extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
HIF‐2α regulates non‐canonical glutamine metabolism via activation of PI3K/mTORC2 pathway in human pancreatic ductal adenocarcinoma

Abstract Hypoxia‐inducible factor‐2α (HIF‐2α) plays an important role in increasing cancer progression and distant metastasis in a variety of tumour types. We aimed to investigate its biological function and clinical significance in human pancreatic ductal adenocarcinoma (PDAC). A total of 283 paired PDAC tissues and adjacent normal tissues were collected from patients who underwent surgery or biopsy at Sun Yat‐sen Memorial Hospital between February 2004 and October 2016. In this study, we noted that HIF‐2α expression was significantly up‐regulated in PDAC, positively associated with disease stage, lymph‐node metastasis and patient survival, and identified as an independent prognostic factor of PDAC patients. We demonstrated that HIF‐2α silencing could reduce proliferation, migration and invasion of PDAC cells in vitro. The similar effect on growth was demonstrated in vivo. Furthermore, we noted that knock‐down of HIF‐2α significantly decreased the expression of glutamate oxaloacetate transaminase 1 (GOT1). Importantly, we confirmed that the PI3K/mTORC2 pathway promoted GOT1 expression by targeting HIF‐2α. Our study validated HIF‐2α was an important factor in PDAC progression and poor prognosis and may promote non‐canonical glutamine metabolism via activation of PI3K/mTORC2 pathway. Targeting HIF‐2α represents a novel prognostic biomarker and therapeutic target for patients with PDAC.

Introduction
Pancreatic cancer is a typical hypoxia digestive solid malignancy. It is so lethal that the 5-year survival rate is only around 5% [1,2]. Aberrant metabolism is considered as one of the hallmarks of cancer [3]. A remarkable phenomenon named Warburg effects is exploited in the clinic for diagnostic purposes, which is exhibited in various tumours [4,5]. Previous research showed that human PDAC utilizes a special metabolism pathway, a non-canonical pathway of glutamine, in which glutamine-derived aspartate is converted into oxaloacetate by aspartate transaminase (GOT1), and this oxaloacetate is converted into malate and then pyruvate, increasing the NADPH/NADP + ratio which can potentially maintain the cellular redox state [6,7]. Reprogramming of energy metabolism is the hallmark progression in the research of malignancy neoplasm [3]. It can balance energy metabolism and biosynthesis to maintain the high malignancy behaviours [8].
Hypoxia is another important feature of solid tumours. It has been convinced that a series of molecular biology change induced by hypoxia are the important reasons of higher malignant, like proliferation, invasion, migration and chemoradiotherapy resistant, relating to hypoxia-inducible factor (HIF) signalling pathway [20,21]. HIFs are composed of two heterodimeric proteins and contain three subunits, HIF-1a, HIF-2a and HIF-3a; among them, HIF-1a and HIF-2a are best studied so far [22]. HIF-1a protein and HIF-2a protein were detected in most types of human tumours, including pancreatic, bladder, colon, breast, glial, hepatocellular, prostate and renal tumours [23]. Different to HIF-1a, HIF-2a is active under mild or physiological hypoxia (<5% O 2 ), and active even after 48-72 hrs [24]. Some important cellular activities are regulated by HIF-2a, such as proliferation, invasion, differentiation and metabolism [25]. Despite that the effect of hypoxia in glycolysis has been verified [26,27], the effect of HIF-2a on non-canonical pathway of glutamine is still unknown in PDAC.
In this study, we investigated HIF-2a expression in PDAC tissues and analysed its correlation with the clinicopathological characteristics and prognosis of PDAC patients. We also studied the function and mechanism of HIF-2a in PDAC cells. Our findings strongly indicate that HIF-2a participates in PDAC carcinogenesis and could regulated non-canonical glutamine metabolism by activation of PI3K/ mTORC2 pathway. We believe that HIF-2a is a potential diagnostic and prognostic marker and a promising therapeutic target.

Patients and clinical samples
The 283 paired PDAC samples were obtained from patients who underwent resection or biopsy at Sun Yat-sen Memorial Hospital between February 2004 and October 2016. No patient received anticancer treatment before surgery. Table 1 presents the detailed data of the clinicopathological characteristics. All samples were evaluated and histologically diagnosed by expert pathologists. All samples were collected with informed consent according to the Sun Yat-Sen University internal review and ethics boards.

Cell culture
The pancreatic cell lines of Capan-2 and Panc-1 were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM (Gibco, Grand Island, NY, USA) with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37°C incubator in a humidified atmosphere of 5% CO 2 . For the hypoxia treatments, cells were incubated in an anaerobic chamber (Invivo2400, Ruskinn Technology, Sanford, Maine, USA). Cells were treated with LY294002

RNA isolation and quantitative RT-PCR (qRT-PCR)
Total RNA was isolated from tissues or pancreatic cell lines using a standard Trizol protocol (Invitrogen, Carlsbad, CA, USA). Total RNA was converted to cDNA by reverse-transcription using oligodT primers and SuperScript II reverse transcriptase (Invitrogen, San Diego, CA, USA). Real-time PCR was performed using SYBR Green reaction mix (Qiagen, Germantown, MD) and analysed on a Roche Light-Cycler system (Roche, Basel, Switzerland). b-Actin was used as the normalization control. The qRT-PCR data were analysed and showed as the fold change (2 ÀDDCT ). The qRT-PCR reaction for each sample was repeated in triplicate. The primer sequences were showed in Table S1.

Cell transfection and viral infection
For transient knock-down experiments, the following small interfering RNAs (siRNAs) were used: HIF-2a siRNA (si-HIF-2a) and synthetic sequencescrambled siRNA (si-control) were purchased from GenePharma Co (Shanghai, China). SiRNAs were transfected into cells using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. After 48 hrs, qRT-PCR was used to measure the efficiency of siRNA knock-down. The primer sequences were showed in Table S1. For stable knock-down of HIF-2a shRNA (sh-HIF-2a) and scrambled control RNA (sh-control) were inserted into the lentiviral vector (LV3H1/ GFP&Puro vector). After transfection of 293T cells for 72 hrs, the viral supernatants were collected. Using a LentiX TM Concentrator overnight at 4°C (Clontech, Mountain View, CA, USA), lentiviral particles were concentrated and tittered to 10 9 TU/ml (transfection unit/ml). Panc-1 and Capan-2 cells (5 9 10 5 cells/well) were seeded in six-well culture plates and cultured in DMEM with 10% FBS and infected with virus and polybrene 24 hrs later. Positive clones were screened with puromycin (2 lg/ml and 5 lg/ml, respectively) for 2-3 weeks. The primer sequences were showed in Table S1.

Boyden chamber cell invasion
Invasion assays were performed using the BD Biocoat Matrigel Invasion Chamber (8 lm; BD Biosciences, SanJose, CA, USA) following the manufacturer's instructions. After transduced with sh-HIF-2a or sh-control, 1 9 10 4 cells were plated in the upper chamber incubated in DMEM without FBS. The bottom chamber contained DMEM with 10% FBS to stimulate invasion. After incubation at 20% or 1% O 2 for 36 hrs, the bottom chamber insert was stained with 0.1% crystal violet, and cells were counted and photographed with a Nikon microscope.

Cell proliferation assays and colony formation assays
Cell counting kit-8 (CCK-8) assay was used to measure cell proliferation according to the manufacturer's instructions. Cells were seeded in 96wells plates (2 9 10 3 cells/well) and cultured at 20% or 1% O 2; CCK-8 solution (10 ll, Dojindo Molecular Technologies, Kyushu, Japan) was added at 0, 24, 48 and 72 hrs, respectively. Then, cells were incubated at 20% or 1% O 2 for 4 hrs. Absorbance was measured at a wavelength of 450 nm using a microplate reader.
To examine HIF-2a on cell growth in Panc-1 and Capan-2 under different levels of O 2 , 1000 sh-HIF-2a or sh-control cells were seeded into the six-well plates. After being incubated for 10-14 days at 37°C at 20% or 1% O 2, the plates were fixed with methanol, stained with 2% crystal violet and counted.

Wound healing assay
Wound healing assay was conducted to examine the capacity of cell migration and invasion. When the cells reached 90-95% confluent, the wound was generated by scratching the surface of the plates with a 10ul pipette tip. Cells then were incubated in DMEM without FBS and then photographed with a Nikon microscope at 0 hr, 24 hrs, 48 hrs.

Cell-cycle analysis
Sh-HIF-2a or sh-control cells were incubated at 20% or 1% O 2 for 48 hrs. After incubated in cold ethyl alcohol at 4°C overnight, cells were analysed with flow cytometer (FACScan; BD Biosciences, Franklin Lake, NJ, USA) equipped with Cell Quest software (BD Biosciences, NJ, USA).

