Continental shelf sediments are globally important for biogeochemical activity. Quantification of shelf-scale stocks and fluxes of carbon and nutrients requires the extrapolation of observations made at limited points in space and time. The procedure for selecting exemplar sites to form the basis of this up-scaling is discussed in relation to a UK-funded research programme investigating biogeochemistry in shelf seas. A three-step selection process is proposed in which (1) a target area representative of UK shelf sediment heterogeneity is selected, (2) the target area is assessed for spatial heterogeneity in sediment and habitat type, bed and water column structure and hydrodynamic forcing, and (3) study sites are selected within this target area encompassing the range of spatial heterogeneity required to address key scientific questions regarding shelf scale biogeochemistry, and minimise confounding variables. This led to the selection of four sites within the Celtic Sea that are significantly different in terms of their sediment, bed structure, and macrofaunal, meiofaunal and microbial community structures and diversity, but have minimal variations in water depth, tidal and wave magnitudes and directions, temperature and salinity. They form the basis of a research cruise programme of observation, sampling and experimentation encompassing the spring bloom cycle. Typical variation in key biogeochemical, sediment, biological and hydrodynamic parameters over a pre to post bloom period are presented, with a discussion of anthropogenic influences in the region. This methodology ensures the best likelihood of site-specific work being useful for up-scaling activities, increasing our understanding of benthic biogeochemistry at the UK-shelf scale.The online version of this article (doi:10.1007/s10533-017-0366-1) contains supplementary material, which is available to authorized users. Continental shelf sediments make up less than 9% of the global seafloor, and yet are responsible for the majority of global benthic biogeochemical cycling of organic matter  programme to choose representative benthic sites on the UK continental shelf. The overarching objectives of the SSB programme were to (i) assess carbon and nutrients cycling and their controls on primary and secondary production in UK and European shelf seas, (ii) to increase our understanding of these processes and their role in wider biogeochemical cycles, and (iii) significantly improve predictive marine biogeochemical and ecosystem models over a range of scales. The approach taken is one of regional-local–regional scaling, which ensures a maintained focus on the wider regional context throughout the project. Such nested sampling designs have been shown to successfully overcome problems associated with spatial scaling . As such, the full methodologies for the techniques used to generate the data presented  are referenced in the appropriate places within the results section and can be found detailed in Online Resource 1. However, the methods used to collect the samples during an 18 month long cruise programme carried out between 2014 and 2015 are now described. All data collected during the SSB programme is archived with the British Oceanographic Data Centre, (http://www.bodc.ac.uk), and corresponding accession/DOI numbers can be found in Online Resource 1. Unless otherwise specified, statistical relationships between sites are determined using the standard error of the mean, based on the central limit theorem.The Shelf Sea Biogeochemistry (SSB) programme is an interdisciplinary programme, with wide ranging objectives, aims and deliverables  for continuous monitoring of near-bed water column parameters. They measured conductivity and temperature, pressure, turbidity, oxygen saturation and chlorophyll fluorescence for bursts of 5 min repeated every 30 min at a sampling frequency of 1 Hz. Measurements of currents and backscatter over approximately the bottom 40 m of the water column were recorded in burst mode for 5 min every hour at a sampling frequency of 1 Hz, a temporal resolution sufficient to quantify turbulence. Intra-tidal Monitoring The National Oceanography Centre (NOC) Liverpool designed the ministable lander to allow shorter-term, higher frequency intra-tidal monitoring of near-bed properties, including velocity, oxygen eddy correlation, water column backscatter (turbidity), bed surface roughness and bedform migration, suspended sediment size, nitrate, temperature, conductivity and depth. Buoys Cefas designed SmartBuoys provide a long term high-frequency time series (at 1 m below sea surface) of salinity, temperature, turbidity, oxygen saturation, chlorophyll fluorescence, photosynthetically active light climate, and water samples for nutrient analysis. The M5 Wexford Coast wave buoy (51.69°N 06.704°W since 2004), part of the Irish Weather Buoy Network provided long-term wave parameters for the region. Lander and Buoy deployment locations and durations can be found in Online Resource 2. Underway data pCO2 and chlorophyll a data were collected while underway throughout the cruise programme. CTD Water column profiles of temperature, salinity, depth, chlorophyll fluorescence and turbidity were collected, along with water samples for sensor calibration, nutrient and Iron analysis using both standard and titanium (ultra-clean) Sea-Bird CTD systems. Fishing Activity Fishing activities and intensities were assessed using the AIS (Automatic Identification System) and Autosub sidescan imaging.Autonomous underwater vehicle survey The Autonomous Underwater Vehicles (AUVs) Autosub3 and Autosub6000  Boxcorer (K16) with 320 mm diameter cylindrical core barrels. In many cases these were then sub-sampled to provide specific sized cores or sediment samples for subsequent experimentation and analysis. Larger sediment samples for faunal analysis were collected using an USNEL-type 500 mm square Scottish Marine Biological Association (SMBA) Box Corer. A Bowers and Conelley Megacorer was used to take multiple (up to 12) simultaneous sediment samples for iron pore-water analysis  camera was used to capture in situ vertical profile images of the top few centimetres of the seabed, including the sediment–water interface  that exhibit: different biogeochemical exchange mechanisms; varied faunal abundance, diversity and function, while staying within a similar hydrodynamic environment. Confounding variables are reduced by adopting a narrow range of depth, temperature and hydrographic variations. To make this selection, a full assessment of the typical conditions within the Celtic Sea is necessary.−1 close to the seaward boundary, but increasing to 1.6 m s−1 in the Bristol Channel  near the shelf break to 1 m (6 s peak wave period) where the Celtic Sea meets the Irish Sea, while extreme values for a return period of 1 year reach significant wave heights in excess of 8–10 m and peak periods of approximately 15 s , although a seasonal pattern is seen in more central regions, with the bulk of activities taking place in spring and summer  within which to limit long-term observational measurements, cruise operations and in situ experimentation. This restricts sampling to an area of minimal topographic and depth variation, away from the shallower coastal regions where bed stresses are higher, and increasingly varied, and away from freshwater inputs which would affect salinity and temperature. The area contains a wide range of sediment and therefore habitat types, and minimises variations in depth and regional hydrodynamics. To further limit potential depth and hydrodynamic variations, an approximately 20 km wide transect running from the south-west to the north-east across this region  was identified. The same selection conditions were met, but the required coverage was reduced to an area of approximately 2500 km2. The next step was to make a full assessment of the spatial heterogeneity within this new, limited, target area and select discrete sampling sites suitable for repeat seasonal sampling, and representative of the dominant habitat types and biogeochemical exchange mechanisms of the shelf.The selected target area provides a constrained region on the inner shelf of approximately 87 × 95 km .Four benthic Landers were deployed at The Celtic Deep 2 (CD2L) and East of the Celtic Deep (ECD) both to the North of the region, Nymph Bank (NB) in the central region and East of Haig Fras (EHF) to the South in areas which have similar hydrodynamic regimes , but a range of bed types. Consideration was also made to existing infrastructure: a SmartBuoy has been located at the Celtic Deep (CD) site since 2009, and was moved to Celtic Deep 2 (CD2) in 2012. In addition, a SmartBuoy was located at the shelf edge (Candyfloss) for assessments of shelf exchanges and links to the pelagic component of the SSB programme (−1), followed by CD2L and ECD (0.36 m s−1) and weakest at NB (0.32 m s−1). There is a similar behaviour for the maximum bed shear stress , but the minimum bed shear stress is significantly higher at ECD (0.02 Nm−2 vs. zero at the other three locations) resulting in an increase of the mean bed shear stress. The tidal ellipses also vary from near circular ellipses at ECD to near rectilinear at EHF matching the expected behaviour of the wider Celtic Sea region, with the polarity of the ellipse anti-clockwise for ECD, CD2L and NB, but clockwise for EHF.Measured tides in the target area Fig.  are domi−1, with a maximum of 22.9 m s−1. There is a strong seasonal signal, with daily mean values of 6.5 m s−1 during the summer, and 10.3 m s−1 in winter. The M5 Wexford coast wave buoy shows winter waves have a mean height of 2.3 m with a maximum recorded height of 8.1 m in January, and summer mean wave height of 1.4 m.Mean daily wind speeds between 2012 and 2015 were 8.1 m s−1 in Summer and Autumn, also typical of offshore shelf waters  and is limited in range in winter (0.2 