Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

Background

The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by β-arrestins, βarr1 and βarr2, which control both their signalling and endocytosis, suggesting that βarrs may also function at primary cilium.

Methodology/Principal Findings

In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.

Conclusions/Significance

Our results show that βarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”.     

Collective invasion of carcinoma cells: When the fibroblasts take the lead

Epithelial to mesenchymal transitions (EMT) have been suggested to be crucial during epithelial cancer cell invasion. However, in a three-dimensional “organotypic” invasion assay squamous cell carcinoma (SCC) cells that retain epithelial characteristics “hitch a ride” with carcinoma associated fibroblasts (CAFs) in order to collectively invade. Thus epithelial cancer cells can utilise the mesenchymal characteristics of CAFs without the need to undergo EMT themselves. This work provides new insight in cancer cell invasion and shows a new role for CAFs as a target for an anti-invasive therapy.Key words: collective invasion, carcinoma associated fibroblast, extracellular matrix, matrix metalloproteinases, RhoCancer cell invasion and metastasis are the main causes of mortality in cancer patients. Understanding how cancer cells move and invade within the surrounding tissue is therefore a key issue. Stromal fibroblasts within a tumor play a crucial role in cancer cell proliferation, survival, angiogenesis as well as invasion (reviewed in ref. ¹). In many cases stromal CAFs are able to produce a wide range of growth factors and cytokines that modulate tumor growth and invasion.^2,3 Their influence in cancer cell invasion and metastasis can also be mediated through the production of MMP''s that promote extra-cellular matrix degradation.⁴It has recently been shown that CAFs can play an unexpected role in SCC invasion.⁵ In a 3D ‘organotypic’ model of invasion that recreates the epidermal/dermal environment CAFs promote the collective invasion of SCC cells.⁶ 3D time-lapse confocal microscopy imaging showed that CAFs were always the leading cell of the invading cohort with the SCC cells following behind. These cohorts closely resembled invading clusters of SCC cells observed in human cancer samples.⁷ CAFs promoted SCC cells collective invasion by remodelling the matrix and making a path that SCC cells can use to invade. This process is clearly shown in Figure 1: a CAF (in red) leads the invasion of a collective chain of SCC cells (green) and makes a path in the surrounding matrix, visualized in grey using confocal reflectance microscopy. Two key experiments helped to understand the role of fibroblasts in this system. Firstly, the separation of the two cell populations by a thin layer of gel without fibroblasts completely abolished SCC invasion and so ruled out the possibility of long distance chemoattractant molecules inducing SCC invasion. Secondly, SCC cells were able to invade into a gel which had previously been remodelled by CAFs that had subsequently been removed. Together these experiments showed that tracks made by the fibroblasts are essential and sufficient to promote collective carcinoma cells invasion. Heterotypic cell contact between both populations was not required, as SCC cells can invade using tracks made by the CAFs even if the CAFs have been removed.Open in a separate windowFigure 1Collective invasion of carcinoma cells led by fibroblast. Confocal time-lapse imaging of carcinoma associated fibroblast (red) leading the way of an invading chain of SCC cells (green) and making path into the surrounding matrix (grey). Panel is 80 x 80 mm and spans 300 minutes, scale are 20 um.Interestingly, inhibition of Rho/ROCK signalling to the actomyosin cytoskeleton or MMPs using small molecule inhibitors blocked SCC invasion even when only CAFs where targeted. Blocking these pathways in carcinoma cells had little or no effect on their invasion. Moreover, inhibition of Rho function specifically in CAFs did not block their invasion into matrices but prevented SCC cells from following. These experiments showed the role of Rho/ROCK and MMPs molecular pathways in track generation by the CAFs and that targeting these pathways in CAFs, but not SCC cells, is critical for preventing cancer invasion. Strikingly, blockade of protease function after CAFs had remodelled the ECM had little effect on the ability of SCC cells to invade. This could explain the relative poor results obtained using MMPs inhibitors as anti-invasive therapies.