Immunohistochemistry and scoring analyses
HIF-2a expression in the PDAC specimens was blind-quantified by two pathologists using a previously described scoring system. Briefly, the immunostaining intensity of each sample was graded as negative = 0, weak = 1, moderate = 2 or strong = 3 (Fig. 1C). The proportion of positively staining cells was assessed as the percentage. The score was then calculated as the intensity score multiplied by the percentage of cells stained (score = intensity x % of positive cells). The samples were classed as low (score < 140) or high (score ≥ 140) HIF-2a expression. Imageswere visualized using a Nikon ECLIPSE Ti (Fukasawa, Japan) microscope system and processed with Nikon software.

Glutamine consumption
Panc-1 and Capan-2 were treated in 3 ml DMEM containing 2.5 mmol/l glutamine in six-well plates, 1 9 10 6 cells were seeded in each well and mitomycin was used to inhibition proliferation. Every 12 hrs replaced the medium, testing the remaining medium volume and the concentration of glutamine in them. The effect of HIF-2a on glutamine consumption was measured after cells were treated at 20%, 1% O 2 for 48 hrs.
NADP + /NADPH ratio measurement NADP + /NADPH ratios were measured using the NADP + /NADPH assay kit (Abcam, ab65349) according to the manufacturer's instructions. Briefly, 10 5 cells were collected on ice in extraction buffer by performing two freeze/thaw cycles (20 min. on dry ice followed by 10 min. at 37°C) and concentration was obtained by comparison with standard curves.

Measurement of intracellular ROS levels
Intracellular ROS was measured using the ROS detection kit (Beyotime Company, S0033, Shang Hai, China) according to the manufacturer's instructions. Briefly, after treatment at 1% O 2 or 20% O 2 for 48 hrs, cells were washed twice in phosphate-buffered saline (PBS) and then incubated with 10 lmol/l DCFH-DA at 37°C for 20 min. Then, DCF fluorescence distribution of 2 9 10 4 cells was detected by fluorescence microplate reader at an excitation wavelength of 488 nm and at an emission wavelength of 525 nm.

Tumour formation assay
The animal care and experimental protocols were approved by the institutional guidelines of Guangdong Province and by the Use Committee for Animal Care. BALB/c nude mice (4-6 weeks old) were subcutaneously injected the left flank with the Panc-1 cells expression the control shRNA and the right flank with the Panc-1 cells expression the shRNA to HIF-2a (7 9 10 6 cells/flank). The volume of each tumour was measured every 3 days. Tumour volume was calculated using the following formula: volume ¼ ðL Â W 2 Þ=2 , where L and W are the longest and shortest diameters, respectively. On the 21th day after injection, mice were killed and tumours were harvested and photographed.

Statistical analysis
Data are presented as the mean AE S.D. of three independent experiments. Cumulative survival time was calculated using the Kaplan-Meier method and analysed by the log-rank test. A multivariate Cox proportional hazards model was used to estimate the adjusted hazard ratios and 95% confidence intervals and to identify independent prognostic factors. All statistical analyses were performed with SPSS19.0 (SPSS Inc, Chicago, IL, USA). Statistical evaluation of data was performed using one-way ANOVA for multiple groups, Student's t-tests for two groups and chi-square test (v 2 test) for non-parametric variables. The threshold for statistical significance was set at *P < 0.05, **P < 0.01.

HIF-2a expression is increased in PDAC and associated with poor prognosis in human PDAC patients
To evaluate the expression of HIF-2a in PDAC and non-tumorous tissues, we first utilized qRT-PCR and Western blot analysis on the cases of PDAC. HIF-2a expression was increased in all of the cases as compared to the adjacent normal tissues (Fig. 1A). Moreover, we used immunohistochemistry analysis to measure the HIF-2a expression in 283 PDAC tissues and 283 paired normal tissues (Fig. 1B). HIF-2a was significantly overexpressed in PDAC tissues as compared with normal tissues and obviously higher in poorly differentiated tissues as compared to well-differentiated tissues (Fig. 1B). Clinicopathological correlation analysis revealed positive correlation between elevated HIF-2a levels with TNM stage, lymph-node metastasis, differentiation and perineural invasion (Table 1).
Next, we examined the correlation between expression of HIF-2a and overall survival (OS) or progression-free survival (PFS). Kaplan-Meier survival analysis noted that HIF-2a overexpression was correlated with reduced OS (HR, 1.678; 95% CI: 1.267-2.222, P < 0.05) and PFS (HR, 1.671; 95% CI: 1.262-2.213, P < 0.05) (Fig. 1D). To further evaluate the prognostic factors associated with OS and PFS in PDAC, we first carried out multivariate Cox regression analyses using gender, age, differentiation, TNM stage, lymph-node metastasis, perineural invasion and HIF-2a level expression as parameters. The results showed that high HIF-2a expression was an independent predictor of OS and PFS in PDAC patients ( Table 2). These findings clearly demonstrated that HIF-2a might be a key factor for poor

HIF-2a knock-down impairs cell growth, migration and invasion of PDAC cells in vitro
Panc-1 and Capan-2 cells were transfected with siRNA targeted HIF-2a or control siRNA, we tested the transfection efficiency of HIF-2a#1 and HIF-2a#2 (Fig. S1). We then stably suppressed HIF-2a in Panc-1 and Capan-2 cells by lentiviral transfection. CCK-8 assays revealed that there was a decrease of cell growth rate in sh-HIF-2a group than sh-control group in Panc-1 and Capan-2 cells ( Fig. 2A). Consistent with our cell growth data, HIF-2a knock-down greatly attenuated the colony numbers of Panc-1 and Capan-2 cells (Fig. 2B). Furthermore, to further investigate the growth inhibition, we compared the cellcycle profiles of HIF-2a down-regulation cells with controls by flow cytometry. As shown in Figure 2C, HIF-2a knock-down dramatically increased the cell population in the G0/G1 phase, whereas it reduced the cell population in the S phases. Together, these data indicated the critical role of HIF-2a on growth of PDAC cells.
Migration and invasion are crucial for cancer progression and result in poor prognosis. We performed wound healing assay to indicate that knock-down of HIF-2a in Capan-2 and Panc-1 cells dramatically attenuated cell motility ( Fig. 3A and B). Moreover, transwell assay was utilized to show that HIF-2a knock-down decreased the migration and invasion in PDAC cells (Fig. 3C and D). These data demonstrated that HIF-2a plays a critical role on the migration and invasion of PDAC cells.

HIF-2a regulates non-canonical glutamine metabolism in chronic hypoxic conditions
To investigate whether hypoxia could affect the metabolism of glutamine in PDAC cells, we measured the glutamine consumption rate.  As shown in Figure 4A, with the reducing oxygen concentration, Panc-1 and Capan-2 depleting glutamine rates were increasing, especially after 48 hrs. Moreover, we evaluated the levels of mRNA and protein of glutamine metabolism enzymes by qRT-PCR and Western blot analysis, respectively ( Fig. 4B and C). These results showed that glutamine metabolism is increased under the reduced oxygen concentration. Next, we examined the effect of HIF-2a on glutamine metabolism in PDAC cells. Si-HIF-2a-transfected cells were separately incubated at 1% O 2 or 20% O 2 for 48 hrs. The glutamine consumption, intracellular ROS levels and NADP + /NADPH ratio were showed in Figure S2A and B. These results demonstrated that HIF-2a knock-down significantly attenuated glutamine consumption rates, increased ROS levels and NADP + /NADPH ratios. We measured the expression of GLS1, GOT1, GOT2 related mRNA and protein after down-regulating HIF-2a by qRT-PCR and western blot analysis. As shown in Figure 4D-F, HIF-2a knock-down induced a significantly decrease expression of GOT1, but had no effect on the expression of GLS1 and GOT2. Si-HIF-2a-transfected Panc-1 and Capan-2 were separately incubated at 1% O 2 for 48 hrs. Other levels of glutamine metabolism enzymes were tested by qRT-PCR and Western blot analysis. We found that HIF-2a had no effect on the expressions of GLUD1, MDH1, ME1 ( Fig. S2C and  D). Taken together, our results showed that HIF-2a could promote non-canonical glutamine metabolism in hypoxic conditions.