and 0.4 m−1).Measured surface temperatures since 2009 ranged between 8.06–19.73 °C (mean 13 °C). Stratification formed in early April in both 2014 and 2015, with re-mixing in mid-December in 2014. This is in keeping with prior observations  and occurred later in the season. Moderate Resolution Imaging Spectroradiometer  satellite data demonstrate that the spring bloom was initiated in early April 2015 coinciding with the onset of stratification, with full bloom conditions observed by mid-April 2015 . However, there was considerable overlap in community composition between closely related sediment types. This was due principally to many benthic species present having broad habitat preferences occurring in multiple sediment habitats. In addition, despite changes in community composition between sediment types, levels of macrofaunal abundance, biomass and diversity remained largely constant across all the samples with perhaps only a slight reduction in these parameters for the sites with the highest fines percentages to the Northeast  within which sampling is constrained, minimising local heterogeneity while ensuring sufficient space to resample the sites without on-going impacts from previous sampling efforts. Process site names represent the order in which they were ground-truthed and are presented according to decreasing fines percentage. The boxes with the highest percentages of fines (A) and sand (G) are used to represent the end-members of the observed spectrum, with the sites H and I displaying intermediate values on the continuum.Each process site is represented by a 0.25 kmThe full benthic Shelf Seas Biogeochemistry programme visited each site four times, to assess seasonal differences across each of the sites, and assess conditions prior to, during and after the spring bloom Table . Much ofThese cruises used a combination of in situ observation, sediment and biological sampling and experimentation to make assessments of biogeochemical processes occurring at each of the sites. While site selection was based on data collected in DY008 and DY021, the data presented below represent typical values averaged over all four cruises, to provide baseline ranges throughout the year for each site, providing the most thorough assessment of site representativeness to the wider target area and Celtic Sea region.2 saturation (p < 0.0001) are apparent which, given the water column similarities between the sites, likely result from differences in the bed sediment or habitat type. Turbidity is highest at ECD, which also corresponds to the highest O2 saturation.The long-term Lander data can be used to assess the hydrodynamic conditions occurring at the process sites Table , to conf2 , although H and I fall into the same textural classification Table . In summThe structure of the near-bed sediment (top 5 cm) was also assessed for each of the sites Table . Depth aSmall-scale seabed topography is provided from acoustic images of the bed measured by the 3D Acoustic Ripple Profiler (ARP) on the miniSTABLE intra-tidal monitoring lander. Results for the four sites show a variation in bed height of up to 4 cm Fig. . Bed strThe analysis described confirms that the four process sites can be considered as statistically different from each other in terms of the sedimentary characteristics (a key scientific variable of the SSB programme), showing a clear and concurrently occurring seasonal signal (key variable), while being similar in terms of hydrodynamic parameters (confounding variables).Assessments were made of key biogeochemical and biological parameters Tables , 6, meas2 consumption nearest the spring bloom. These seasonal signals are discussed further in Hicks et al.  in all cases, except for organic nitrogen between H and G. These trends are maintained with similar magnitudes when considered seasonally, except for site G, where the core used for analysis had a much higher fines content than typical for this site. Oxygen penetration depths are significantly different only between I and G, although total oxygen consumption rates are significantly different in all cases except between I and H. It should be noted, however, that total oxygen consumption rates are calculated based on the combination of data from three different analytical methods providing total oxygen uptake rates, diffusive oxygen uptake rates and oxygen penetration depths, and are discussed in more detail in Hicks et al.  and Smits et al. . Chlorop4+ ranges between 0.23 and 145 μM across all sites and cruises. The concentrations at Sites A, H and I generally increase from the sediment surface to 10 cm depth, and are relatively stable below 10 cm .Pore water nutrient concentrations were measured in triplicate usually down to 20 cm using a depth variable resolution. Data averaged for 0–10 cm are presented Table . The con cm Fig. . At SiteTOxN is usually at a maximum in the top 2 cm except at Site G where values at depth are occasionally higher than at the surface, with a maximum value of 16.6 μM. Nitrite ranged between 0.07 and 8.27 μM and is generally evenly distributed throughout the top 20 cm. The differences between sites are not statistically significant, however, this is likely due in part to large ranges resulting from measurements averaged over the different seasons  and decrease sharply across the oxic surface layer . Average surface (0–2 cm depth) porewater Fe concentrations are highest at site I, lowest at site H and intermediate at site A  using two similar sampling methods . The range of fluxes measured at each site for all nutrients was such that there was no significant difference when considered spatially between sites. Both nitrite and TOxN fluxes are lowest on average at site A and increased through sites I and H, with the highest average fluxes at site G. The greatest range in nitrite and TOxN fluxes are at site H (−0.035 to 0.132 and −0.586 to 0.649 mmol m−2 d−1 respectively). The fluxes of ammonium are highly variable at all four sites, and site I is the only one to be negative overall with an average flux −0.003 mmol m−2 d−1. Sites G and H have the highest fluxes of ammonium (>0.04 mmol m−2 d−1) with the greatest range at site H. Silicate fluxes are on average highest at site A (1.212 mmol m−2 d−1) almost double that of the other sites. Site H and I silicate fluxes are very similar with the lowest fluxes at site G (0.531 mmol m−2 d−1). Phosphate fluxes are highest at Site A, which has a negative flux (into the sediment) on average (−0.018 mmol m−2 d−1) and has the smallest range of fluxes compared to the other three sites.On average, the fluxes of all macronutrients are positive, indicating a general release of macronutrients from the sediments into the water column. However, both negative and positive nutrient fluxes are measured at all sites, except for silicate fluxes at site A, which were consistently positive , and 3-times lower at the site with the coarsest sediments, site H (2.70 ± 5.54 × 10−3 mmol m−2 d−1). However, the range in Fe flux calculations is also greatest at site A, and equal to the range across all sites, while the range is smallest at site H. It is important to note that our assessment of diffusive Fe flux requires a simplification of benthic exchange processes. For example, the roles of advection and bioturbation/bioirrigation at these sites are not accounted for directly in the presented results, and yet they can serve to enhance the transport of Fe .Diffusive iron (Fe) fluxes are positive at all sites ranging from 0.01 to 54.4 × 10Note that some shallow burrowing infauna were also collected, but for clarity all fauna collected in the trawls will be termed as epifauna.At all sites, epifaunal organisms are rather sparsely distributed Table . Average2). Three phyla dominated the three sites: (1) Cnidaria are the most dominant at Site I and H and the third dominant at Site G; (2) Arthropoda is the second dominant at all sites; and (3) Echinodermata is the dominant at Site G and the third dominant at Site H and I.Autosub3 seabed photographs were also analysed to estimate faunal density and biomass during DY034. At the time of survey, near-bottom water column turbidity at Site A prevented the acquisition of useful seabed photographs. All megabenthos and demersal fish were counted, measured and identified to the lowest taxonomic level possible (Table 2 NIOZ boxcorer (macrofauna).All sites contain very few large infaunal species with no single sample containing more than a couple of individuals. It is concluded that, due to their low densities, large (>1 cm) infaunal organisms are not a substantial part of the benthic fauna in the study area and that adequate sampling of the benthic fauna is provided by the Jennings trawl (large epifauna) and the 0.08 mMacrofaunal abundance is highest at sites I and H. Site A has slightly lower average abundance, significantly lower than H and G (p < 0.05) whilst site (G) has less than 50% of the abundance of the other three sites (p < 0.0001).