⁸ Rho/ROCK function was dispensable in SCC cells; however, depletion of the small GTPase Cdc42 and its effector MRCK disrupted the acto-myosin cortex of carcinoma cells and blocked their capacity to invade in response to CAFs.In order to invade and metastasise, carcinoma cells can switch from an epithelial state to a more mesenchymal phenotype.⁹ This process, called EMT, allows epithelial cancer cells to adapt their behaviour and confers the capacity to remodel the ECM on the cancer cells.¹⁰ However, in patient tissue samples, it has been observed that carcinoma cells can invade without undergoing an EMT, these cancer cells do not upregulate mesenchymal markers and retain cell to cell contact during their invasion.¹¹ This work explains how carcinoma cells that have not undergone EMT could invade a 3D matrix. These cells use the mesenchymal characteristics of the stromal fibroblasts to remodel the ECM and consequently follow behind invading fibroblasts. In tumours of mesenchymal origin CAFs are not required for invasion; work from Friedl and colleagues, clearly shows that HT1080 fibrosarcoma cells could lead collectively invading chains of cancer cells The authors showed how the leading cell of the collective chain remodels collagen fibres into tracks as it invades through the action of MT1-MMP (MMP14).¹²In normal conditions, epithelial cells and dermal fibroblasts are in complete homeostasis and separated by a basement membrane (Fig. 2A). In addition, normal dermal fibroblasts are unable to promote SCC invasion. Understanding how CAFs are activated will be an important step forward. A desmoplastic response is observed in many tumours indicating a change in behaviour of fibroblasts.¹³ During wound healing or fibrosis, fibroblasts are in an active state that has been suggested to be similar to cancer activation.¹⁴ TGFβ has been shown to be a key player in fibroblasts activation and could support cancer progression.¹⁵ However, TGFβ was not responsible for SCC cells invasion since a TGFβ inhibitor had no effect in carcinoma cells collective invasion induced by the CAFs in the 3D invasion assay (Cedric Gaggioli and Steven Hooper, unpublished data). Interestingly, a probe that binds only to the active form of the small GTPase Rho showed that the activity of this protein was increased in CAFs compared to normal fibroblasts in tissue samples. Elevated expression of α5 integrin was also present in these cells and this has been implicated in Rho activation in a number of systems.^16–18 Consistent with this observation, depletion of integrin a5 in CAFs reduced their ability to promote the invasion of SCC cells. Alternatively, CAFs could also be derived from endothelial cells through a process called endothelial to mesenchymal transition¹⁹ (EndMT), or from cancer cells through EMT.²⁰ These processes could be responsible for CAFs generation in the tumor stroma resulting in matrix remodelling and tracks generation in order for the carcinoma cells to collectively invade the surrounding tissue and metastasize (Fig. 2B).Open in a separate windowFigure 2Model of carcinoma cells collective invasion. (A) Schematic representation of a normal epithelium. Epithelial cells (light blue) and normal fibroblasts (pink) are separated by a basal membrane and are in a perfect homeostasis. Cross talk between both cell types occurs through adhesion and chemokine secretion. (B) Schematic representation of carcinoma cells collective invasion. CAFs (red) take the lead of a collective invading chain of SCC cells (brown). Invasion of CAFs is MMPs dependent but Rho/ROCK independent. However, track generation by CAFs is Rho/ROCK/MLC dependent. SCC cells require the small GTPase Cdc42 and its effector MRCK in order to collectively invade trough those tracks (black).This study opens a new field of investigation for collective cancer cell invasion. This work highlights carcinoma associated fibroblasts as new therapeutic targets which will be a new direction in cancer cell invasion and metastasis therapy.     

