ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and extracellular vesicle recycling

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.

First person – Katharina Vestre

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Katharina Vestre is first author on ‘ Rab7b regulates dendritic cell migration by linking lysosomes to the actomyosin cytoskeleton’, published in JCS. Katharina is a PhD student in the lab of Cinzia Progida at the Department of Biosciences, University of Oslo, Norway, investigating the coordination between intracellular traffic and the cytoskeleton, and how this affects processes such as cell division and migration.

Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments

A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant’s early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~ 10% increase), decreased IgG content in intracellular vesicles (~ 9% decrease), and a trend towards decreased IgG recycling (~ 55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~ 60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.

Tight junction protein ZO-2 modulates the nuclear accumulation of transcription factor TEAD

Tight junction protein Zonula occludens 2 (ZO-2) presence at the nucleus inhibits the transcription of genes regulated by TEAD transcription factor. Here, we analyzed if the movement of ZO-2 into the nucleus modulates the nuclear concentration of TEAD. In sparse cultures of ZO-2 knock down (KD) MDCK cells, nuclear TEAD diminished, as in parental cells transfected with a ZO-2 construct without nuclear localization signals, indicating that ZO-2 facilitates the entry of TEAD into the nucleus. Inhibition of nPKCδ in parental cells triggers the interaction between ZO-2 and TEAD at the cytoplasm and facilitates TEAD/ZO-2 complex nuclear importation. Using proximity ligation, immunoprecipitation and pull-down assays TEAD/ZO-2 interaction was confirmed. Nuclear TEAD is phosphorylated and its exit in parental cells is enhanced by activation of a ZO-2 nuclear exportation signal by nPKCε, while the nuclear accumulation of ZO-2 triggered by the mutation of ZO-2 nuclear export signals, induces the no change in TEAD nuclear concentration. In summary, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD through a process regulated by nPKC δ and ε, and provide a novel role of ZO-2 as a nuclear translocator of TEAD.

The Protein Phosphatase PP2A Plays Multiple Roles in Plant Development by Regulation of Vesicle Traffic—Facts and Questions

The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP–GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole plant development. Overall, PP2A has essential roles in membrane interactions of plant cell and it is crucial for plant development and stress responses.

Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~10% increase), decreased IgG content in intracellular vesicles (~9% decrease), and a trend towards decreased IgG recycling (~55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.

Engineering self-organized criticality in living cells

Complex dynamical fluctuations, from molecular noise within cells, collective intelligence, brain dynamics or computer traffic have been shown to display noisy behaviour consistent with a critical state between order and disorder. Living close to the critical point can have a number of adaptive advantages and it has been conjectured that evolution could select (and even tend to) these critical states. One way of approaching such state is by means of so called self-organized criticality (SOC) where the system poises itself close to the critical point. Is this the case of living cells? It is difficult to test this idea given the enormous dimensionality associated with gene and metabolic webs. In this paper we present an alternative approach: to engineer synthetic gene networks displaying SOC behaviour. This is achieved by exploiting the presence of a saturation (congestion) phenomenon of the ClpXP protein degradation machinery in E. coli cells. Using a feedback design that detects and then reduces ClpXP congestion, a critical motif is built from a two-gene network system, where SOC can be successfully implemented. Both deterministic and stochastic models are used, consistently supporting the presence of criticality in intracellular traffic. The potential implications for both cellular dynamics and designed intracellular noise are discussed.