HIF-2a knock-down suppresses tumorigenicity and non-canonical glutamine metabolism of PDAC cells in vivo
To further explore the promotion of HIF-2a in tumour progression and non-canonical glutamine metabolism, the HIF-2a stable knockdown or sh-control cells were subcutaneously injected into nude mice. We measured the tumour growth activity and showed that tumour growth in the sh-HIF-2a group was significantly decreased compared with the sh-control group (Fig. 5A). The size and weight of tumours from the HIF-2a knock-down group were significantly lower than those of tumours from the sh-control group ( Fig. 5B and C).  Immunostaining revealed that knock-down HIF-2a could decrease the positive rate of Ki67 compared with the control group (Fig. 5D). Furthermore, we measured the inhibition of HIF-2a and GOT1 in the xenotransplanted tumours derived from the sh-HIF-2a and sh-control groups, respectively. The results noted that sh-HIF-2a groups had lower expression of GOT1 expression than the sh-control group ( Fig. 5E and F). Taken together, our results demonstrated that HIF-2a knock-down suppressed tumorigenicity and non-canonical glutamine metabolism of PDAC cells in vivo.

HIF-2a regulates non-canonical glutamine metabolism via activation of PI3K/mTORC2 pathway
The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway plays a significantly role in pancreas accelerating the formation of PDAC, activating in premalignant pancreatic lesions. Previous studies demonstrated that mTORC2 regulates the transcription levels of HIF-2a in multiply cancer [15,[17][18][19]. To explore whether PI3K/mTOCR2 plays an important role in regulating HIF-2a and non-canonical glutamine metabolism in PDAC, HIF-2a and GOT1 mRNA and protein levels were measured after the pharmacologic inhibitions suppressed the activity of target proteins. Panc-1 and Capan-2 were exposed to rapamycin for 48 hrs at 1%, 3% O 2 . As shown in Fig. 6A and B, inhibiting mTORC1 did not have significant influence on HIF-2a expression. Interestingly, our results noted the positive correlation between mTORC2 and HIF-2a ( Fig. 6C and D). Panc-1 and Capan-2 were treated with dual mTORC1/mTORC2 inhibitor PP242 for 48 hrs at 20%, 1%, 3% O 2 . HIF-2a, GOT1 mRNA expression and protein were determined by qRT-PCR and Western blot, respectively. The results demonstrated that inhibition of mTOR2 activity by PP242 down-regulated the expression of HIF-2a and GOT1 in mRNA and protein levels ( Fig. 6C and D). GDC-0068 is a highly selective pan-AKT inhibitor. Panc-1 and Capan-2 were exposed to GDC-0068 for 48 hrs at 1%, 3% O 2 . As shown in Figure S3, inhibiting AKT has no effect on HIF-2a expression. Moreover, we found pharmacologic inhibition of PI3K activity with LY294002 attenuated the expression of HIF-2a and GOT1 (Fig. S4). Taken together, these data indicated that HIF-2a regulates non-canonical glutamine metabolism by activation of PI3K/mTORC2 pathway.

Discussion
PDAC is a fatal and poor prognostic gastrointestinal carcinoma [1]. Metabolic Reprogramming is an important feature of the tumour environment [6]. The recovery of non-canonical pathway of glutamine is a hallmarks progression [6]. Pancreatic cancers are clinically and histologically characterized as hypovascular tumours [28,29]. They both play critical role in development and progression of pancreatic cancer. Identification the relationship between non-canonical pathway of glutamine and HIFs is crucial for the management of PDAC, and the pathway regulate non-canonical glutamine metabolism in hypoxia may provide avenues for therapeutic intervention. Numerous and severe hypoxic regions in PDAC were verified to be related with progression aggressiveness and poor prognosis [30]. HIFs are important in hypoxia that can transcript many genes to code protein and play a role in many aspects of cancer biology [31]. HIF-1a and HIF-2a protein were detected in most types of human tumours, including pancreatic, bladder, colon, breast, glial, hepatocellular, prostate and renal tumours [23]. In our study, we validated that HIF-2a was overexpressed in PDAC tissues and was associated with TNM stage, lymph-node metastasis, differentiation and perineural invasion of PDAC individuals. HIF-2a was a potential target of poor prognosis which was also revealed from worse overall survival and progression-free survival.
After analysing with clinical samples, we utilized PDAC cells to search for further support. We found that knock-down of HIF-2a led to decrease in proliferation and antagonistic effects on cell-cycle progression of PDAC cells. Moreover, the similar effect on growth of HIF-2a was also demonstrated in vivo. Metastasis plays a crucial role on tumour progression. We revealed that knock-down of HIF-2a can repress migration and invasion of PDAC cells. Despite that the effect of HIF-2a on tumour metastasis in vivo was not observed in our trial, we demonstrated that lymph-node metastasis was connected with high level of HIF-2a in PDAC tissues.
To meet the demands of energy and biosynthesis under hypoxia, tumour cells must develop metabolic responses like high glycolytic rate and hexosamine biosynthetic pathway activation [27]. Our results demonstrated that in PDAC cell lines, the consumption of glutamine was increased under reduced hypoxia concentration, especially in prolonged hypoxia. Meanwhile the key enzyme expression of glutamine metabolism pathway was also increased.
Considering HIFs were important factors in hypoxia environment, we suggested that HIF-2a could regulate glutamine metabolism in prolonged hypoxia. As expected, after HIF-2a was knocked down, the consumption of glutamine was reduced, while the intracellular ROS level and NADP + /NADPH ratio were increased compared with sh-control group. The expression of GOT1 was inhibited by HIF-2a knock-down, which is a key enzyme of non-canonical pathway of glutamine. The similar phenomenon was demonstrated in vivo. This is the first time to show HIF-2a could regulate non-canonical pathway of glutamine, Fig. 6 mTORC2 not mTORC1 regulates HIF-2a, GOT1 in PDAC under prolonged hypoxia. (A and B) HIF-2a mRNA and protein levels determined by qRT-PCR (mRNA) and Western blot (protein) after treatment of Panc-1 and Capan-2 cells with mTORC1 inhibitor rapamycin for 48 hrs at 3% or 1% O 2 . b-Actin was used as loading control. (C and D) Panc-1 and Capan-2 cells were treated with mTORC1/mTORC2 inhibitor PP242 and cultured for 48 hrs at 20%, 3% or 1% O 2, and HIF-2a, GOT1 mRNA and protein levels were determined by qRT-PCR (mRNA) and Western blot (protein). b-Actin was used as loading control. Data are presented as mean AE S.D. from three independent experiments. *P < 0.05, **P < 0.01. which is a typical pathway of metabolism in PDAC. Our preliminary work revealed that tumour expression of GOT1 was significantly linked with tumour dedifferentiation, vascular invasion and lymphatic invasion [32]. We revealed HIF-2a was one of the upstream genes of GOT1 and regulate GOT1 to impact PDAC malignancy behaviours.
We future explored a potential pathway to regulate GOT1 via HIF-2a. Nearly 100% of pancreatic adenocarcinomas have a K-RAS mutation [33,34]. Activated K-RAS promotes the PI3K signalling pathway and the serine/threonine kinase mTOR [35]. It has been verified the reprogramming of glutamine metabolism is mediated by oncogenic KRAS [6]. In our study, we demonstrated that pharmacologic inhibition of PI3K or mTORC2 can repress HIF-2a transcription and downregulate GOT1. In contrast, the expression of HIF-2a was independent of mTORC1 or AKT. It revealed that PI3K/mTORC2 pathway played a significant role in non-canonical glutamine metabolism of PDAC via HIF-2a.
In conclusion, we determined that HIF-2a is an independent prognostic factor for PDAC and promotes the progression of PDAC both in vitro and in vivo. In prolonged hypoxia environment, HIF-2a can regulate non-canonical pathway of glutamine, and PI3K/ mTORC2 is one of the pathways to regulate HIF-2a to promote non-canonical glutamine metabolism. Moreover, we wondered whether HIF-2a might be a potential target for the treatment of PDAC.