Impaired β-cell regeneration after partial pancreatectomy in the adult Goto-Kakizaki rat, a spontaneous model of type II diabetes

In the Goto-Kakizaki (GK) rat, a genetic model of type II diabetes, there is a restriction of the beta-cell mass as early as fetal age, which is maintained reduced in the adult animal. In order to investigate the beta-cell growth potential in the adult hyperglycemic GK rat, and to determine whether it differs from non-diabetic Wistar (W) rats, we have performed 90% pancreatectomy (Px) in 8- to 10-week-old male animals. Spontaneous beta-cell regeneration and involvement of beta-cell replication, beta-cell neodifferentiation from ductal precursor, and beta-cell apoptosis were evaluated by immunocytochemistry and morphometry at different time points: day 0 (D0), D2, D7, and D14 after Px. In GK rats, deterioration of the diabetic state with severe and chronic hyperglycemia was evident as soon as D2, while in W/Px, normoglycemia to moderate hyperglycemia was observed. In W/Px rats, the total beta-cell mass gradually increased on D2, D7, and D14, as compared to non-Px W rats. By contrast, in GK/Px rats, there was only a non-significant tendency to increased total beta-cell mass, as compared to related non-Px group. Adult GK rats displayed lower beta-cell proliferation rates compared to W. In response to Px, early increase of beta-cell proliferation was present in both W/Px and GK/Px rats on D2, but it returned to non-Px values in GK rats on D7 and D14, while in W/Px rats beta-cell proliferation was maintained increased as compared to non-Px W rats. The very low apoptotic beta-cell frequency on D0, D2, D7, and D14, in both W and GK, either non-Px or Px, did not allow us to conclude that any significant differences exist between the different groups. beta-cell neoformation from ducts, and more specifically from foci of regeneration, was found to be less activated in GK/Px rats as compared to W/Px. Together, these results suggest that in the adult hyperglycemic GK rat undergoing Px, beta-cells still have the capacity to regenerate, but with a lower efficiency as compared to non-diabetic W rats. This defect in the GK rat is the result of both genetic predisposition contributing to an altered beta-cell neogenesis potential already present in the neonatal period, and environmental factors (chronic hyperglycemia) leading to a reduced beta-cell proliferative capacity specific to the adult animals.     

Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 × 10⁸ purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10¹⁰ and 10¹² phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance

The ongoing COVID‐19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS‐CoV‐2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo‐EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri‐bispecific fusion constructs that exhibit up to 100‐ and 1,000‐fold increase in neutralization potency, respectively. Cryo‐EM of the sybody‐spike complex additionally reveals a novel up‐out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS‐CoV‐2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS‐CoV‐2 escape mutants.     

The expression of Met/hepatocyte growth factor receptor gene in giant cell tumors of bone and other benign musculoskeletal tumors

Overexpression of the hepatocyte growth factor receptor (Met/HGF receptor), a transmembrane tyrosine kinase encoded by the MET proto-oncogene, is involved in transformation and invasive behavior of human carcinomas and sarcomas. We have previously found that bone sarcomas express high levels of Met/HGF receptor while in some cases the ligand HGF is co-expressed with the receptor, activating an autocrine loop. In this study, we analyzed 40 biopsy samples of a collection of giant cell tumors and other rare benign tumors of bone for expression of the MET proto-oncogene. These included nonossifying fibromas, osteoblastomas, desmoplastic fibromas of bone, chondroblastomas, and giant cell tumors of bone. Snap frozen samples were tested for the MET and HGF gene expression by immuno-histochemistry and Western blotting with anti-MET antibodies and RT-PCR. Over 50% of all cases scored positive for MET expression being constantly positive in recurrent or locally aggressive lesions. Sporadic co-expression of the Met/HGF receptor and ligand is also demonstrated. Met/HGF receptor expression in benign bone neoplasms suggests its early involvement in sarcomagenesis.     

