Revealing the origins of vortex cavitation in a Venturi tube by high speed X-ray imaging.

Hydrodynamic cavitation is useful in many processing applications, for example, in chemical reactors, water treatment and biochemical engineering. An important type of hydrodynamic cavitation that occurs in a Venturi tube is vortex cavitation known to cause luminescence whose intensity is closely related to the size and number of cavitation events. However, the mechanistic origins of bubbles constituting vortex cavitation remains unclear, although it has been concluded that the pressure fields generated by the cavitation collapse strongly depends on the bubble geometry. The common view is that vortex cavitation consists of numerous small spherical bubbles. In the present paper, aspects of vortex cavitation arising in a Venturi tube were visualized using high-speed X-ray imaging at SPring-8 and European XFEL. It was discovered that vortex cavitation in a Venturi tube consisted of angulated rather than spherical bubbles. The tangential velocity of the surface of vortex cavitation was assessed considering the Rankine vortex model.

A TGF-ß-responsive enhancer regulates SRC expression and epithelial-mesenchymal transition-associated cell migration.

The non-receptor tyrosine kinase SRC is overexpressed and/or hyperactivated in various human cancers, and facilitates cancer progression by promoting invasion and metastasis. However, the mechanisms underlying SRC upregulation are poorly understood. In this study, we demonstrate that transforming growth factor-ß (TGF-ß) induces SRC expression at the transcriptional level by activating an intragenic the SRC enhancer. In the human breast epithelial cell line MCF10A, TGF-ß1 stimulation upregulated one of the SRC promotors, the 1A promoter, resulting in increased SRC mRNA and protein levels. Chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that the SMAD complex is recruited to three enhancer regions ∼15 kb upstream and downstream of the SRC promoter, and one of them is capable of activating the SRC promoter in response to TGF-ß. JUN, a member of the activator protein (AP)-1 family, localises to the enhancer and regulates TGF-ß-induced SRC expression. Furthermore, TGF-ß-induced SRC upregulation plays a crucial role in epithelial-mesenchymal transition (EMT)-associated cell migration by activating the SRC-focal adhesion kinase (FAK) circuit. Overall, these results suggest that TGF-ß-induced SRC upregulation promotes cancer cell invasion and metastasis in a subset of human malignancies.

Src activation in lipid rafts confers epithelial cells with invasive potential to escape from apical extrusion during cell competition.

Abnormal/cancerous cells within healthy epithelial tissues undergo apical extrusion to protect against carcinogenesis, although they acquire invasive capacity once carcinogenesis progresses. However, the molecular mechanisms by which cancer cells escape from apical extrusion and invade surrounding tissues remain elusive. In this study, we demonstrate a molecular mechanism for cell fate switching during epithelial cell competition. We found that during competition within epithelial cell layers, Src transformation promotes maturation of focal adhesions and degradation of extracellular matrix. Src-transformed cells underwent basal delamination by Src activation within sphingolipid/cholesterol-enriched membrane microdomains/lipid rafts, whereas they were apically extruded when Src was outside of lipid rafts. A comparative analysis of contrasting phenotypes revealed that activation of the Src-STAT3-MMP axis through lipid rafts was required for basal delamination. CUB-domain-containing protein 1 (CDCP1) was identified as an Src-activating scaffold and as a Met regulator in lipid rafts, and its overexpression induced basal delamination. In renal cancer models, CDCP1 promoted epithelial-mesenchymal transition-mediated invasive behavior by activating the Src-STAT3-MMP axis through Met activation. Overall, these results suggest that spatial activation of Src signaling in lipid rafts confers resistance to apical extrusion and invasive potential on epithelial cells to promote carcinogenesis.

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses.

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells.

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain-containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.

Direct cross-sectional imaging using X-ray Compton scattering: application to commercial batteries.

A synchrotron-based technique using Compton scattering imaging is presented. This technique has been applied to a coin battery (CR2023), and the cross-sectional image has been obtained in 34 ms without sample rotation. A three-dimensional image of the whole structure has been reconstructed from 74 cross-sectional images taken consecutively by scanning the incident, wide X-ray beam along one direction. This work demonstrates that quick cross-sectional imaging of regions of interest and three-dimensional image reconstruction without sample rotation are feasible using Compton scattering imaging.

ß-catenin-promoted cholesterol metabolism protects against cellular senescence in naked mole-rat cells.