−2) is considerably (2–3×) higher at site A than at the other three sites. This indicates that the average body size of macrofauna is larger at site A than at the other three sites.In direct contrast to abundance, wet weight biomass (g m2 core (a measure of α-diversity) is highest in the intermediate sites H and I, with significantly lower diversity seen at sites A (p < 0.001) and G (p < 0.0001). However, the cores taken at site G are much more variable in terms of species composition and this higher variability in species between replicate samples (β-diversity) meant that the total number of species identified at site G is the same as site I and only a little less than site H. Site A displays relatively low diversity compared to the other sites.The average number of species per 0.08 mMacrofauna abundance and biomass data were combined with published trait information describing modes of sediment reworking and mobility , maximum  and mean  mixed depths of particle redistribution are presented in Table Nephrops norvegicus and Goneplax rhomboides. Bioturbation activity is observed to peak in August with sediment surface mixing occurring to a depth of approximately 8 mm.Macrofaunal bioturbation activity was measured through quantification of redistribution of fluorescent particle tracers and absolute changes in concentrations of the inert tracer sodium bromide respectively . Activity levels are very low Fig.  across tNote that only data from the first two cruises (DY008 and DY021) are presented here.3 Ind m−2 and maximum values of >1200 × 103 Ind m−2. Sites I, G and H are very similar in terms of meiofauna abundance, with average values lying between 550 and 600 × 103 ind m−2, however the differences are significant (p < 0.05). Muddy sediments are known to harbour greater densities of nematodes , and G and H are similar . As with abundance values, biomass values lie within the ranges observed for European subtidal areas  site A and I are very similar , and, like abundance and biomass, considerable similarity was found for site pairs A and I (p = 0.635), and G and H (p = 0.054), whilst all other pairwise comparisons show significant differences (p ≤ 0.05).Porosity Table  is a majBacterial 16S rRNA genes dominate the total microbial assemblages within coastal sediments, with reports of only 2% of 16S rRNA genes affiliated with archaea DeLong . Our datHabitat variations across the four sites echo the differences in sediment variation seen within the constrained target area, and confirmed that the process study sites represent significantly different habitats. These differences were also reflected in the bulk biogeochemical properties of the bed, although seasonal variability in pore water concentrations and nutrient fluxes are sufficient to mask spatial variability between the sites.We have described the way the four process study sites, which encompass the range of sediment and habitat variation seen in UK shelf seas, were identified within a constrained target area of the Celtic Sea, for investigation within the benthic component of the SSB programme. The sites differ significantly in terms of sediment, habitat type and bed structure, whereas differences in confounding physicochemical variables are minimal and seasonal changes (e.g. the phenology and magnitude of the spring bloom) occurred concurrently across the sites. This provides discrete, exemplar process study sites across the appropriate range of bed types to represent the wider region, for targeted field campaigns as part of the SSB programme.Logistical limitations to in situ observations, sampling and experimentation are unavoidable, and decisions must often be made early in the project planning stages regarding site selection. In shelf sea environments, which are both spatially and temporally variable at a range of scales, this site selection process becomes particularly important; especially where results are intended to be up-scaled and used to represent or model systems at shelf or regional scales.In these cases, as in the SSB Programme, the key to addressing such issues is to consider these scaling necessities from the outset, and to assess regional scales and variability during the site selection process . To address this, sufficient replication is required to determine the variability within the data, in order to interpret whether any temporal/seasonal changes observed fall within the natural spatial variability of the sites  justified the use of the surface sediment coverage model  were similar across sites, thereby minimising the impact of these confounding variables.Throughout the selection process, it was essential to maintain a clear focus on the scientific objectives of the programme, set out in the overarching aims of the SSB programme. However, the shelf is a complicated system, and local environmental conditions such as bottom water temperature, oxygen and nutrient concentrations and pelagic primary production inputs are all known to affect biogeochemical cycling within shelf sediments .Marine observations and experiments often aim to investigate conditions relative to a defined baseline condition, to quantify change  for each of the four process sites. This index is derived from the relative distribution of individuals across five ecological groups spanning a range of sensitivities to disturbance  which gained protection from a known long term monitoring position of which fishermen were aware.An additional consideration when interpreting the data collected in a programme such as this is that data collection focuses on a limited window of time—in this case a little over a year. Spatial patterns are likely to change over time, and the interactive effects of spatial and temporal changes are likely to mean that each site evolves along a different trajectory ;Determination of whether existing, larger scale models and predictions of shelf-scale heterogeneity (used in step 1) are accurate; essential for subsequent up-scaling.Step Two: A full assessment of the variability within this target area allows:Fully encompass the range of spatial heterogeneity occurring across the target area;Be sufficiently different in terms of the key scientific variables;Be sufficiently similar in terms of confounding variables;Be small enough to minimise within-site heterogeneity, which can then be addressed through sufficient replication;Have sufficient replication across scales to have sufficient statistical power to find hypothesised differences among metrics.Be large enough to reduce over-sampling during repeat, seasonal visits.Step Three: The final choice of process study sites requires them to:In relation to the SSB programme, following the above procedure led to the selection of four exemplar process study sites that span the full range of variability exhibited on the UK shelf. These sites are significantly different in terms of their sediment and habitat type, yet are highly similar in terms of confounding variables e.g. hydrodynamic forcing, water depth, temperature, and salinity. We contend that the proposed site selection procedure ensures a very strong likelihood of site-specific work being useful for up-scaling activities and thus increasing our understanding of benthic biogeochemistry at the UK-shelf scale.Online Resource 1Detailed methodologies and data availability (DOCX 175 kb)Online Resource 2Long Term Observation deployment positions and operation timescales (DOCX 78 kb)Online Resource 3Spatial survey sediment characteristics, organised by  % Fines < 63 μm (DOCX 174 kb)Online Resource 4Bathymetric maps generated from Autosub3 (a) site A, (b) site G, (d) site I and Autosub6000 (d) site H multibeam data . Water depth ranged from 101-106 m at Site A, with the study box having a general depth of 103 m; 96-101 m at Site G, study box general depth of 98 m; 106-107 m at site I, study box general depth of 107 m; 103-109 m at Site H, study box general depth of 105 m (TIFF 4187 kb)Online Resource 5Sidescan of the 4 study sites: (a) Site A, (b) Site G, (c) Site H and (d) Site I. All scale bars represent 100 m. The parallel white lines are the nadir and represent lines with no data. Note that at Site G, the white vertical band represents an area where no data were collected. The presence of repeating backscatter ‘stripes’ at Site G is clear and appear to be matched by bathymetric variations suggestive of sedimentary bedforms. Presumed “trawl marks”  are particularly notable at Site I, but also present at sites A, G and H (TIFF 5871 kb)Online Resource 6−2.d−1) averaged over all seasons: Boxes represent mean and SE, with max and min whiskers (DOCX 783 kb)Mean Nutrient Fluxes (mmol.mOnline Resource 7Nephrops norvegicus and (d) Goneplax rhomboides; Echinodermata Asteroidea (b) Astropecten irregularis, (f) Luidia sarsii; and, Cnidaria (c) Cnidaria spp. type 01, (f) Bolocera spp. type 01 (TIFF 4930 kb)Example of density dominant fauna across sites G, H and I. Taxa were determined to the lowest taxonomic level whenever possible (otherwise a morphotype was assigned). Arthropoda (a) Online Resource 8Site specific species abundance (DOCX 125 kb)Below is the link to the electronic supplementary material.


Runoff from high-elevation debris-covered glaciers represents a crucial water supply for millions of people in the Hindu Kush-Himalaya region, where peak water has already passed in places. Knowledge of glacier thermal regime is essential for predicting dynamic and geometric responses to mass balance change and determining subsurface drainage pathways, which ultimately influence proglacial discharge and hence downstream water availability. Yet, deep internal ice temperatures of these glaciers are unknown, making projections of their future response to climate change highly uncertain. Here, we show that the lower part of the ablation area of Khumbu Glacier, a high-elevation debris-covered glacier in Nepal, may contain ~56% temperate ice, with much of the colder shallow ice near to the melting-point temperature (within 0.8 °C). From boreholes drilled in the glacier’s ablation area, we measured a minimum ice temperature of −3.3 °C, and even the coldest ice we measured was 2 °C warmer than the mean annual air temperature. Our results indicate that high-elevation Himalayan glaciers are vulnerable to even minor atmospheric warming. Temperate glacier ice  yields greater ice velocities than cold ice (below Tm), both through the more rapid deformation of warmer ice under a given stress, and through basal motion, which is facilitated by the presence of meltwater at the ice-bed interface and within subglacial sediments, if present4. Temperate ice will also exhibit enhanced ablation rates and yield greater proglacial discharge than cold ice, aided by the increased importance of a subglacial drainage system to evacuate meltwater5. Millions of people in the foothills of the Hindu