Adenoviral pVII proteins are multifunctional, highly basic, histone-like proteins that can bind to and transport the viral genome into the host cell nucleus. Despite the identification of several nuclear localization signals (NLSs) in the pVII protein of human adenovirus 5 (HAdV5), the mechanistic details of nuclear transport are largely unknown. Here we provide a full characterization of the nuclear import of precursor (Pre-) pVII protein from an ancient siadenovirus, frog siadenovirus 1 (FrAdV1) using a combination of structural, functional and biochemical approaches. Two strong NLSs (termed NLSa and NLSd) interact with importin (IMP)b and IMPa respectively, and are the main drivers of nuclear import. A weaker NLS (termed NLSb) also contributes, together with an additional signal (NLSc) which we found to be important for nucleolar targeting. Expression of Pre-pVII wild-type and NLS defective derivatives in the presence of selective inhibitors of different nuclear import pathways revealed that, unlike its human counterpart, FrAdV1 Pre-pVII nuclear import is dependent on IMPa/b and IMPb1. Clearly, AdVs evolved to maximise the nuclear import pathways for the pVII proteins, whose subcellular localization is the result of a complex process. Therefore, our results pave the way for an evolutionary comparison of the interaction of different AdVs with the host cell nuclear transport machinery.

Contractile vacuole complexes (CVCs) are complex osmoregulatory organelles, with vesicular (bladder) and tubular (spongiome) subcompartments. The mechanisms that underlie their formation and maintenance within the eukaryotic endomembrane network are poorly understood. In the Ciliate Tetrahymena thermophila, six differentiated CORVETs (class C core vacuole/endosome tethering complexes), with Vps8 subunits designated A-F, are likely to direct endosomal trafficking. Vps8Dp localizes to both bladder and spongiome. We show by inducible knockdown that VPS8D is essential to CVC organization and function. VPS8D knockdown increased susceptibility to osmotic shock, tolerated in the wildtype but triggering irreversible lethal swelling in the mutant. The knockdown rapidly triggered contraction of the spongiome and lengthened the period of the bladder contractile cycle. More prolonged knockdown resulted in disassembly of both the spongiome and bladder, and dispersal of proteins associated with those compartments. In stressed cells where the normally singular bladder is replaced by numerous vesicles bearing bladder markers, Vps8Dp concentrated conspicuously at long-lived inter-vesicle contact sites, consistent with tethering activity. Similarly, Vps8Dp in cell-free preparations accumulated at junctions formed after vacuoles came into close contact. Also consistent with roles for Vps8Dp in tethering and/or fusion were the emergence in knockdown cells of multiple vacuole-related structures, replacing the single bladder.

The progression of tumors is heavily influenced by mechanical properties of their microenvironment. In this work, we designed micropatterned models with varying distances and shapes to investigate the differences between metastatic breast cancer cells MDA-MB-231 and non-metastatic MCF-7 in reconfiguring the extracellular matrix to promote cell migration induced by long-distance cell mechanics. Both cancer cells were able to rearrange type I collagen fibres to form collagen threads, in which MDA-MB-231 consistently migrated more rapidly than MCF-7, ranging from matrix model with differently arrayed spacings to complex polygonal models. MDA-MB-231 displayed higher capability of reorganizing fibre bundles at longer distance (800 μm). Further looking for differences in cellular mechanical mechanisms, siRNA knockdown inhibiting either integrin β1 or Piezo1 decreased fibre assembly. Metastatic MDA-MB-231 showed inhibited migration with integrin knockdown, whereas scattered migration with Piezo1 knockdown indicating cells losing distant mechanosensation. MCF-7 with reduced E-cadherin by siRNA transfection showed less significant differences in migration compared to MDA-MB-231 that didn’t express E-cadherin. MDA-MB-231 expressed little E-cadherin mRNA indicating mutagenetic change in genome. In summary this work has explored the differences between malignant and benign breast cancer cells, particularly in terms of fibre matrix remodelling and cell migrations, along with significant differences in E-cadherin expressions, of which had an important effect on cell migration. The results of this study provide new research approaches for therapeutic advances in breast cancer.

The complex and dynamic interplay between internalization, anterograde transport, recycling and degradation determines the density of functional G protein-coupled receptors (GPCRs) at the cell surface and, consequently, the magnitude of their associated physiological responses. As opposed to most members of the GPCR superfamily, the delta opioid receptor (DOP) is only weakly expressed at the neuronal plasma membrane, thus representing a critical limitation for its use as a therapeutic target. Although DOP appears as a promising candidate for the development of better-tolerated analgesics, the molecular and cellular mechanisms underlying the regulation of its cell surface expression remain poorly characterized. This work investigates the constitutive ( i.e. ligand-independent) trafficking of DOP, an understudied cellular process potentially involved in the control of plasma membrane-localized receptors. In HEK293 cells stably expressing Flag-tagged DOP, we first confirmed that this GPCR is constitutively internalized through a clathrin-dependent and b-arrestin-independent mechanism. Immunofluorescence experiments with selected Rab protein isoforms indicated that internalized DOP was mainly colocalized with the early endosome marker Rab5, as well as the rapid recycling endosome marker Rab4. Co-transfection with Rab5 dominant-negative mutant inhibited the intracellular distribution of the receptor, indicating that its constitutive endocytosis is Rab5-dependent. DOP cell surface expression and ligand-induced signaling were also significantly reduced following Rab4-specific DsiRNA treatments, suggesting a role for this small GTPase in the regulation of DOP constitutive recycling. Mapping of the major region of interaction between DOP and both Rabs revealed that Rab4 binds the third intracellular loop of DOP, whereas Rab5 seems to preferentially interact with the distal region of the C-terminal end of DOP. Altogether, these results show for the first time that DOP constitutive internalization and recycling are critical to maintain its cell surface bioavailability and responsiveness to agonists.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5,000-8,000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFb homodimeric coreceptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT Endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.