The naked mole-rat (NMR; Heterocephalus glaber) exhibits cancer resistance and an exceptionally long lifespan of approximately 30 years, but the mechanism(s) underlying increased longevity in NMRs remains unclear. In the present study, we report unique mechanisms underlying cholesterol metabolism in NMR cells, which may be responsible for their anti-senescent properties. NMR fibroblasts expressed ß-catenin abundantly; this high expression was linked to increased accumulation of cholesterol-enriched lipid droplets. Ablation of ß-catenin or inhibition of cholesterol synthesis abolished lipid droplet formation and induced senescence-like phenotypes accompanied by increased oxidative stress. ß-catenin ablation downregulated apolipoprotein F and the LXR/RXR pathway, which are involved in cholesterol transport and biogenesis. Apolipoprotein F ablation also suppressed lipid droplet accumulation and promoted cellular senescence, indicating that apolipoprotein F mediates ß-catenin signaling in NMR cells. Thus, we suggest that ß-catenin in NMRs functions to offset senescence by regulating cholesterol metabolism, which may contribute to increased longevity in NMRs.

CDCP1 promotes compensatory renal growth by integrating Src and Met signaling.

Compensatory growth of organs after loss of their mass and/or function is controlled by hepatocyte growth factor (HGF), but the underlying regulatory mechanisms remain elusive. Here, we show that CUB domain-containing protein 1 (CDCP1) promotes HGF-induced compensatory renal growth. Using canine kidney cells as a model of renal tubules, we found that HGF-induced temporal up-regulation of Src activity and its scaffold protein, CDCP1, and that the ablation of CDCP1 robustly abrogated HGF-induced phenotypic changes, such as morphological changes and cell growth/proliferation. Mechanistic analyses revealed that up-regulated CDCP1 recruits Src into lipid rafts to activate STAT3 associated with the HGF receptor Met, and activated STAT3 induces the expression of matrix metalloproteinases and mitogenic factors. After unilateral nephrectomy in mice, the Met-STAT3 signaling is transiently up-regulated in the renal tubules of the remaining kidney, whereas CDCP1 ablation attenuates regenerative signaling and significantly suppresses compensatory growth. These findings demonstrate that CDCP1 plays a crucial role in controlling compensatory renal growth by focally and temporally integrating Src and Met signaling.

p18/Lamtor1-mTORC1 Signaling Controls Development of Mucin-producing Goblet Cells in the Intestine.

Mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in controlling cell growth and metabolism in response to nutrients and growth factors. The activity of mTORC1 is dually regulated by amino acids and growth factor signaling, and amino acid-dependent mTORC1 activity is regulated by mTORC1 interaction with the Ragulator-Rag GTPase complex, which is localized to the surface of lysosomes via a membrane-anchored protein, p18/Lamtor1. However, the physiological function of p18-Ragulator-dependent mTORC1 signaling remains elusive. The present study evaluated the function of p18-mediated mTORC1 signaling in the intestinal epithelia using p18 conditional knockout mice. In p18 knockout colonic crypts, mTORC1 was delocalized from lysosomes, and in vivo mTORC1 activity was markedly decreased. Histologically, p18 knockout crypts exhibited significantly increased proliferating cells and dramatically decreased mucin-producing goblet cells, while overall crypt architecture and enteroendocrine cell differentiation were unaffected. Furthermore, p18 knockout crypts normally expressed transcription factors implicated in crypt differentiation, such as Cdx2 and Klf4, indicating that p18 ablation did not affect the genetic program of cell differentiation. Analysis of colon crypt organoid cultures revealed that both p18 ablation and rapamycin treatment robustly suppressed development of mucin-producing goblet cells. Hence, p18-mediated mTORC1 signaling could promote the anabolic metabolism required for robust mucin production in goblet cells to protect the intestinal epithelia from various external stressors.Key words: mTORC1, p18/lamtor1, intestinal epithelium, goblet cells, mucin.

Atg5-mediated autophagy controls apoptosis/anoikis via p53/Rb pathway in naked mole-rat fibroblasts.

The naked mole-rat (NMR, Heterocephalus glaber) is the longest-living known rodent species, with a maximum lifespan of over 30 years. NMRs exhibit negligible senescence, exceptional resistance to cancer, and high basal autophagy activity compared with mouse. The molecular mechanisms and physiological roles underlying the high basal autophagy activity in NMRs remain to be elucidated. We identified that the Atg12-Atg5 conjugate, a critical component of autophagosome formation, was highly expressed in NMR skin fibroblasts (NSFs) compared with that in mouse skin fibroblasts. Phenotypic analysis of Atg5 knockdown NSFs revealed that high basal autophagy activity in NSFs was associated with abundant expression of the Atg12-Atg5 conjugate. Atg5 knockdown in NSFs led to accumulation of dysfunctional mitochondria, and suppressed cell proliferation and cell adhesion ability, promoting apoptosis/anoikis accompanied by upregulation of the apoptosis-related genes, Bax and Noxa. Furthermore, inhibition of the p53/Rb pro-apoptotic pathway with SV40 large T antigen abolished Atg5 knockdown-induced increases in apoptosis/anoikis. Taken together, these findings suggest that high basal autophagy activity in NMR cells, mediated by Atg5, contributes to suppression of p53/Rb-induced apoptosis, which could benefit the longevity of NMR cells.