Development of accessory reproductive glands and its control by the corpus allatum in adult male Melanoplus sanguinipes

Changes in the protein content of and the rate at which labelled protein appears in the accessory reproductive glands (ARG), fat body, and haemolymph were studied in normal and allatectomized (CA^?) males of the migratory grasshopper, Melanoplus sanguinipes. In addition, the effects of treatment of CA^? insects with synthetic juvenile hormone (SJH), copulation, and removal of the ARG were examined.In normal males the protein content of the ARG increases linearly during the first 14 days after emergence. Incorporation of label by the ARG is maximal at day 7 and then decreases until, at day 14, it is the same as at day 1. The protein content of the fat body and haemolymph increases up to day 10 then declines, whereas changes in the uptake of label by the fat body and haemolymph parallel those of the ARG.Removal of the corpora allata (CA) prevents the normal increase in protein content of the ARG, but the protein content of the fat body and haemolymph increases steadily throughout the 14 days. Incorporation of label into the ARG, fat body, and haemolymph remained low throughout the experiment. Treatment of CA^? insects with SJH, or copulation, stimulates the uptake of label by the ARG, fat body, and haemolymph and also results in an increase in their protein content.Removal of the ARG leads to an increase in the protein content of the fat body and haemolymph. Uptake of label by the fat body remains low after the operation. Although the rate at which labelled protein appears in the haemolymph is high initially, it declines steadily to day 14.We conclude that the CA regulate ARG development. It is suggested that the fat body, under CA control, synthesizes proteins which are incorporated into secretions of the ARG. Further, it is proposed that the primary effect of copulation is activation of the CA.     

Physiological and proteome study of sunflowers exposed to a polymetallic constraint

The new energy requirements of the growing world population together with the actual ecological trend of phytoremediation have made challenging the cultivation of energetic crops on nonagricultural lands, such as those contaminated with trace elements. In this study, phenotypical characterization and biochemical analyses were combined to emphasize the global response of young sunflowers (Helianthus annuus L.) grown in hydroponic media contaminated with different Cd, Ni, and Zn concentrations. Leaves and roots of sunflowers reaching the stage “2‐extended leaves” and exposed to different trace metal concentrations were harvested and analyzed by 2D‐DIGE in order to study in depth the molecular responses of the young plants upon the polymetallic exposure. Proteomics confirmed the observed global reduction in growth and development. If photosynthetic light reactions and carbon metabolism were the most affected in leaves, in roots significant disruptions were observed in proteins involved in respiration, oxidative balance, protein and gene expression, and in the induction of programmed cell death. Elemental analyses of the plantlets indicated a profound impact of the treatment resulting in misbalance in essential micronutrients. Altogether, this study highlights the sensitivity of the sunflower to a polymetallic pollution and indicates that its use as a remediative tool of trace element polluted soils is limited.     

Dissociation and re-association of RNA polymerase with DNA during osmotic stress response in Escherichia coli

The thermodynamic association of RNA polymerase (RNAP) with DNA is sensitive to salt concentration in vitro. Paradoxically, previous studies of changes in osmolarity during steady-state cell growth found no dependence between the association of RNAP to DNA and K⁺ concentration in Escherichia coli. We reevaluated this issue by following the interaction of RNAP and genomic DNA in time-course experiments during the hyper-osmotic response. Our results show that the interaction is temporally controlled by the same physical chemistry principle in the cell as in vitro. RNAP rapidly dissociates from the genome during the initial response when the cytoplasmic K⁺ accumulates transiently, and concurrently the nucleoid becomes hyper-condensed. The freed RNAP re-associates with the genome during a subsequent osmoadaptation phase when organic osmoprotectants accumulate as K⁺ levels decrease. RNAP first surrounds the hyper-condensed nucleoid forming a sphere of RNAP before it progressively moves in to the center of the nucleoid. Our findings reinterpret the dynamic protein–DNA interactions during osmotic stress response. We discuss the implications of the dissociation/association of RNAP for osmotic protection and nucleoid structure.