Supporting information
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Retromer-driven membrane tubulation separates endosomal recycling from Rab7/Ypt7-dependent fusion

How does a Rab function in both recycling and fusion? An endosomal subcomplex of the SNX-BAR retromer can bind to Ypt7 and compete with the HOPS complex. Assembly of the full retromer then results in displacement of Ypt7. These data explain how domain formation and Ypt7 participation can be coordinated.

back to the Golgi by formation of membrane tubules (Seaman, 2012;Burd and Cullen, 2014). Rab7 and Ypt7 are required for endosomal recycling and directly bind to retromer (Rojas et al., 2008;Seaman et al., 2009;Balderhaar et al., 2010;Liu et al., 2012;Priya et al., 2015). However, the Rab itself was not present on the tubules (Arlt et al., 2015b). Ypt7-GTP also interacts with the vacuolar homotypic fusion and vacuole protein sorting (HOPS) tethering complex, which is required for fusion of late endosomes with lysosomes in both yeast and metazoan cells (Balderhaar and Ungermann, 2013).
Early and late endosomes can be distinguished morphologically by electron microscopy (Prescianotto-Baschong and Riezman, 2002;Griffith and Reggiori, 2009), and the transition through these compartments can be traced by endocytosed cargo (Toshima et al., 2006;Arlt et al., 2015a). This analysis revealed that this cargo colocalized simultaneously with ESCRTs, retromer, the endosomal fusion machinery, and trace amounts of Ypt7, suggesting that the endosomal cargo-sorting machinery operates in parallel on maturing endosomes (Arlt et al., 2015a). Considering that Ypt7 binds both retromer and the HOPS tethering complex, which mediates fusion (Wickner, 2010), Ypt7 availability could be a possible explanation of how maturation is sensed.
Here we address the key question of how a dually used Rab like Rab7/Ypt7 can support two distinct events on endosomes. Using purified, full-length retromer subcomplexes, CSC and SNX-BAR, we dissect the consecutive assembly of retromer complexes on membranes. We demonstrate that retromer complex formation on membranes releases Ypt7, which then becomes available for fusion. Our data can explain how a single endosomal Rab coordinates fission and fusion events during maturation.
In agreement, Mon1-Ccz1 colocalizes with Vps21 to endosomes (Rana et al., 2015). In yeast, Rab5 inactivation requires the BLOC-1 complex and its interacting GTPase-inactivating protein Msb3 (Lachmann et al., 2012;John Peter et al., 2013;Rana et al., 2015). This change in Rab seems to determine both fusion specificity and recruitment of sorting machineries. One such sorting machinery is the retromer complex, which, together with either SNX or SNX-BAR proteins, mediates recycling of transmembrane cargo receptors (B) Overproduced CSC and SNX-BAR complexes were purified via the C-terminal TAP tag on Vps35 (CSC) or the SNX subunit Vps5. SNX-GFP had in addition a C-terminal GFP on Vps17; a CSC-GFP Vps29 was tagged similarly. Complexes were analyzed via SDS-PAGE, and gels were stained with Coomassie. (C, D) Gel filtration of SNX-BAR and CSC. TEV eluates from IgG columns were applied to gel filtration as described in Materials and Methods. TEV indicates the migration of the tobacco etch virus protease used to elute the protein from IgG beads. (E) Interaction of purified CSC and SNX-BAR with Rabs. C-terminal GST-tagged Vps21 and Ypt7 Rabs charged with either GDP or GTPγS were immobilized on GSH beads and incubated with purified SNX-BAR and CSC complexes. Eluted proteins were analyzed by SDS-PAGE and Western blotting with antibodies against the calmodulin-binding peptide (cbp; top of displayed gel) or directly analyzed by Coomassie staining (bottom). (F, G) Effect of purified CSC complex on vacuole fusion. Purified vacuoles from the two tester strains were incubated in the presence of ATP for 90 min at 26°C in the presence of the indicated amount of purified CSC (F). In G, the nonspecific GAP Gyp1-46 and the Ypt7 GEF Mon1-Ccz were included in the vacuole fusion reaction. Fusion reactions contained either no CSC or two different amounts of inhibitory CSC concentrations based on the titration in F. HOPS was titrated into each assay as indicated, and fusion was assayed after 90 min at 26°C (see Materials and Methods). filtration columns (Figure 1, C and D). Complexes were analyzed by negative stain microscopy and class averaged, and the overall architecture was determined ( Figure 2). For the CSC, we obtained an elongated particle of 15 nm with multiple small extensions ( Figure 2, A-C). Taking the human CSC model as a template, we were able to place the crystal structures of Vps29-Vps35 and Vps26 into the density of our structure, unraveling a striking similarity of the human and yeast CSC complexes ( Figure 2D). Indeed, the recently solved structure of a fragment of human Vps35 in complex with Vps26 that was published while this work was under review nicely agrees with our findings on the yeast complexes (Lucas et al., 2016). For the SNX-BAR dimer, we observed a large particle of 7 × 15 nm with possible symmetry along the vertical axis (Figure 2, E-G). Our attempt to model the available structure of the human SNX9 into the density failed due to the missing structural information on the large N-terminal segments that precede the PX and SNX-BAR domains. Because the SNX complex functions in the context of membranes, the obtained structure likely reflects the inactive cytosolic form.

Membrane activity of the full-length Vps5-17 SNX-BAR complex
Purified human SNX-BAR proteins can tubulate liposomes, which requires the BAR domain, binding to PI-3-P, and an amphipathic helix (Carlton et al., 2004;van Weering et al., 2012). We used our purified Vps5-17 heterodimer to address its functionality, using liposomes with a simple mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with 1.5 mol% PI-3-P and observed massive tubules of several micrometers by cryo-electron microscopy ( Figure 3A). In contrast, the CSC complex did not affect the shape of liposomes ( Figure 3A).
To determine membrane activity in a more accessible system, we turned to giant unilamellar vesicles (GUVs). GUVs have been used extensively to determine membrane activity of peripheral membrane proteins (e.g., Itoh et al., 2005;Saarikangas et al., 2009;Meinecke et al., 2013;Numrich et al., 2015). When added to the GUVs, the SNX-BAR heterodimer generated tubules emanating from the GUV surface ( Figure 3B), in agreement with our ultrastructural analysis ( Figure 3A). On longer incubations, GUVs began to shrink due to the consumption of membranes by tubulation (as seen in Figure 3C). As before, the CSC did not affect GUV morphology ( Figure 3B), although we detected some clustering of GUVs at higher concentrations ( Figure 4I). To monitor the localization of purified SNX-BAR and CSC complexes directly, we tagged either Vps29 (for CSC) or Vps17 (for SNX-BAR) with green fluorescent protein (GFP), which does not impair their function in vivo (Arlt et al., 2015b). Both complexes were found on GUV membranes (Figure 3, C and D), and SNX-BAR-GFP caused similar tubulation of GUVs as observed for the untagged complex ( Figure 3B, quantified in H). When we left out PI-3-P from our GUV preparations, both SNX-BAR binding to the GUV surface and tubulation were lost ( Figure 3E). The SNX-BAR heterodimer thus requires PI-3-P binding for membrane recruitment and tubulation activity.
We cooverexpressed all retromer subunits in a single yeast strain. Overproduced yeast SNX-BAR retromer could be purified as a heteropentamer but had the tendency to aggregate or disassemble during subsequent assays (Supplemental Figure S1). In contrast, both full-length SNX-BAR and CSC complexes were stable on gel filtration and reasonably clean enough to be used in subsequent assays ( Figure 1, B and D).
As a first assay for functionality, we tested their interaction with endosomal Rabs, which were immobilized as glutathione S-transferase (GST) fusion proteins on glutathione beads. In agreement with previous findings (Rojas et al., 2008;Seaman et al., 2009;Balderhaar et al., 2010;Liu et al., 2012;Harrison et al., 2014;Priya et al., 2015), only CSC interacted with Ypt7-GTP ( Figure 1E) and could inhibit the Ypt7-dependent vacuole fusion assay in a dose-dependent manner ( Figure 1F). For the latter assay, vacuoles were purified from two tester strains that upon ATP-dependent in vitro fusion restored maturation of the immature alkaline phosphatase (Pho8) present in one vacuole population by supplying the protease present in second vacuole population (Haas et al., 1994). The inhibition by CSC was efficiently quenched by purified HOPS complex ( Figure 1G), suggesting that both compete for Ypt7 on vacuoles. Note that we included in this competition assay an unspecific GAP, Gyp1-46, and excess of the Ypt7-GEF Mon1-Ccz1 to make possibly inaccessible Ypt7 available for HOPS or CSC binding . Our data thus imply that CSC is able to recognize Ypt7 in the context of membranes.