Ubiquitination of Src promotes its secretion via small extracellular vesicles.

Upregulation of the Src tyrosine kinase is implicated in the progression of cancer. The oncogenic potential of Src is suppressed via several negative regulation systems including degradation via the ubiquitin-proteasome pathway. Here, we show that ubiquitination of Src promotes its secretion via small extracellular vesicles (sEVs) to suppress its oncogenic potential. In MDCK cells expressing a modified Src that can be activated by hydroxytamoxifen, activated Src was transported to late endosomes/lysosomes and secreted via sEVs. The secretion of Src was suppressed by ablation of Cbl E3-ligase, suggesting the contribution of ubiquitination to this process. Activated Src was ubiquitinated at multiple sites, and Lys429 was identified as a critical site for sEV-mediated secretion. Mutation of Src at Lys429 (R429) caused resistance to ubiquitination and decreased its secretion via sEVs. The activated R429 mutant was also transported to late endosomes/lysosomes, whereas its incorporation into intraluminal vesicles was reduced. Activation of the R429 mutant induced a greater FAK activation than that of wild-type Src, thereby potentiating Src-induced invasive phenotypes, such as invadopodia formation and invasive activity. These findings demonstrate that ubiquitination of activated Src at Lys429 promotes its secretion via sEVs, suggesting a potential strategy to suppress the oncogenic function of upregulated Src.

Sphingolipid/Pkh1/2-TORC1/Sch9 Signaling Regulates Ribosome Biogenesis in Tunicamycin-Induced Stress Response in Yeast.

Reduced ribosome biogenesis in response to environmental conditions is a key feature of cell adaptation to stress. For example, ribosomal genes are transcriptionally repressed when cells are exposed to tunicamycin, a protein glycosylation inhibitor that induces endoplasmic reticulum stress and blocks vesicular trafficking in the secretory pathway. Here, we describe a novel regulatory model, in which tunicamycin-mediated stress induces the accumulation of long-chain sphingoid bases and subsequent activation of Pkh1/2 signaling, which leads to decreased expression of ribosomal protein genes via the downstream effectors Pkc1 and Sch9. Target of rapamycin complex 1 (TORC1), an upstream activator of Sch9, is also required. This pathway links ribosome biogenesis to alterations in membrane lipid composition under tunicamycin-induced stress conditions. Our results suggest that sphingolipid/Pkh1/2-TORC1/Sch9 signaling is an important determinant for adaptation to tunicamycin-induced stress.

Src mediates TGF-ß-induced intraocular pressure elevation in glaucoma.

Glaucoma, a progressive and irreversible optic neuropathy, is one of the leading causes of vision impairment worldwide. Elevation of intraocular pressure (IOP) due to transforming growth factor-ß (TGF-ß)-induced dysfunction of the trabecular meshwork is a risk factor for glaucoma, but the underlying molecular mechanisms remain elusive. Here, we show that Src kinase is involved in TGF-ß-induced IOP elevation. We observed that dasatinib, a potent Src inhibitor, suppressed TGF-ß2-induced IOP in rat eyes. Mechanistic analyses in human trabecular meshwork cells showed that TGF-ß2 activated Src signaling and concomitantly increased cytoskeletal remodeling, cell adhesion, and extracellular matrix (ECM) accumulation. Src was activated via TGF-ß2-induced upregulation of the Src scaffolding protein CasL, which mediates the assembly of focal adhesions, cytoskeletal remodeling, and ECM deposition. Activation of Src suppressed the expression of tissue plasminogen activator, thereby attenuating ECM degradation. Furthermore, the Src inhibitor ameliorated TGF-ß2-induced changes in the contractile and adhesive characteristics of trabecular meshwork cells, and ECM deposition. These findings underscore the crucial role of Src activity in TGF-ß-induced IOP elevation and identify Src signaling as a potential therapeutic target in glaucoma.

Effect of insufficient temporal coherence on visibility contrast in X-ray grating interferometry.

X-ray grating interferometry, which has been spotlighted in the last decade as a multi-modal X-ray imaging technique, can provide three independent images, i.e., absorption, differential-phase, and visibility-contrast images. We report on a cause of the visibility contrast, an effect of insufficient temporal coherence, that can be observed when continuous-spectrum X-rays are used. This effect occurs even for a sample without unresolvable random structures, which are known as the main causes of visibility contrast. We performed an experiment using an acrylic cylinder and quantitatively explained the visibility contrast due to this effect.

Complementation analysis reveals a potential role of human ARV1 in GPI anchor biosynthesis.