Kush-Himalaya region depend on glacier melt as part of their water resources6, yet measurements of the internal characteristics and dynamics of mountain glaciers, particularly their internal temperature field, are scarce. At higher elevations, rising surface temperatures may cause peak meltwater to elapse in the next 30 years, leading to a long-term reduction in the glacial contribution to downstream water resources7. It is therefore increasingly important to determine glacier thermal regimes to better forecast 21st Century glacier retreat and meltwater production.A glacier’s thermal regime exerts a strong influence on its dynamics, mass balance, and thus its response to climatic change – a particular concern with rising atmospheric temperatures8 while, to our knowledge, none includes an empirically-constrained thermal regime10. The only higher-order dynamic model that has been applied to a debris-covered Himalayan glacier11 calculated englacial and subglacial temperatures by solving for thermal fluxes12 that were estimated in the absence of field data. Consequently, predictions of future mass loss vary and contain large uncertainties; for example, projections of glacier mass loss in the Everest region by 2100 range between 10% and 99%11.Most spatially-distributed numerical models of Himalayan glacier motion include only an unrefined representation of glacier dynamics14, complicating direct measurement of internal ice temperature. Seasonal variations in subglacial hydrology inferred from satellite-derived surface velocities suggest the presence of temperate ice at the base of high-elevation debris-covered glaciers16. Limited field measurements of ice temperatures have been made, but only reached shallow depths (<~20 m) where ice temperature is influenced by seasonal variations in air temperature17. Measurements in this zone do not therefore reflect longer-term and deeper ice temperatures. For example, a single temperature measurement of −5.3 °C was made at 2.7 m depth on Khumbu Glacier in 197418; drilling reached 20.3 m depth where the borehole froze shut, which was interpreted to indicate a perennially cold shallow ice zone19. A shallow borehole drilled on Rongbuk Glacier (located north of Mt. Everest) in 1966 gave an ice temperature of −4 °C at 3 m depth and −2.1 °C at 10 m depth; this gradient was used to infer temperate ice at depth for other glaciers south and east of Mt. Everest that are at slightly lower elevations20. Ice temperatures have been modelled for East Rongbuk Glacier and matched to empirical measurements from three boreholes21: one from ice core analysis high in the accumulation area22, the other two from shallow boreholes in the ablation area, but no other methodological data were provided21. More recent work on four high-elevation Himalayan glaciers found that ice temperature on the Gyabrag Glacier (north-west of Mt. Everest) was −8.0 °C at a depth of 10 m, ~3–4 °C warmer than the mean annual air temperature (MAAT)23. However, temperatures remain unknown below the shallow seasonally-influenced layer, particularly at depths where the thermal conditions would be most relevant for modelling ice flow.Debris-covered glaciers have a more complex surface topography and differing mass loss processes compared to clean-ice glaciers25. Our thermistor naming convention (e.g. S1_5.0) has two parts: ‘S*’ refers to the site; the suffix denotes the depth (in metres) of each thermistor below the surface.Here, we present ice temperature profiles measured along Khumbu Glacier, which originates high on the Nepali side of Mt. Everest and currently terminates at ~4,850 m a.s.l. Fig. . Borehol26. Beneath this curve, thermistors settle towards the undisturbed temperature of the surrounding ice27. Ice temperatures range from −0.47 °C (S1_5.0) to −3.3 °C (S3_24.6). Ice is warmer at Site 1 than Site 3, and in general becomes warmer with depth along each borehole. The uppermost thermistors at Sites 2 and 3 (S2_2.6 and S3_4.7) record increasing temperatures between June and mid-October.Borehole temperature time series Fig.  show an Tm (see Methods). Ice temperatures are generally colder near to the glacier surface and increase approximately linearly with depth . All ice temperatures at Site 3 . These temperatures are plotted along Khumbu Glacier from the icefall to the terminus  is most strongly seasonally influenced, reflecting its shallow location and thinner overlying debris layer  is influenced by seasonal air temperature variations, responding to rising air temperatures through the monsoon . Therefore, despite the debris layer, at least the uppermost 10 m of the ablation area is notably influenced by seasonal surface temperaturesyer Fig. . The sha27. If this overturning is real, the mechanism remains to be explained, but is most likely to be related to the advection of a relatively cold ice layer.Our ice temperature measurements reveal that Khumbu Glacier is polythermal; with cold ice in the upper part of the ablation area and temperate ice at depth in the lower part of the ablation area Fig. . The col29. The temperate layer on Khumbu Glacier would thus comprise ~56% of the ablation area ice volume (Site 3 to terminus). The accumulation area of Khumbu Glacier is above 6,200 m a.s.l. in the Western Cwm of Mt. Everest where the MAAT is below −9 °C31. Thus, ice is expected to form at or below −9 °C, as on the northeast side of Mt. Everest22. As this cold ice is advected downglacier, it will be warmed from below by geothermal heating, from above by warm air at lower elevations , and from within by deformation, refreezing and meltwater flow33. The emergence of ice in the lower part of the ablation area removes colder near-surface ice17. These processes explain the generally increasing ice temperatures downglacier and at depth  at Site 1, 31 m depth at Site 2, and 255 m at Site 3. This suggests a substantial layer of temperate ice, similar to results from lower-elevation valley glaciers elsewhere in the world18. As this measurement was taken in the shallow ice layer affected by seasonal temperature variations, a temperature recorded in November (shortly following the warm season24) likely represents close to the warmest temperature the ice reaches during the year. Our equivalent thermistor in this layer (S3_4.7) recorded a minimum temperature of −3.1 °C in late June and a maximum of −2.3 °C in late October at the end of the warm season  may be compared with the single nearby measurement of −5.3 °C recorded at 2.7 m depth and at a similar time of year to our measurements  in 1974son Fig. . Assumin34, this might partially explain the presence of warmer ice at depth farther downglacier. Alternatively, amplified climate warming at high elevations2 may be penetrating deeper into the glacier. Since ice located above the CTS at Site 2 was within 0.8 °C of Tm, and at Site 1 all measurements were within 0.5 °C of Tm  and a thin (<0.5 m) local debris layer that could be cleared prior to drilling. At Sites 1 (2 boreholes) and 2 (ten boreholes), drilling ceased due to the presence of debris in the borehole. At Site 1, we believe that the drill may have reached the bed, but this is difficult to confirm with the available observations. At Site 2, ten boreholes were drilled to 12–22 m depth at locations with surface elevation varying by ~10 m, suggesting a spatially extensive and possibly continuous debris layer beneath the surface. The borehole at Site 3 was drilled to the maximum length achievable (~155 m) with our equipment at 5,200 m elevation. Borehole inclinometry revealed that it was drilled off-vertical (reading a maximum of 30° at the base). Thus, although the borehole length was 155 m, the depth of the borehole base was 132 m beneath the glacier surface. The sensor depths presented here have been corrected to reflect the true depth (rather than borehole length). Inclinometer data were not available for the boreholes at Sites 1 and 2, but the deviation at Site 3 is lower towards the surface so no thermistor at Sites 1 or 2 is likely to be more than ~1 m in error.Thirteen boreholes were drilled into Khumbu Glacier in May 2017 at three sites in the ablation area . Resistance was converted to temperature using a Steinhart and Hart43 polynomial fitted to the manufacturer’s calibration curve, with a further correction using a freezing-point offset for each thermistor obtained from an ice-bath calibration. Previous studies using such thermistors46 suggest that with this secondary calibration, an accuracy of ±0.05 °C can be achieved. We therefore consider the thermistors to be accurate to ±0.05 °C at 0 °C, but accept that this value represents an indication of uncertainty rather than a maximum limit.The longest borehole at each site was selected to be instrumented with strings of thermistor sensors. The thermistor string contained negative temperature coefficient thermistors (Honeywell UNI-curve 192-502-LET-AOI) connected by a multicore cable, spaced more closely at depth. Higher up the cable, thermistors were spaced more evenly  according to the expected length of the borehole which was based on measuredUncertainty in vertical depth for each thermistor was estimated as the sum of error in the exact location of cable splicing (±0.2 m) and cable stretch upon lowering into each borehole (+0.5% of sensor depth). These are indicated in Fig. 27 fitted to the raw thermistor data shown in Fig. T is the borehole ice temperature at time t, Q is the heat released by drilling per unit length of the borehole; k is the thermal conductivity of pure ice at 0 °C (2.1 W m−1 K−1), T0 is the undisturbed ice temperature, and s is the time in seconds until the start of the freezing curve.The undisturbed ice temperature for each thermistor that froze in was estimated by taking the minimum of the running mean for one hour during the night (to avoid the slight noise-related influence from solar charging during the day), late