Structure of SNX-BAR and CSC complexes
Structures of human CSC and SNX-BAR have been solved, including the Vps29-Vps35 interface and isolated Vps26 (Collins et al., 2005(Collins et al., , 2008Shi et al., 2006;Hierro et al., 2007), which were used to model the full-length complex (Hierro et al., 2007). For the SNX complexes, structures of the BAR domains of SNX9 and SNX1, the human orthologue of Vps5, are available (Pylypenko et al., 2007;van Weering et al., 2012). To obtain insights into the architecture of the full-length CSC and SNX complexes, we applied purified complexes to gel  (Collins et al., 2005(Collins et al., , 2008Shi et al., 2006;Hierro et al., 2007) fitted into the density of yeast retromer. Scale bars, 50 Å.
Our initial experiments suggested that CSC might not require additional proteins for membrane binding ( Figure 3D). However, high concentrations of CSC might have masked crucial membrane requirements for recruitment. Indeed, when we reduced the CSC concentration twofold, CSC-GFP binding to membranes was abolished ( Figure 4A). When we added the SNX-BAR complex to this reaction, CSC was efficiently bound to the GUV membrane and began to accumulate in SNX-BAR-induced tubules ( Figure 4B). This reaction depended on PI-3-P on the GUV membrane ( Figure 4C), in agreement with the PI-3-P-dependent recruitment of the SNX-BAR complex ( Figure 3E). We then included Ypt7 and the cargo Vps10 in the reaction. Ypt7 ( Figure 4D) or Vps10 ( Figure 4G) was sufficient to recruit the CSC complex to membranes also in the absence of PI-3-P ( Figure 4F). Of interest, the presence of its ligands seems to suppress CSC-mediated aggregation of GUVs ( Figure 4I). As before, addition of the SNX-BAR complex caused the previously observed tubulation of membranes that contained CSC-GFP (Figure 4, E and H; quantified in K). Our data are thus consistent with the idea that CSC and SNX-BAR can bind membranes independently of each other but are found in the same resulting tubular structure. Either Ypt7-GTP or the cargo can strongly enhance CSC binding to membranes, whereas SNX-BAR-dependent tubulation requires PI-3-P. Of importance, CSC is then part of the forming tubule.

SNX-BAR retromer assembly displaces Ypt7 from the retromer
On endosomes, cargo is likely a limiting factor for CSC recruitment. Ypt7/Rab7 binding might therefore be necessary to provide sufficient binding sites to concentrate the CSC on endosomal membranes. SNX-BAR complex with GFP-tagged Vps17 was incubated with GUV membranes and examined as in B. (D) CSC can bind GUVs. CSC with GFP-tagged Vps29 was added to GUVs and analyzed as before. (E) PI-3-P is required for SNX-BAR membrane association. GUVs lacking PI-3-P were incubated with the same concentration of SNX-BAR complex as in C. (F, G) Effect of surfacebound Ypt7-GTP or Vps10 on SNX tubulation efficiency. GUVs carrying DGS-NTA were incubated with His-tagged Ypt7-GTP or Cy5-labeled Vps10 for 10 min at room temperature before SNX-BAR-GFP was added. Samples were examined by fluorescence microcopy. (H) Quantification of the tubulation efficiency of CSC and SNX-BAR complexes. Data are represented as mean ± SD.
Western blotting. As before, SNX-BAR (Vps17-GFP) did not bind to Ypt7, whereas CSC (Vps29-GFP) selectively bound to Ypt7-GTP ( Figure 5A). Strikingly, binding of CSC to Ypt7-GTP was lost as soon as equimolar SNX-BAR was present in the pulldown reaction ( Figure 5, A and B), suggesting that assembly of the retromer complex displaces Ypt7. We then used just the Nterminal fragment of Vps5, which has been suggested as a possible interaction site to the CSC (Collins et al., 2005), and also observed loss of CSC binding to Ypt7 (Supplemental Figure S1C). These observations suggest that the interface between SNX-BAR and CSC overlaps with the Ypt7 binding site.
To monitor the displacement of Ypt7 from CSC on membranes, we used a modified GUV-binding assay. As shown before, CSC can be recruited to GUVs by either Ypt7 or Vps10 (Figure 4, D and G). We therefore used Vps10-loaded GUVs to bind CSC to the membrane and then added Ypt7 as a fluorescently labeled but otherwise untagged protein. If SNX-BAR indeed competes with Ypt7 for CSC binding, Ypt7 should be displaced from GUVs if we add the SNX-BAR complex to the reaction. We initially tested Ypt7 binding to the GUV surface and observed CSC-( Figure 5, C and D) and GTP-dependent ( Figure 5F) recruitment. Of importance, addition of the SNX-BAR complex to this reaction resulted in a quantitative loss of Ypt7 from the GUV surface ( Figure 5, D vs. E), in agreement with Ypt7 displacement from the CSC on assembly of the entire SNX-BAR retromer complex on membranes.

DISCUSSION
Our data provide a molecular explanation for how Ypt7 can participate in two competing interactions on the endosomal surface ( Figure 5H). The GEF Mon1-Ccz1 promotes Ypt7 activation on endosomes. 1) CSC then binds Ypt7-GTP and cargo such as the Vps10 receptor, which enhances its ability to localize to endosomes. 2) Endosomes also carry the SNX-BAR complex that interacts with PI-3-P. Assembly of the retromer pentamer from the SNX-BAR and CSC subcomplexes 3) moves cargo into tubular structures and 4) displaces Ypt7 from the CSC. Ypt7-GTP then becomes available for the vacuolelocalized HOPS tethering complex, which promotes SNARE-dependent fusion of the mature endosomes with vacuoles.
Our findings have several important implications. Displacement of Ypt7 from endosomal tubules can explain why Ypt7 remains on the donor membrane when retromer tubules are formed (Arlt et al., 2015b). The findings are also consistent with the different vacuole phenotypes of the vps5∆ (SNX∆) and vps35∆ (CSC∆) strains (Liu et al., 2012). Whereas vacuoles in a strain lacking the CSC subunit membranes. We previously observed by in vivo analyses that Ypt7 remains on the vacuolar rim when SNX-BAR-and CSC-positive tubules emanate from vacuoles (Arlt et al., 2015b). This exclusion could be due to a selective inactivation on tubules by a retromerrecruited GAP (Seaman et al., 2009) or a consequence of the assembly between CSC and SNX-BAR subcomplex during tubule formation. We addressed the latter possibility using Rab pull down with Ypt7-GTP as a readout. For this, the SNX-BAR complex was titrated in a defined molar ratio relative to CSC to the pull-down reaction, and Ypt7-associated CSC was subsequently evaluated by with this, only a small fraction of Ypt7 was found on maturing endosomes (Arlt et al., 2015a), which could initially bind to the CSC before it becomes available for HOPS.
Our data agree with the findings in mammalian cells that CSC and SNX-BAR complexes exist as two separate complexes (Haft et al., 2000;Pylypenko et al., 2007;van Weering et al., 2012). In yeast as well, the SNX-BAR complex seems to localize earlier than the CSC to maturing endosomes (Arlt et al., 2015a), and both can be found on endosomal domains (Chi et al., 2014). We showed here that CSC binds to membranes containing either cargo, Ypt7-GTP or the SNX-BAR complex, in agreement with three binding sites along the Vps29-35-26 trimer (Collins et al., 2005;Liu et al., 2012;Seaman, 2012). The SNX-BAR complex requires PI-3-P for its endosomal recruitment and can efficiently tubulate membranes. Our in vitro assays suggest that assembly of the functional retromer complex during tubule formation occurs from two distinct subcomplexes also in yeast.
In agreement with this analogy, our electron microscopy analysis reveals a striking similarity of the yeast CSC to the human complex ( Figure 2). We could easily model all available structures and the Vps35 model into our structure and also show additional segments that agree nicely with the recently solved structure of the CSC (Vps35 and Vps26; Lucas et al., 2016). We can also solve the overall structure of the SNX-BAR complex, which likely reflects the inactive cytosolic conformation of the complex. Because the most complete SNX9 structure with its PX and BAR domain still lacks the first 200 residues (Pylypenko et al., 2007), we refrained from modeling the fragment into our density. The overall assembly of the SNX-BAR retromer cannot yet be deduced from our data, as the heteropentameric complex was not suitable for electron microscopy (EM) analysis.
Of interest, although several SNX proteins can act alone to tubulate membranes (van Weering et al., 2012), their activity seems lower than our full-length Vps5-17 complex, which generated long tubules. We do not yet know whether the tubulation of the SNX-BAR complex has been facilitated by the labile GUV surface, although we also detected strong tubulation of liposomes ( Figure  3A). This activity must be controlled in vivo, where the formation of tubules should be a consequence of available cargo and retromer assembly, as also suggested by mutant analyses (Seaman and Williams, 2002). Our present data show the qualitative interdependence between cargo, PI-3-P, and Ypt7 on recruiting retromer subcomplexes. We have, for instance, not yet resolved how cargo is concentrated into retromer-decorated tubules. Further analysis of the organization of assembled retromer on endosomal tubules, as also postulated recently (Lucas et al., 2016), and in particular its cross-talk with cargo, will be important for our understanding of retromer function.
The availability of Ypt7-GTP for fusion is likely only one critical parameter that determines maturation of the endosome. It is unclear how the activity of the ESCRT complexes on ubiquitinated cargo determines fusion competence of endosomes. It appears as if all endosomes are cleared of ESCRT cargoes before fusing with vacuoles. Similarly, the Rab5-like Vps21 is a substrate of the BLOC-1associated Msb3 protein, and no Vps21 is found on vacuoles in wildtype cells (Lachmann et al., 2012;John Peter et al., 2013;Rana et al., 2015). We consider it likely that retromer, ESCRT, and COR-VET act in parallel on endosomes to promote cargo sorting and fusion (Burd and Cullen, 2014;Arlt et al., 2015a). Only if all cargo is taken care of should fusion of late endosomes with the vacuole/lysosome be initiated. The availability of functional retromer will allow us to resolve this question in future studies.
Vps35 are round, they are fragmented in a vps5∆ strain, suggesting that the free CSC complex might compete with HOPS for Ypt7, as also observed in our fusion assay (Figure 1, F and G). In agreement FIGURE 5: Binding of SNX-BAR to CSC releases Ypt7. (A) Rab interaction of CSC with Ypt7 in the presence of SNX-BAR. Purified CSC and SNX-BAR complexes were incubated with GST-Ypt7 charged with GDP or GTP. For the competition assay, CSC was preincubated with Ypt7 for 15 min before the addition of the indicated relative molar amount of SNX-BAR complex. Incubations on further analysis were as in Figure 1E. (B) Quantification of Rab-CSC interaction; n = 3. (C-G) Interaction of Ypt7 with CSC is reduced upon SNX-BAR addition. GUVs carrying His-tagged Vps10 were incubated with ATTO-labeled Ypt7-GTP or -GDP without His tag (C). In D, GFPtagged CSC (Vps29-GFP) was added. In E and G, untagged SNX-BAR complex was added to the reaction in a fourfold excess over CSC. In F and G, Ypt7-GDP was used instead of Ypt7-GTP. All images were processed equally. (H) Model of SNX-BAR and CSC interaction with PI-3-P, cargo, and Ypt7. Assembly of the retromer complex displaces Ypt7, thus separating a retromer-positive tubule and a Ypt7-positive membrane. For further details, see the text. Ostrowicz et al., 2010). In brief, strains were grown in rich yeast extract/peptone medium supplemented with 2% galactose for 60 h at 30°C. Cells were lysed in buffer (50 mM Tris/HCl, pH 8.0, 300 mM NaCl, 1.5 mM MgCl 2 ) supplemented with 1 mM dithiothreitol (DTT) and protease inhibitor FY (Roche, Munich), and lysate was cleared by centrifugation at 4°C for 5 min at 3500 × g and then for 1 h at 50,000 × g. Cleared lysate was incubated with immunoglobulin G (IgG)-Sepharose beads (GE Healthcare) at 4°C for 1 h, followed by elution using tobacco etch virus (TEV) protease at 16°C for 90 min, and then used in subsequent assays, including EM. Where indicated, purified complexes were subjected to size-exclusion chromatography on a Superose 6 10/300 column (GE Healthcare).
GUVs were prepared using an electroformation protocol as described in Romanov et al. (2012). Briefly, the desired lipid mix (concentration 2 mM in 500 µl) was dissolved in a chloroform:methanol (2:1) solution, and 3 µl was spotted onto prewashed indium tin oxide-coated slides. Organic solvent was removed in vacuum, and slides were assembled to create a chamber for 500 µl of 300 mM sucrose, followed by electroformation using Vesicle Prep Pro (Nanion, Munich, Germany) in a 3-h cycle. GUVs were centrifuged onto a sucrose cushion at 120 × g for 20 min at 4°C using sedimentation buffer (1 mM HEPES/KOH, pH 7.4, 267 mM glucose, 1 mM DTT). The cushion consisted of sedimentation and swelling buffer (1 mM HEPES/KOH, pH 7.4, 240 mM sucrose, 1 mM DTT) mixed 1:1. GUVs were taken up into a final volume of 5 µl, resulting in a final concentration of 0.2 mM.

