular nanovesicles (INVs). The tight regulation of trafficking pathways by cell signaling, where kinases phosphorylate key small GTPases in the trafficking pathway enable coordination of cell response to the extracellular milieu. Integrin heterodimer expression and trafficking differ in different tissues and contexts. In this Chapter, we discuss recent studies on integrin trafficking and its contribution to normal physiological and pathophysiological states.