in the time series (to ensure the settled temperature was as close to the true temperature as possible). Some thermistors, for example at Site 2 Fig. , still sTm, was calculated at the depth of each thermistor using the Clausius-Clapeyron equation of melting-point depression46:Ttp and ρtr are the triple point temperature (273.16 K) and the pressure of water (611.73 Pa) respectively, γ is the Clausius-Clapeyron constant and ρ is the ice overburden pressure, which can be approximated as:ρi is the density of ice (900 kg m−3), g is the gravitational acceleration (9.81 m s−2) and h is the height of the overlying ice column given here by sensor depth (m). Values of the Clausius-Clapeyron constant range from that of pure (air-free) water/ice (0.0742 K MPa−1)30, a small content of soluble impurities and air within ice (0.079 K MPa−1)47 to that for pure ice and air-saturated water (0.098 K MPa−1)48. These values were all tested for the best match of each Tm to the freezing-point of each thermistor27: the constant for pure ice and air-saturated water provided the closest fit and was used in the analysis here. However, the theoretical Tm values still differed from the true freezing temperature of the thermistors by 0.04 to 1.1 °C, suggesting that a further factor depressed the freezing-point. One possible explanation is the presence of solutes and impurities within the ice, the concentrations of which are currently unknown within Khumbu Glacier.The pressure-dependent melting-point temperature, Tm were used along with the undisturbed ice temperatures to estimate the CTS depth46. At Sites 2 and 3, a line of best fit through all the undisturbed thermistor values . This line intersected Tm at 20 m depth. For reference, when a line of the same gradient as Site 3 was used, it intersected Tm at 30 m depth.The values of


Intercellular distribution of the plant hormone auxin largely depends on the polar subcellular distribution of the plasma membrane PIN-FORMED (PIN) auxin transporters. PIN polarity switches in response to different developmental and environmental signals have been shown to redirect auxin fluxes mediating certain developmental responses. PIN phosphorylation at different sites and by different kinases is crucial for PIN function. Here we investigate the role of PIN phosphorylation during gravitropic response. Loss- and gain-of-function mutants in PINOID and related kinases but not in D6PK kinase as well as mutations mimicking constitutive dephosphorylated or phosphorylated status of two clusters of predicted phosphorylation sites partially disrupted PIN3 phosphorylation and caused defects in gravitropic bending in roots and hypocotyls. In particular, they impacted PIN3 polarity rearrangements in response to gravity and during feed-back regulation by auxin itself. Thus PIN phosphorylation, besides regulating transport activity and apical-basal targeting, is also important for the rapid polarity switches in response to environmental and endogenous signals. Intercellular auxin transport and metabolism are responsible for changes in cellular auxin concentration4, which leads to different auxin responses7. In recent years, detailed characterization of auxin transport proteins and their regulators has broadened our knowledge of polar auxin transport, auxin gradient formation and mechanisms of differential growth and organogenesis9. The most prominent auxin transporters are AUX1/LIKE AUX1 auxin importers11, P-glycoproteins of the ATP-binding cassette transporter family of auxin exporters12, and polarly localized PIN auxin exporters13. Although all of these proteins are involved in passing auxin across the plasma membrane out of or into the cell, it seems that PIN auxin efflux carriers14 are predominant in mediating the directionality of the intercellular auxin flow by virtue of their polar subcellular localization in auxin-transporting cells16.The plant hormone auxin, indole-3-acetic acid (IAA), controls plant growth and development by modulating fundamental cellular processes such as cell division, expansion, and differentiation21. Three of these kinases, PINOID (PID), WAG1 and WAG224 play a crucial role. They are at least partially functionally redundant25. Overexpression of these kinases leads to a basal-to-apical (rootward-to-shootward) shift in PIN polarity that causes disruption of the auxin maxima and to the collapse of the root meristem and agravitropic root growth23. On the other hand, pid, wag1, wag2 single or multiple mutants show more preferential basal PIN localization causing deprivation of auxin from the shoot meristem and a pin-like inflorescence phenotype or more basal localization of the otherwise apically localized PIN2 in root epidermis leading to agravitropic root growth26. In accordance with the model that more PID-dependent phosphorylation leads to a preferentially apical PIN localization, phosphomimicking or phosphodead mutations of serine/threonine amino acids within the PIN hydrophilic loop show more apical or basal localization respectively.One of the major regulatory mechanisms of PIN polar targeting is phosphorylation. Several studies demonstrated that serine/threonine protein kinases from the AGCVIII kinase family phosphorylate the hydrophilic loop of PIN proteins, which correlates with the change in PIN polar localizationArabidopsis cells in root and co-localizes with several PIN proteins such as PIN1, PIN2 and PIN4. It was shown that D6PK can interact directly with PIN1 protein and phosphorylate it28.Recently, a related subfamily of AGCVIII kinases involved in auxin transport and plant development has been identified. D6 protein kinase (D6PK) localizes to the basal membrane of 30. A similar phenomenon of PIN3 relocation, albeit slower, has been observed during the hypocotyl phototropic response31. Recently, a subsequent second re-polarization event during hypocotyl bending has been identified, which is important for resetting the asymmetry in the PIN polar distribution; ultimately leading to the termination of the bending32. This second repolarization is likely related to the auxin feed-back on PIN polarity as seen in so called auxin canalization processes of leaf venation and vascular tissue regeneration34. Although PIN3 and also PIN737 relocalization events are likely to be involved in redirecting auxin fluxes to create particular growth responses, insight into the underlying mechanism of relocation is still limited.Changes in polar subcellular localization of PINs seem to be an essential mechanism for redirecting auxin fluxes in response to different environmental stimuli. For example, the apolar distribution of PIN3 becomes polar after gravitropic stimuli, leading to relocalization of PIN3 towards the gravity vector and correlating with changed auxin fluxesIn this study we examined potential phosphorylation sites in the PIN3 hydrophilic loop and their role in tropic responses. We identified sites that play a role in both PIN3 polarity rearrangements during gravitropic responses, thus demonstrating a crucial role for PIN phosphorylation in polarity switches in response to external signals such as gravity or endogenous signals such as auxin itself.37. PIN phosphorylation by PID has an impact on PIN polarity and auxin transport directionality in different developmental contexts by regulating apical-basal PIN localization19. We tested whether PID contributes to the gravitropic response and also to the gravity-induced relocation of PIN3 in roots and hypocotyls. It is known22 that root bending in PID overexpressing line (35S::PID) is defective , agreeing with previously reported mild gravitropic defects28. This argues against an important role of D6PK in root gravitropism, and suggests that the contribution of D6PK for the regulation of PIN activity, either in columella cells for redirection of auxin fluxes, or in epidermis for shootward auxin transport, is not crucial.Further, we tested the potential involvement of PIN phosphorylation by D6PK kinaseOverall these results show that PID/WAG, but not D6PK, play an important role in gravity-mediated PIN3 repolarisation and in gravitropic bending both in roots and shoots. Higher PID expression inhibits PIN3 polarization and gravitropic bending whereas decreased PID/WAG expression leads to increased PIN3 polarization.in vitro and in vivo28. Therefore, we investigated the putative phosphorylation sites in the PIN3 loop. Two putative phosphorylation clusters, P1 and P2, both containing three serines   to mimic the non-phosphorylated state or by aspartic acid to mimic the constitutively phosphorylated status .Previous studies showed that PID directly phosphorylates the central hydrophilic loop of PIN proteins both 21) Fig. , were chpin3–4 mutant to evaluate their impact on PIN3 function. The transformed plants did not exhibit any strong and obvious developmental defects. Levels of mutated PIN3 transcript in plants were evaluated by qPCR and only lines exhibiting similar expression level as PIN3 in wild type were used for further experiments  as compared to the wild-type, but the PIN3-YFP-P2D mutant variant exhibited the least rescue showing defective gravitropic bending comparable to the pin3-4 mutant  and P2 (adapted from18) that have been shown to be phosphorylated in vivo41, and prepared different phosphorylation variants including phosphomimic and phosphodead. Prepared mutant variants in the pin3-4 mutant background displayed only minor defects in overall development. More specifically, they show defects in the root and hypocotyl gravitropic growth and gravity-induced PIN3 relocation that are in particular observed in the phosphomimic variants. The hypocotyl experiments also revealed a defect in the auxin-mediated PIN3 relocation, again apparent in the phosphomimic variants. These phenotypes of phosphomimic variants correspond with PIN3 relocation and bending phenotypes in root and hypocotyl in 35S::PID line suggesting