GUV assays and fluorescence microscopy
To determine protein binding and membrane deformation by retromer complexes, purified CSC or SNX-BAR complexes were used at a final concentration of 100-150 nM in the reaction. The complexes were first added to 5 µl of the concentrated GUVs (0.2 mM) in a final volume of 30 µl in phosphate-buffered saline (PBS) and then

Strains and reagents
All reagents were purchased from Sigma-Aldrich, unless noted otherwise. All Saccharomyces cerevisiae strains were generated from the respective parent BY strain by integration of PCR-amplified cassettes by homologous recombination as described previously (Longtine et al., 1998;Puig et al., 2001;Janke et al., 2004). Strains are listed in Table 1.

Plasmid generation
The C-terminal fragment (corresponding to amino acid residues 1416-1579) of VPS10 was amplified from genomic DNA of strain BY4727 and cloned into BamHI, SalI-digested pET32c expression vector to generate plasmid pET32c(-Trx)-VPS10 (1415-1579). The N-terminal fragment of Vps5 (residues 1-280) was amplified by PCR from genomic DNA and cloned into a pET24b expression vector using restriction sites NdeI and XhoI.

Vacuole fusion assay
Vacuole purification and fusion were performed as previously described (LaGrassa and Ungermann, 2005). In brief, vacuoles were purified from BJ3505 and DKY6281 strains and subjected to vacuole fusion in the presence of ATP. The indicated amounts of CSC and HOPS were added to the fusion reaction, and fusion was determined after 90 min of incubation at 26°C.
SNX-BAR and CSC complexes were purified from yeast using tandem affinity purification (TAP) as described (Puig et al., 2001;  incubated for 15 min before examination by microscopy. Preloading of GUVs with GTP-or GDP-loaded Ypt7 or Vps10 was carried out by incubating GUVs for 20 min at room temperature with 0.5 µM each protein. GUVs were imaged on an Olympus IX-71 inverted microscope using 60×/numerical aperture (NA) 1.40 and 100×/NA 1.49 objectives, InsightSSI illumination, a scientific complementary metaloxide semiconductor camera (PCO), and SoftWoRx software (Applied Precision). Usually, z-stacks of 0.2 µm in 15 optical slices to cover a total of 3 µm were acquired, followed by deconvolution using SoftWoRx software and image processing in ImageJ (National Institutes of Health, Bethesda, MD). GUV morphology was quantified by counting at least 50 GUVs per sample in three independent experiments.

Rab pull-down assay
The glutathione-Rab pull-down experiment was carried out as described (Markgraf et al., 2009). Recombinant GST-tagged Rab proteins were loaded with 1 mM GDP/GTP in 20 mM HEPES/ NaOH, pH 7.4, and 20 mM EDTA and subsequently incubated with GST beads (pretreated with the same buffer containing 5% bovine serum albumin) for 1 h at 4°C. Beads were washed, and retromer complexes (20-40 µg) were added for 1.5 h in 20 mM HEPES/NaOH, pH 7.4, 300 mM NaCl, and 1 mM MgCl 2, followed by washing in buffer and elution by addition of 20 mM EDTA, 0.1% Triton X-100. Proteins in eluates were precipitated by trichloroacetic acid and analyzed by SDS-PAGE and Western blotting.

Electron microscopy and image processing
Purified protein samples were negatively stained with 0.035 mg/ ml uranyl formate according to a previously described protocol (Ohi et al., 2004). The micrographs were collected with a chargecoupled device camera (Tvips, 4k × 4k) integrated into a Jeol JEM-1400 with LaB6 cathode at 120 kV. After manual selection of the single particles, reference-free and reference-based alignment, as well as K-means and ISAC classification, were performed with EMAN2 and SPARX. For three-dimensional (3D) reconstruction of the CSC, random conical tilt data were collected at −50 and 0°. We selected 2707 particle pairs using EMAN2 e2RCTboxer tool. All untilted particles were aligned and classified with EMAN2 and SPARX. A 3D model was calculated from different classes using the corresponding tilted particles. The resulting model was submitted to 3D multireference projection matching against the data set of 9875 single particles using SPARX. The 3D model of SNX was calculated with the VIPER algorithm. We aligned 2245 particles and classified K-means into 50 classes. These provided the template for ab initio 3D structure determination with sxviper (SPARX). The initial model was refined subsequently with the single particles.
For liposome cryo-EM, retromer complexes and liposomes were incubated for 1 h at room temperature before a 4-µl sample was applied on a Quantifoil 2/1 holey carbon grid, blotted from both sides with Whatman1 for 3.5 s, and plunged into liquid ethane using Cryoplunge3 with GentleBlot technology. The vitrified samples were imaged with a Jeol JEM-3200FSC electron microscope operating with a field emission gun at 200 kV. An in-column omega energy filter was used to improve image contrast by zero-loss filtering with a slit width of 15 eV. Micrographs were recorded for 3 s as 15 frame stacks with a K2 camera (Gatan) under minimal-dose conditions (20 e -/Å 2 ; Li et al., 2013).