ARV1 is involved in regulating lipid homeostasis but also in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. Here, we examined whether human ARV1 can complement the role of yeast ARV1 in GPI biosynthesis. Overexpression of human ARV1 could rescue the phenotypes associated with GPI anchor synthesis defect in the yeast arv1Δ mutant. The results suggest that Arv1 function in GPI biosynthesis may be conserved in all eukaryotes, from yeast to humans.

Roles of raft-anchored adaptor Cbp/PAG1 in spatial regulation of c-Src kinase.

The tyrosine kinase c-Src is upregulated in numerous human cancers, implying a role for c-Src in cancer progression. Previously, we have shown that sequestration of activated c-Src into lipid rafts via a transmembrane adaptor, Cbp/PAG1, efficiently suppresses c-Src-induced cell transformation in Csk-deficient cells, suggesting that the transforming activity of c-Src is spatially regulated via Cbp in lipid rafts. To dissect the molecular mechanisms of the Cbp-mediated regulation of c-Src, a combined analysis was performed that included mathematical modeling and in vitro experiments in a c-Src- or Cbp-inducible system. c-Src activity was first determined as a function of c-Src or Cbp levels, using focal adhesion kinase (FAK) as a crucial c-Src substrate. Based on these experimental data, two mathematical models were constructed, the sequestration model and the ternary model. The computational analysis showed that both models supported our proposal that raft localization of Cbp is crucial for the suppression of c-Src function, but the ternary model, which includes a ternary complex consisting of Cbp, c-Src, and FAK, also predicted that c-Src function is dependent on the lipid-raft volume. Experimental analysis revealed that c-Src activity is elevated when lipid rafts are disrupted and the ternary complex forms in non-raft membranes, indicating that the ternary model accurately represents the system. Moreover, the ternary model predicted that, if Cbp enhances the interaction between c-Src and FAK, Cbp could promote c-Src function when lipid rafts are disrupted. These findings underscore the crucial role of lipid rafts in the Cbp-mediated negative regulation of c-Src-transforming activity, and explain the positive role of Cbp in c-Src regulation under particular conditions where lipid rafts are perturbed.

c-Src-induced activation of ceramide metabolism impairs membrane microdomains and promotes malignant progression by facilitating the translocation of c-Src to focal adhesions.

The proto-oncogenic tyrosine kinase c-Src is up-regulated in various human cancers, implicating its role in tumour progression. Upon activation, c-Src translocates to focal adhesions and initiates intracellular signalling cascades that promote malignant transformation, but the underlying mechanisms for c-Src translocation remain unclear. In the present study we show that c-Src up-regulation perturbs sphingolipid/cholesterol-enriched membrane microdomains by activating ceramide synthesis, resulting in promotion of c-Src translocation. Using an inducible c-Src expression system in Csk (C-terminal Src kinase)-deficient fibroblasts, we found that translocation of c-Src to focal adhesions/podosomes occurs in the later stages of cell transformation. Activated c-Src is liberated from microdomains and promotes the phosphorylation of FAK (focal adhesion kinase) and cortactin localized to focal adhesions/podosomes. In parallel with these events, anabolic metabolism of ceramides is activated by up-regulation of the de novo synthesis pathway. Inhibition of ceramide conversion into glucosylceramide promotes liberation of c-Src from microdomains, and inhibition of de novo ceramide synthesis restores the microdomain distribution of c-Src and suppresses malignant phenotypes such as increased cell motility and anchorage-independent cell growth. These results suggest that c-Src-induced activation of ceramide synthesis impairs the integrity of microdomains and contributes to malignant progression by promoting the translocation of c-Src to focal adhesions/podosomes.

Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast.

Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.

Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray.

Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10-20 keV (ΔE/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

Perturbation of sphingolipid metabolism induces endoplasmic reticulum stress-mediated mitochondrial apoptosis in budding yeast.

Sphingolipids are a class of membrane lipids conserved from yeast to mammals which determine whether a cell dies or survives. Perturbations in sphingolipid metabolism cause apoptotic cell death. Recent studies indicate that reduced sphingolipid levels trigger the cell death, but little is known about the mechanisms. In the budding yeast Saccharomyces cerevisiae, we show that reduction in complex sphingolipid levels causes loss of viability, most likely due to the induction of mitochondria-dependent apoptotic cell death pathway, accompanied by changes in mitochondrial and endoplasmic reticulum morphology and endoplasmic reticulum stress. Elevated cytosolic free calcium is required for the loss of viability. These results indicate that complex sphingolipids are essential for maintaining endoplasmic reticulum homeostasis and suggest that perturbation in complex sphingolipid levels activates an endoplasmic reticulum stress-mediated and calcium-dependent pathway to propagate apoptotic signals to the mitochondria.