that PIN3 phosphorylation at least partly mediated by PID and associated dephosphorylation by phosphatases are required for both gravity-mediated PIN3 relocations and bending.Here we have demonstrated that PIN phosphorylation and PID/WAGs kinases activity are involved in the gravity-induced relocation of the PIN3 protein in root columella and hypocotyl endodermal cells. Downregulation of PID/WAGs increases gravity-induced PIN3 relocation and consequently gravitropic bending, whereas upregulation of PID reduces PIN3 relocation and bending. To confirm the role of phosphorylation in this process and to assess which potential phosphorylation sites might be important, we chose two clustered sites, P1 , that is important for subcellular trafficking and PIN3-mediated developmental processes, such as auxin efflux activity, root growth, and root gravitropism42. Nevertheless, the PIN3-M3 phosphodead mutant variant was demonstrated to be still phosphorylated by PID or WAG1 in vitro21.A similar study about phosphorylation sites in the PIN3 hydrophilic loop has identified a different phosphorylation site, M3  holoenzyme, also targets and regulates PIN subcellular localization43. In roots, dephosphorylation by PP6 complex led to changes in PIN polarity from apical to basal45, thus the role of phosphatases in controlling PIN polar targeting during gravitropic responses should not be ignored. Given the complexity of the number of putative phosphorylation sites in PIN proteins and different kinases and phosphatases, it seems apparent that multiple overlapping PIN phosphorylation mechanisms will be involved in regulation of different aspects of PIN-dependent auxin transport. Nonetheless, our work provides evidence demonstrating that these phosphorylation processes regulate PIN polarity switches and thus auxin fluxes redirections in response to both environmental and endogenous regulations.In addition to the role of PID kinase in regulating both PIN transport activity and its polar localization, the related D6PK kinase has been also shown to phosphorylate PIN proteins recognizing overlapping residues as PID kinase, but being more specifically involved in regulating PIN activityPIN3::PIN3-YFP46; pin3–4 (SALK_005544); 35S::PID-2122; wag1/wag2/pid18. All seeds were grown on agarose plates containing 1/2 strength Murashige and Skoog medium with 1% sucrose. Seeds were vernalized for 3 days at 4 °C and consequently grown at 18 °C under 16-h-light/8-h-dark photoperiod. For hypocotyl experiments, after stratification the germination was induced by placing the plates in the light for 5–6 hours that were then transferred to darkness and kept at 18 °C for 4 days. For root or hypocotyl gravitropic stimulations, plates with 4-day-old seedlings were turned 90°, scanned at every time point by scanner and the angles were measured by ImageJ. For root length measurements 5 day old seedling were used, for hypocotyl length measurements 4 day old dark grown seedlings. The emerged lateral root assay was performed on 14 day old seedlings and LR primordia were counted with a differential interference contrast microscope BX51 (Olympus). Each experiment was conducted at least in triplicate. For the statistical evaluation, the t-test was done with the Excel statistical package.The published transgenic and mutant lines were: 46 was used for transgene construction. Four different DNA fragments  possessing different mutations (Table Arabidopsis was accomplished via Agrobacterium tumefaciens (strain PMP90)-mediated infiltration by floral dip. All transformed lines were analyzed and at least 3 independent transgenic lines for each construct with similar expression level were used in this study.The binary vector pK7m42GW containing PIN3::PIN3-YFP sequencens Table  were synFor confocal microscopy, a Zeiss LSM 710 confocal scanning microscope or Zeiss LSM 710 vertical confocal scanning microscope were used. To monitor the gravitropic response, plates were scanned 24 h after gravistimulation. Images were processed in Zeiss ZEN software and ImageJ. Each experiment was performed at least three times.http://rsb.info.nih.gov/ij). Quantification of gravity-induced PIN3-YFP relocalization in columella cells was performed on maximal intensity projections of Z-scans of columella cells by measuring the signal intensity at the apical membranes (marked with dark colors) and comparing with signal intensity of basal membranes (marked with light colors) of the cells on the periphery of columella before and after gravistimulation  or 35S::PID-FLAG17 cloned into pGREEN. pINTAM3-PID was induced with 1 µM 4OH-tamoxifen (Sigma-Aldrich) and 5 µM beta-estradiol for 24 h before harvesting. All infiltrations were performed with p19 to reduce silencing48. Samples were extracted based on Abas & Luschnig49, with modifications for a quicker extraction in order to detect phosphorylated protein versions. Frozen tissue was ground in liquid nitrogen and collected into extraction buffer containing PhosSTOP (Roche), centrifuged for 3 minutes at 300 g (4 °C) and the supernatant (soluble and membrane fractions) was immediately solubilized with 2% lithium dodecyl sulfate and 10 mM DTE. Samples were centrifuged at 20 000 g for 30 min (4 °C) and the supernatant precipitated by chloroform/methanol50. Proteins were separated by SDS-PAGE, blotted and probed with anti-GFP monoclonal mouse antibody (Roche), stripped and probed with anti-FLAG M2 monoclonal mouse antibody (Sigma-Aldrich). As N. benthamiana leaves contain a strong non-specific signal from anti-FLAG antibody at about 90 kD, only the lower half of the blot was used for anti-FLAG. For Phos-tag analysis, 25 µM Phos-tag (WAKO) was incorporated into the gel. Phosphorylated bovine casein, ovalbumin, PID and AtPIN1 were used as positive controls for Phos-tag.PIN3-YFP and mutant variants were transiently expressed in 3H-IAA (12uL 3H-IAA + 10 mL AM + agar) was applied to the apical part of the hypocotyls. After 6 hours, hypocotyls were collected, homogenized in liquid nitrogen and incubated overnight in Opti-Fluor scintillation cocktail (Perkin Elmer). Amount of transported 3H-IAA was then measured in a scintillation counter (Hidex 300SL) for 300 s with three technical repetitions. Negative control was performed by inserting the droplet above the decapitated hypocotyl to account for any diffusion though the agar. Additional negative control was performed using 10 μM NPA, to inhibit auxin transport.Etiolated seedlings were prepared as described above, except plates were kept at 21 °C for 5 days. Etiolated hypocotyls were decapitated to exclude the effect of auxin biosynthesis in cotyledons and a droplet of AM + agar (1.25%) with 51. Expression levels were normalized to the non-auxin-responsive genes β-TUBULIN (At5g12250), EEF (At1g30230) and CDKA (At3g48750).Total RNA was extracted with the RNeasy kit (QIAGEN). Poly(dT) cDNA was prepared from total RNA with Superscript III (Invitrogen).Quantitative RT-PCR was done with LightCycler 480 SYBR Green I Master reagents (Roche Diagnostics) and a LightCycler 480 Real-Time PCR System (Roche Diagnostics). Data were analyzed with qBASE v1.3.4Supplemental figures and table


The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells. Mitophagy is crucial for mitochondrial quality control and maintenance of cellular homeostasis. Here the authors identify an E3 ubiquitin ligase, HUWE1, that collaborates with LC3-interacting protein AMBRA1 to induce mitochondrial clearance. This high-quality clearance system is fundamental for preserving cellular homoeostasis and for critical processes, such as inflammation and cell death or diseases, including cancer and neurodegeneration2. The main mitophagy pathway is driven by the stabilization of the mitochondrial kinase PINK1, resulting in the recruitment of the E3 ubiquitin ligase PARKIN to damaged mitochondria, and in a ubiquitylation cascade targeting several outer mitochondrial membrane (OMM) proteins. Indeed, ubiquitylation events are fundamental during mitophagy, contributing to the normal turnover of mitochondrial proteins in basal conditions3, and promoting the recognition of UBD (ubiquitin binding domain)-containing proteins, which allow mitochondria selective autophagy4. Optineurin (OPTN) and NDP52 are the the main mitophagy receptors, acting as bridges between ubiquitin-tagged mitochondria and the autophagosome-associated protein, MAP1LC3/LC3 (microtubule-associated proteins 1A/1B light chain 3), thus leading to mitochondria engulfment into autophagosomes, upon mitochondrial membrane depolarization6. More recently, PHB2 (Prohibitin-2), an inner mitochondrial membrane protein, has also been demonstrated to be involved in selective mitochondria removal, cooperating with PARKIN in mammals7.Mitophagy is an evolutionary-conserved mechanism that allows damaged or undesired mitochondria removal by an autophagosome–lysosome pathway9; (ii) Bcl2-L-13 is the mammalian homologue of Atg32, and it stimulates mitochondria fragmentation and therefore mitophagy10; and lastly, (iii) FUNDC1 allows mitochondrial clearance upon hypoxia11. Of note, these mitophagy receptors are post-translationally modified in order to regulate their interaction with LC3 during mitophagy12.Besides the PINK1/PARKIN system, the OMM proteins NIX/BNIP3L, Bcl2-L-13 and FUNDC1 are also fundamental to trigger mitophagy in mammals, by interacting directly with LC3 and regulating mitochondrial clearance. In particular, (i) NIX/BNIP3L promotes mitochondria removal during reticulocytes differentiation13. Here, we describe HUWE1 as the novel E3 ubiquitin ligase that collaborates with AMBRA1 to induce mitochondrial clearance, by inducing mitofusin 2 (MFN2) degradation. Moreover, since AMBRA1 exhibits (i) mitochondria localization14, (ii) a LIR (LC3-interacting region) motif and (iii) the capacity to induce mitophagy13, we decided to investigate whether AMBRA1 could be defined as a