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The species of Haplothrips (Thysanoptera, Phlaeothripinae) and related genera recorded from the Hawaiian Islands

Abstract An illustrated identification key is provided to 17 species of Thysanoptera: Phlaeothripinae from Hawaii that are members of the Tribe Haplothripini, together with a further species that is similar in general appearance to members of that Tribe. Of these 18 species, 13 are considered introduced from other parts of the world, but five appear to be endemics. Known only from Hawaii, Haplothrips fissus syn. n. is considered to have been based on a teratological specimen and is placed as a synonym of the Hawaiian endemic Haplothrips davisi. Both this species and two further endemics, Haplothrips rosai and Priesneria doliicornis, are possibly mycophagous rather than phytophagous. The Indonesian species Haplothrips sesuvii syn. n. is recognised as a synonym of Haplothrips robustus from Australia, although both names have been used in Hawaii. Two further species that are presumed to be Hawaiian endemics, Apterygothrips remotus and Haplothrips williamsi, remain known only from the original specimens.

Introduction
The Tribe Haplothripini constitutes one of three major lineages within the subfa mily Phlaeothripinae (Mound and Minaei 2007), and Haplothrips is by far the largest genus in this group, with 243 species listed worldwide (ThripsWiki 2017). Most Haplothrips species breed in flowers, and the recorded hosts involve a wide range of genera, particularly in the families Asteraceae and Poaceae. In contrast, some species of Haplothripini are predatory, and one or more species endemic to Hawaii are possibly mycophagous. The information presented here concerning Haplothripini recorded from the Hawaiian Islands is part of a projected overview of the thrips fauna of this island chain. The first part of this overview included an extensive list of references to publications on Thysanoptera from Hawaii, and concerned the 99 species of the sub-order Terebrantia (Mound et al. 2016). A very high proportion of this fauna comprises taxa that have been introduced relatively recently, with less than 10% of Terebrantia species, and only five of the 18 species considered here, likely to be native to these islands.
The members of the Haplothripini share the following character states: antennae 8-segmented; head usually with a maxillary bridge clearly developed; prosternum with paired basantra well-developed; fore wings with a constriction medially; tergite IX of males with setal pair S2 short and stout; male sternite VIII usually without a pore plate. However, as is common in the diagnoses of other major groups of Thysanoptera -Phlaeothripidae, each of these character states is absent in one or more species of Haplothripini. One of the species included here, Bamboosiella cingulata, is included because it is similar in size and shape to species of Haplothrips and Karnyothrips, and is often taken with such species from grasses. However, the genus Bamboosiella is not considered a member of the Haplothripini (Okajima 2006;Mound and Minaei 2007). A further Haplothripini species, Leptothrips mali (Fitch), that is listed in the "Hawaiian all-species checklist database" (Bishop Museum 2002), is not included here because the record is indicated as based on a quarantine interception. There is no evidence that this North American predatory species has ever been found living on any of the Hawaiian Islands. Members of the genus Leptothrips are readily distinguished from almost all other Phlaeothripinae by the striate metanotum (Fig. 11). Full nomenclatural information about Thysanoptera is available on the web (ThripsWiki 2017).

1
Maxillary stylets restricted to mouth cone, not retracted anterior to dorsal posterior margin of head (Fig. 3); prosternal basantra weakly developed [body strongly bicoloured, head, thorax and abdominal segments IX-X brown but segments I-VII and all tibiae yellow (Fig. 1) Fore tibia inner margin with sub-apical tubercle and small setal-bearing tubercle (Fig. 16); prosternal basantra longer than wide (Fig. 9) .....Podothrips lucasseni -Fore tibia with no tubercle at inner apical margin; prosternal basantra usually wider than long (Fig. 10)  Remarks. Described originally from Australia, with the synonym sakimurai from Oahu by Moulton (1937), this species is widespread throughout the tropics, and has been referred to frequently as Antillothrips cingulata. It is sometimes abundant, living in association with the leaves of tussocks of grass, but adults disperse and alight on other plants, and on Oahu have been recorded from lettuce leaves. The species is similar in size and colour to Karnyothrips melaleucus, with the head, thorax and abdominal apex dark brown but abdominal segments I-VII yellow. However, the maxillary stylets are short and restricted to the mouth cone (Fig. 3), and the prosternal basantra are only faintly indicated, whereas these prosternal sclerites are large in Karnyothrips species (cf Figs 8-10). The male remains unknown, and the genus is not regarded as a member of the Haplothripini (Okajima 2006).

Androthrips ramachandrai Karny
Remarks. Described from India, this species has been reported from warmer parts of the Americas living in the leaf galls of Gynaikothrips species on Ficus trees (Boyd and Held 2006). In common with other members of the genus Androthrips, it has enlarged fore femora with a small tubercle at the base on the inner margin (Fig. 5). In contrast to some similar members of this genus the hind tibiae are dark brown, although antennal segments III-V are yellow. It is a predator of gall-inducing thrips (Melo et al. 2013), and in Australia has been found in galls induced by various species of Phlaeothripidae. On Hawaii, in the vicinity of Hilo, it has been found twice on the leaves of Ficus.
Apterygothrips remotus (Bianchi) Remarks. Described in the genus Pseudocryptothrips, but subsequently redescribed and transferred to Apterygothrips by Sakimura and Bianchi (1977), this species apparently remains known only from the original three females that were collected on Haleakala, Maui. These specimens were described as apterae, lacking ocelli, with the mesopresternum reduced to a pair of small lateral triangles, antennal segment III with one sense cone and segment IV with two, and all the major setae with their apices pointed.

Dolichothrips franae Mound & Okajima
Remarks. Species of the genus Dolichothrips have the mouth cone unusually long and pointed (Fig. 8). D. franae has previously been known only from Oahu and Kauai, where it was living on the leaves of Macaranga tanarius. However, eight females have been studied that were collected in October 2014 from Hibiscus tiliaceus on Oahu, and a breeding population was found on Hawaii in May 2016 on the leaves of Hibiscus brakenridgei. This thrips species is known only from females, and it is likely to have been introduced to the Hawaiian Islands from somewhere in Southeast Asia (Mound and Okajima 2015). In contrast to the following species, the pronotum is extensively sculptured (Fig. 6).

Dolichothrips indicus (Hood)
Remarks. Although described from India, this species has been found widely across the Pacific, including Japan, Taiwan, Guam, New Caledonia, and French Polynesia (Mound and Okajima 2015 Remarks. The single female from which fissus was described is identical in structure with specimens of davisi, except that the two sub-median dorsal anal setae are bifurcate at a point approximately two-thirds along their length (Fig. 14). This condition is here interpreted as teratological, possibly resulting from damage as a pupa, although such aberrations are more commonly associated with damage to the anterior rather than the posterior end of phlaeothripid pupae. Described from nearly 20 specimens taken on Hawaii, both sexes of davisi were collected in July 2016 on Hawaii on Mauna Kea, from dead Acacia koa bearing lichens, and on Maui on Haleakala at approximately 2500m, from dead Styphelia with lichens. Although similar in general appearance to rosai, the two sense cones on each of antennal segments III and IV are stouter, segment VIII is broad at the base (Fig. 19), the major setae on tergite IX are longer than the tube (Fig. 14), and the male lacks a sternal pore plate. The pronotal anteromarginal and anteroangular pairs of setae are equally long, but they vary in length from scarcely three to more than five times as long as the pronotal discal setae. In the field, the adults could be mistaken for Karnyothrips flavipes, although the antennae and hind tibiae are dark brown, and in both sexes the fore tarsi lack a prominent tooth.

Haplothrips gowdeyi (Franklin)
Remarks. Widely found in tropical countries around the world, this species is usually recognisable by the pale yellow colour of antennal segments III-VI that are in sharp contrast to the dark brown of the rest of the body. However, on the Hawaiian Islands there is a second species, Haplothrips kurdjumovi, that is quite similar in colour and general appearance. Despite the superficial similarities, gowdeyi has antennal segment III scarcely longer than wide, and thus almost sub-spherical (Fig. 20), and this segment bears two sense cones. These differences are discussed further below. Although reported as breeding in the inflorescences of grasses, gowdeyi also breeds in other flowers including Asteraceae (Mound and Wells 2015). Despite being widespread and common in tropical countries there have been no detailed studies on its biology.