receptor, and to better characterize this pathway. We thus found that the activity of the mitophagy receptor AMBRA1 is regulated by a phosphorylation upstream of its LIR motif, mediated by the IKKα kinase.We previously demonstrated that the LC3-interacting protein AMBRA1 plays a role in the selective degradation of ubiquitylated mitochondria, transducing both canonical PINK1/PARKIN-dependent and -independent mitophagyAltogether, these findings highlight AMBRA1, HUWE1 and IKKα as three novel and crucial proteins for mammalian mitophagy regulation, following mitochondrial membrane depolarization in a PINK1/PARKIN-free context.13, we searched for a novel putative E3 ubiquitin ligase, which could control mitochondrial protein ubiquitylation in cooperation with AMBRA1 during the mitophagy process.Since we have previously shown that AMBRA1 regulates the dismissal of ubiquitylated mitochondria in PINK1/PARKIN-independent mitophagyActA , which stimulates mitophagy13 in HeLa cells grown in two different isotope labelling media (light and heavy). The immunoprecipitated samples of the negative control (light medium lysate) and the experimental sample (heavy medium lysate) were mixed and then analysed by MS analysis15. Interestingly, this screening led us to identify a single E3 HECT-Ubiquitin ligase, HUWE1 , whose role in macroautophagy/mitophagy was yet undisclosed -based mass spectrometry (MS) analysis in order to detect AMBRA1-interacting proteins, upon mitophagy stimulation, in a PARKIN-free cellular system. Thus, we immunoprecipitated Myc-AMBRA1sed Fig. .Fig. 1 HActA. Total lysates were immunoprecipitated with an anti-Myc antibody or an IgG control and the immune-complexes were analysed by western blotting. We found that AMBRA1ActA was able to co-immunoprecipitate with HUWE1  in order to induce mitophagy. Total lysates were immunoprecipitated using an anti-HUWE1 antibody or IgG control and the immune-complexes were analysed by western blot. As shown in Fig. WT preferentially binds HUWE1 following mitophagy induction.In order to confirm these MS results, we performed a co-immunoprecipitation experiment in HeLa cells expressing a vector encoding for Myc-AMBRA1WE1 Fig. . Since tGiven the evidence that HUWE1 is an AMBRA1-interacting protein preferentially upon mitophagy stimulation, we then focussed on a putative role of HUWE1 in mitophagy.16, we checked whether it was present at the mitochondria following mitophagy induction. By performing a mitochondria–cytosol fractionation in HeLa cells expressing Myc-AMBRA1ActA or an empty vector (PcDNA3), we found that AMBRA1ActA expression enables HUWE1 localization at the mitochondria . As shown in Fig. ActA expression13, such as COXII and COXIV, is rescued in HUWE1-interfered cells. To confirm these data, we performed a confocal microscopy analysis in cells co-expressing both GFP-ShHUWE1 or GFP-ShCtr together with AMBRA1ActA, and we measured the mitochondria amount in transfected green fluorescent protein (GFP)-positive cells. As expected, following AMBRA1ActA expression with GFP-ShCtr, we observed the formation of mitochondria aggregates (mito-aggresomes) around the nuclei, and a decrease in mitochondria staining; by contrast, GFP-ShHUWE1 transfected cells presented a delay in both mito-aggresomes formation and mitochondrial clearance  HeLa cells5. As shown in Supplementary Fig. ActA transfection, we also observed a reduction of acidic mt-mKeima upon HUWE1 downregulation. These data confirm that HUWE1 depletion impairs AMBRA1-mediated mitophagy. Finally, we also confirmed these data with the wild-type AMBRA1, following mitophagy induction. In fact, we transfected HeLa cells with Myc-AMBRA1WT in combination or not with siRNA-HUWE1. Then, we treated cells with O/A . By western blot analysis, we observed a decrease of mitochondrial markers, COXII and COXIV, in the mitophagy context . By analysing MFN2 protein levels in Myc-AMBRA1ActA transfected cells, we observed a decrease in this OMM protein. Interestingly, MFN2 decrease is rescued by downregulating HUWE1 or by treating cells with MG132  exhibits a LIR motif on its sequence for LC3B binding, (iv) is able to regulate PINK1/PARKIN-independent mitophagy and (v) controls the PINK1/PARKIN-mediated mitophagy13, we investigated whether AMBRA1 can act as a mitophagy receptor.Next, since AMBRA1 (i) operates as a cofactor for HUWE1 activity in order to trigger mitophagy, (ii) displays mitochondrial localization5. Indeed, these cells are deficient for five mitophagy receptors, i.e., OPTN, NDP52, NBR1, P62 and TAX1BP1. In these cells, only the expression of NDP52 or OPTN  is able to rescue the mitophagy pathway. In fact, the treatment of these cells with mitochondrial uncouplers does not induce mitophagy . Since the phosphorylation status of a serine residue (Ser) flanking the LIR motifs of known mitophagy receptors, such as FUNDC111, BNIP320, NIX/BNIP3L21, Bcl2-L-1310 and OPTN22, controls their binding with LC3, we investigated whether AMBRA1 could be post-translationally modified in a serine site in proximity of its LIR motif13.A key process in the induction of selective autophagy is the interaction between mitophagy receptors and Atg8 (autophagy-related protein 8) protein family membersS1014A) and a phospho-mimetic mutant (AMBRA1S1014D). By performing co-immunoprecipitation experiments, we analysed AMBRA1–LC3B interaction in basal conditions and upon mitophagy stimulation (O/A treatment), obtained by overexpressing Myc-AMBRA1WT and AMBRA1S1014A or AMBRA1S1014D in HeLa cells. First, we confirmed that AMBRA1 interacts mostly with LC3B following mitophagy stimuli13. Moreover, we observed that alanine substitution at residue 1014 reduced the binding between AMBRA1 and LC3B, following O/A treatment  as a putative phosphorylation site close to the AMBRA1-LIR motif Fig. . Second,ent Fig. . By contThese results highlight that the phosphorylation status of AMBRA1 on S1014 influences the interaction between AMBRA1 and LC3B during mitophagy.WT compared with its mutants on S1014. To this aim, we transfected HeLa cells with the empty vector PcDNA3 as a control, and vectors encoding Myc-AMBRA1WT or Myc-AMBRA1S1014A or Myc-AMBRA1S1014D and checked, by western blot analysis, the levels of mitochondrial proteins COXII and COXIV.Next, in order to ascertain whether S1014 is relevant in AMBRA1-induced mitophagy, we performed functional experiments to evaluate the mitophagy activity of AMBRA1WT showed a decrease in COXII and COXIV levels, while cells expressing the AMBRA1S1014A mutant showed a lower decrease in mitochondrial marker levels compared with AMBRA1WT or AMBRA1S1014D  induced mito-aggresome formation and a clear decrease of mitochondria amount to a larger extent than AMBRA1WT , HeLa cells transfected with AMBRA114D Fig. . Of noteorm Fig. . Also, w1WT Fig. . Moreove4-(trifluoromethoxy)phenylhydrazone (FCCP), O/A) or autophagy conditions ). As shown in the graph -S1014-AMBRA1 serum). We tested its efficiency by comparing the level of phospho-S1014 in basal versus mitophagy . In particular, phosphorylation of S1014 , we precisely calculated KD values for LC3B interaction with unphosphorylated (P0) and phosphorylated (P1) AMBRA1-LIR peptides. We also mapped the CSP on the LC3B sequence and three-dimensional structure  experiments in which synthetic peptides spanning the AMBRA1-LIR at various phosphorylation states (P0: steady state and P1: phosphorylation state) were titrated into all six mATG8 proteins, including LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2  simulations, evaluating conformations of the steady-state structure of AMBRA1 when compared to the phosphorylated structure. In this case, we used the P62-LIR/LC3B experimental structure as a template for modelling. As shown in the plot of Fig. Taken together, these results give a proof of concept that phosphorylation of S1014 is fundamental to stabilize the conformation of AMBRA1-LIR in the binding pocket of LC3B, thus promoting its interaction with LC3B  and its mitophagy activity.24. One of the candidates with the highest score was the IKKα kinase , a serine–threonine kinase belonging to the IKK family that includes IKKα, IKKβ, IKKγ, IKKε and TBK1 proteins25. The predicted IKKα consensus motif26 was localized upstream of the LIR motif of AMBRA1 27 in order to avoid a kiss and run action between the wild-type kinase and AMBRA1, and then we immunoprecipitated the IKKα mutant with an anti-HA antibody and, finally, we checked for AMBRA1–IKKα complex by western blot analysis. As shown in Fig. K44M), which acts as a dominant negative mutant27, on AMBRA1 phosphorylation at S1014. To this end, we over-expressed vectors encoding Flag-IKKαWT or HA-IKKαK44M in HeLa cells, and we induced mitophagy by using O/A. As shown in Fig. WT to its kinase-dead mutant. To this end, we transfected HeLa cells with a vector encoding Myc-AMBRA1WT, Flag-IKKαWT or HA-IKKαK44M and immunoprecipitated individually these three proteins from the mitochondrial fraction. Then, we combined the immunoprecipitated kinases (IP-IKKαWT or IP-IKKαK44M) with the immunoprecipitated substrate (IP-AMBRA1). The western blot analysis showed that the phosphorylation signal at AMBRA1–S1014 was present only in the IP-IKKα fraction  softwareRA1 Fig. , suggestion Fig. . SubsequThese results prove that IKKα is the kinase responsible for AMBRA1 phosphorylation at S1014 upon mitophagy induction, and thus it represents a novel upstream factor accountable for mitophagy activation.ActA, and treated or not with the IKKα inhibitor (BAY-117082) by detecting COXII and COXIV levels by western blot analysis. We observed that BAY-117082 treatment was able to rescue mitochondrial clearance following AMBRA1ActA overexpression  of IKKα in mitophagy.In analogy with other mitophagy receptors, the phosphorylation of AMBRA1 at the S1014 site, flanking its LIR motif, is a crucial event for the engulfment of ubiquitin-tagged mitochondria