Haplothrips graminis Hood
Remarks. Described originally from Texas, and the synonymic species fusca Moulton from Oahu, this grass-living thrips is known to be widespread through Central America (Mound and Marullo 1996

Haplothrips kurdjumovi Karny
Remarks. Described from Russia and widespread in Central Europe across Asia, this species is also introduced to North America and New Zealand (Minaei and Mound 2008). It appears to have been first identified from Hawaii in 2011, but in July 2016 considerable numbers of females were found at Volcano on Carex inflorescences as well as in the flowers of Pyracantha and Rubus. Moreover, two females were collected on Oahu at Palikea and the Mokuleia Trail. In the field it is easily mistaken for gowdeyi, because of the dark legs and almost clear yellow of antennal segments III-VI. Under a stereo microscope it is clear that antennal segment III is longer than wide and slightly asymmetric (Fig. 21), and on slide mounted specimens the presence of only a single sense cone on this segment can be confirmed. The species is reported to be a predator of mites and Lepidoptera eggs, but probably also feeds on floral tissues.

Haplothrips leucanthemi (Schrank)
Remarks. This species, under the name niger, was reported from Maui by Sakimura (1990), based on specimens taken on Haleakala at an altitude of 6500-8500 ft from the grass Anthoxanthum odoratum, both in 1963 and 1977. However, the host plants of this thrips are not likely to be any species of Poaceae. Bisexual populations occur widely in Europe in the flowers of certain species of Asteraceae, particularly Chrysanthemum leucanthemum, the ox-eye daisy. A uni-sexual form that is commonly referred to as H. niger, occurs in the flowers of red clover, Trifolium pratense. Efforts by workers in Europe to distinguish the two forms morphometrically have been less than successful (Mound and Minaei 2007), and niger is currently considered a synonym. The species is unusual within the genus Haplothrips because of the short postocular setae on the head that do not extend to the posterior margin of the compound eyes (Fig. 4). Although not collected recently on the Hawaiian Islands, this European species is known from southern Australia, New Zealand, North America, Chile and Argentina (Hoddle et al. 2012).

Haplothrips rosai Bianchi
Remarks. Described from three female and three male micropterae collected on Hawaii in the vicinity of Volcano, a single male of this species was taken in July 2016 on the lower slopes of Haleakala, Maui. In general appearance if is similar to davisi, but the pronotal anteromarginal pair of setae are no longer than the pronotal discal setae (Fig. 15), the major setae on tergite IX are shorter than the tube, and the male has a conspicuous transverse pore plate on the eighth sternite. Antennal segments III and IV each bear two rather weak and slender sense cones, and segment VIII is unusual in being constricted to the base. As with davisi, this apparently endemic species is probably mycophagous.

Remarks.
This new synonymy is based on a comparison of a paralectotype of sesuvii from Java, on loan from the Senckenberg Museum, Frankfurt, with more than 50 specimens of robustus from across Australia, some of which were earlier compared with the holotype of this species in the Natural History Museum, London (Pitkin 1973;Mound and Minaei 2007). These two species were distinguished from each other by zur Strassen (1983), who provided seven sets of measurements, including wing width, maxillary bridge width and lengths of setae on tergites VIII and IX, each of which was larger in robustus than in sesuvii. However, although zur Strassen provided details of the type specimens of sesuvii that he studied, he did not indicate from what specimens of robustus his comparative measurements were taken. Measurements of the paralectotype of sesuvii fit well within the range of structural variation that occurs in robustus females from across Australia, and this variation is not unusual for a polyphagous, widespread species. Although known from Indonesia, Australia, New Hebrides, and Oahu, no male of this species is known, and the possibility exists that it may have been introduced from somewhere in Africa (Mound and Minaei 2007). In the Hawaiian Islands this thrips is recorded only from Oahu. The first record (Bianchi 1947), under the name sesuvii, was from Sesuvium portulacastrum [Aizoaceae] but specimens have been studied that were collected on Oahu by Sakimura at various dates between 1946 and1970 from Lipochaeta [Asteraceae] and Atriplex [Chenopodiaceae]. Amongst the Hawaiian thrips fauna it is the only species with broad fore wings that are constricted medially but lack duplicated cilia on the distal posterior margin (Fig. 18).

Haplothrips williamsi Moulton
Remarks. This species apparently remains known only from the original series of eight females taken at the western end of Hawaii in 1929. The original description was rather superficial, but the holotype and one paratype have now been re-examined. Based on these specimens, williamsi is one of the very few amongst the 240 species of Haplothrips that has two sense cones on antennal segment III but on segment IV has only three (instead of four) sense cones. The head (Fig. 13) and body are light brown with the tube darkest. The legs are almost clear yellow as is antennal segment III in contrast to the uniformly dark brown of segments IV-VIII. The antennal sense cones are rather stout with rounded apices. The maxillary stylets are retracted to the postocular setae, and one third of the head width apart with a distinct maxillary bridge. The fore tarsus has a slight thickening at the inner apex that might be interpreted as a particularly minute tooth. The metanotum lacks sculpture medially, and the median major setae are finely pointed.
In contrast, most of the major setae are capitate including S1 on tergite VIII, but S2 on VIII and all three pairs on IX are finely acute and longer than the tube. This species shows interesting similarities to the endemic species davisi and rosai, in the the fore tarsus and the conical form of antennal segment III, also the elongate acute setae on tergite IX.

Karnyothrips flavipes (Jones)
Remarks. This is one of the most commonly collected Phlaeothripidae on the Hawaiian Islands, where it has been taken widely, living on dead branches and twigs. Although common and widespread on the islands, it is rarely found in large numbers, a characteristic that agrees with its habits as a predator of other small arthropods (Jaramilo et al. 2010). Small and dark brown, with a slightly elongate head (Fig. 12), it is usually recognisable from the slightly yellow colour of antennal segment III, and the hind tibiae that are yellowish on at least the distal half. The number of antennal sense cones varies in this species, with segment IV bearing either three or four, although segment III consistently bears two sense cones (Okajima 2006). The fore tarsus bears a prominent curved tooth at the inner apex (Fig.  12), and the major setae S1 and S2 on tergite VIII are capitate, as is S1 on the ninth tergite.

Karnyothrips longiceps (Hood)
Remarks. There is a slide of this species in the USNM collection, Beltsville, taken in quarantine from Hawaii at San Pedro, California in March, 1969, and this is possibly the basis for longiceps being listed from Hawaii. However, two micropterous females of this species were collected from dead Acacia koa on Hawaii in July 2016, one at Hakalau, Mauna Kea, the other at Kipuka Ki, Hawaii Volcanoes National Park. Described originally from Illinois, USA, it appears to be widespread south through Central America to Chile and Peru (Mound and Marullo 1996). It is the same size and colour as flavipes, but the fore tarsal tooth is minute and the hind tibiae almost completely yellow. Apart from the presence of two pairs of sigmoid setae on the tergites, longiceps could equally well be placed in Apterygothrips, since these two genera are not clearly distinguished by the available generic diagnoses.
Although not taken as frequently as K. flavipes, this worldwide bicoloured predatory thrips occurs widely on the Hawaiian Islands. It is found more frequently on grasses than on dead branches, and is similar in general appearance to Bamboosiella cingulata (Figs 1-2). However, in contrast to that species the maxillary stylets are deeply retracted and close together medially in the head, the prosternal basantra are well-sclerotised, the mid and hind legs are clear yellow, and tergite VIII is yellow at least in part.

Podothrips lucasseni (Krüger)
Remarks. Described from Java, with one synonym from Hawaii and another from Thailand, this species is widespread in tropical Asia from India to northern Australia (Ritchie 1974), but specimens have also been studied from South Africa and Madagascar. It is a predatory species on coccoids living on Poaceae, and is particularly associated with sugar cane. A brown species with clear yellow tibiae, it is one of the species of Podothrips in which the fore wings lack duplicated cilia. The fore femora inner margin is slightly rugose, and in large individuals is sometimes rather swollen. But it is unusual within the genus in having a pair of small triangular sclerites lateral to the pelta that represents the first abdominal tergite. As in other members of the genus Podothrips, the prosternal basantra are longer than wide (Fig. 9), and there is a small tubercle on the inner apex of the fore tibiae (Fig. 16).

Priesneria doliicornis (Bianchi)
Remarks. Described in Karnyothrips but transferred by Pitkin (1973) to the genus Priesneria because of the curious third antennal segment (Fig. 23), the relationships of this species remain in some doubt. Two species with similar antennae are described in the genus Karnyothrips from southern Japan (Okajima 2006), of which antennalis has one sense cone on segment III and three on segment IV, whereas inflatus is particularly similar to doliicornis in having two on both III and IV. A third species with similar antennal structure has been described in Karnyothrips from southern China (Wang et al. 2013), but that has two sense cones on segment III and four on IV. The curious shape of antennal segment III, and the number of sense cones on III and IV, may not be good indicators of relationships in the Karnyothrips group. Bianchi described doliicornis from 10 females collected on Hawaii, of which nine were micropterous and one macropterous. Two micropterous females were collected in July 2016 at sites near Volcano, Hawaii, one on dead branches of Metrosideros, and one on dead branches of Dodonaea.