into autophagosomes. Since we demonstrated that HUWE1 downregulation results in a block of AMBRA1-mediated mitophagy, we hypothesized that HUWE1 activity could influence AMBRA1 phosphorylation at S1014 upon mitophagy induction. Thus, we transfected HeLa cells with a siRNA-Ctr or siRNA-HUWE1 or with a vector encoding HUWE1, and then treated cells with O/A .Interestingly, we observed that the downregulation of HUWE1 induced a significant reduction in AMBRA1–S1014 phosphorylation upon mitophagy induction. By contrast, HUWE1 overexpression resulted in increased phosphorylation of AMBRA1 at the same site Fig. . In analTaken together, these findings clearly define an epistatic role of HUWE1 in IKKα- and AMBRA1-mediated mitophagy.28 and cardiac29 ischaemia/reperfusion (I/R) injury; also, FUNDC1 is a well-known regulator of mitophagy under hypoxia11, and its implications in ischaemia have been recently elucidated31. Of note, the intramitochondrial protein ATPIF1, during ischaemia-preconditioning context, has been reported to regulate selective degradation of potentially toxic mitochondria  by collapsing their membrane potential (ΔΨm)33.Following the evidence on AMBRA1-mediated mitophagy in relay with HUWE1 and IKKα, we checked the physiopathological relevance of this functional interplay in models of ischaemia. Indeed, there is general consensus that mitophagy can be triggered in similar conditions as it happens via receptors. For instance, NIX/BNIP3L mediates mitochondrial removal during cerebral34; this evidence prompted us to investigate whether AMBRA1 itself could drive mitophagy during I/R injury.Ischaemia and its prodromal phases are characterized by detrimental oxidative stress, which we showed to be counteracted by AMBRA1-induced mitophagy33. Total lysates were then subjected to western blot analysis of P-S1014-AMBRA1, recording that AMBRA1 is neatly phosphorylated between 30 min and 1 h of OGD/RX treatment ent Fig. . Neverth/RX Fig. . In anotria Fig. . In sum,ActA strongly favours the interaction between HUWE1 and MFN2, thus acting as a cofactor for HUWE1 E3 ubiquitin ligase activity, and leading subsequently to MFN2 ubiquitylation and then to its degradation. The data highlight a novel important paradigm for HUWE1 E3 ubiquitin ligase activity regulation and open a wide range of studies to understand whether AMBRA1 may control HUWE1 activity in other contexts. Of note, HUWE1 depletion abolishes both MFN2 degradation and AMBRA1-mediated mitophagy in HeLa cells. Also, it has been established that MFN2 degradation is an important event for mitophagy induction35. In line with this evidence, we showed that MFN2 is degraded in the early steps of AMBRA1-mediated mitophagy (18 h post transfection of Myc-AMBRA1ActA). Moreover, HUWE1 depletion induces a clear reduction of total mitochondria ubiquitylation following AMBRA1ActA expression. Since we found that MFN2 degradation correlates with AMBRA1-mediated mitophagy induction, it will be necessary to better understand in the future whether or not this event is a critical step for AMBRA1-mediated mitophagy. Also, although MNF2 is a favourite substrate of HUWE1, it will be important to investigate whether HUWE1 could ubiquitylate other mitochondrial substrates in order to control mitophagy induction.In the present study, we demonstrated that the mitophagy receptor AMBRA1 acts at two critical steps during mitochondrial membrane depolarization-induced mitophagy. First, we identified the E3 ubiquitin ligase HUWE1 as an important co-operator of AMBRA1 in order to induce mitophagy, following mitochondrial membrane depolarization. In particular, we demonstrated that AMBRA137. Interestingly, HUWE1 has been defined as a unique BH3-containing E3 ubiquitin ligase38; it would be interesting to characterize the binding between AMBRA1 and HUWE1 by checking, in particular, whether a BH3 mechanism is involved in this context. Of note, both HUWE1 and AMBRA1 can interact with the anti-apoptotic factor MCL1 (induced myeloid leukaemia cell differentiation protein Mcl-1)38. In this scenario, it could also be interesting to define the relationship among AMBRA1–HUWE1-dependent mitophagy, mitochondrial dynamics and apoptosis.We recently demonstrated that AMBRA1 is a BH3 (BCL2-homology domain 3)-like protein, containing a BH3 domain necessary for its interaction with BCL2 family proteins39. It is opportune to underline that AMBRA1 also controls c-Myc expression, cooperating in the PP2A-mediated dephosphorylation of c-Myc40. It could thus be interesting to analyse the correlation between AMBRA1–HUWE1 and c-Myc regulation upon mitophagy stimulation. As previously demonstrated, mitochondria are fundamental in cellular homoeostasis and the HUWE1–AMBRA1 axis can represent a focal point of interconnection between healthy and tumour cells.The HUWE1–AMBRA1 axis in mitophagy defines a new pathway for damaged mitochondrial removal related to a novel E3 ligase, yet unwound in this field, but since AMBRA1 and HUWE1 are two large multi-domain proteins, they may cooperate in several processes, such as cancer, apoptosis and cell proliferation. In fact, HUWE1 has been described as a tumour suppressor gene controlling the proto-oncogene c-Myc regulation. HUWE1 participates to c-Myc degradation, leading to a reduction in tumour diffusion41. Since we demonstrated that AMBRA1-dependent mitophagy is not controlled by the PINK1/PARKIN pathway, we propose here an alternative system that regulates mitophagy. The removal and proteolysis of OMM proteins appears to be necessary for mitophagy42. Since the PINK1/PARKIN pathway is mutated in one of the most diffuse neurodegenerative disturb, Parkinson’s Disease44, AMBRA1–HUWE1-dependent mitophagy could offer a novel target therapy to counteract this disease as it may preserve cellular vitality. The mitophagy programme is a highly intricate system comprising different characters. In fact, PINK1-related mitophagy receptors present UBDs in their sequences that allow the interaction with Ser65-phosphoUb-containing mitochondria45. By contrast, mitochondrial proteins, such as NIX, FUNDC1 and Bcl2-L-13, lacking an UBD, can anyway promote mitophagy of ubiquitylated mitochondria. In our current work, we added a piece to this heterogeneous puzzle, describing AMBRA1 as a new UBD-free mitophagy receptor, which acts following mitochondrial membrane depolarization. Indeed, we revealed that the expression of AMBRA1 is sufficient to restore mitochondrial clearance in cells depleted for PINK1/PARKIN-related mitophagy receptors, such as NDP52, OPTN, TAX1BP1, P62 and NBR1. In analogy with mitophagy receptors Bcl2-L-13, FUNDC1 and NIX/BNIP3L, we demonstrated that AMBRA1 is also able to stimulate mitochondrial clearance in the absence of any PINK1 phospho-ubiquitin signals. Indeed, we previously proved that AMBRA1 was able to induce mitophagy in PINK1−/− MEFs cells13.In addition, it is noteworthy that MFN2 degradation is also mediated by PARKIN activity24. Based on this, we cannot exclude that other factors  could also affect the interactions between full-length AMBRA1 and mATG8 proteins. Moreover, since GABARAPs seem to be more involved in the PINK1/PARKIN-dependent mitophagy pathway than LC3s46, it will be very interesting to investigate, in the future, whether AMBRA1 requires a preferentially binding to GABARAPs or to LC3s in mitophagy, depending on a particular context (PINK1/PARKIN dependent or independent) or on other processes .Moreover, we have proved that AMBRA1-mediated mitophagy activity is tightly dependent on its phosphorylation state. In particular, following mitophagy induction, the phosphorylation on S1014 increases the AMBRA1–LC3B binding thus promoting an enhancement in AMBRA1-mediated mitophagy. Moreover, we have revealed that the HUWE1 ubiquitylation signal is fundamental not only in tagging mitochondria for mitophagy, but also in activating AMBRA1 for mitophagy. In fact, we observed that downregulation of HUWE1 results in a reduction of AMBRA1-activated phosphorylation state, necessary for its interaction with the autophagosome marker LC3B. We can speculate that a fine dual relationship occurs between AMBRA1 and HUWE1: the former is necessary for HUWE1 recruitment to the mitochondria; the latter, once at the mitochondria, ubiquitylates OMM protein(s), thus inducing mitophagy and producing a signal for AMBRA1–S1014 phosphorylation, flanking its LIR motif. This event causes in turn a structural rearrangement necessary to make the AMBRA1-LIR motif more accessible to LC3B. In particular, we detected that the negative charge on S1014 of AMBRA1 produces an increase in electrostatic interactions in the proximity of the tryptophan site (W1020) on the AMBRA1-LIR motif. As shown in Supplementary Fig. 47. We here demonstrated a novel role of IKKα, independently of its role in NF-κB pathway in mitophagy. Interestingly, Criollo et al.48 proved that the IKK complex was able to promote macroautophagy activation in an NF-κB-independent manner.Our results clearly define the importance of post-translational modifications on the phosphorylated site, in proximity of the identified LIR motif of AMBRA1. Several reports demonstrated the central role of kinase-mediated phosphorylation in cargo receptor-mediated autophagy25. Interestingly, one of these proteins, TBK1, plays a pivotal role in controlling selective autophagy, such as xenophagy and mitophagy49. Here, we demonstrated that, in analogy with TBK1, which is mainly described to be involved in the phosphorylation of the mitophagy receptor OPTN upstream of its LIR motif in xenophagy23 and in mitophagy5, IKKα is able to phosphorylate the mitophagy receptor AMBRA1 flanking its LC3-interacting region during mitochondrial clearance. It seems that such mechanisms are evolutionary conserved and we hypothesize that similar to OPTN, AMBRA1 could also be involved in other selective autophagy processes .Although the IKK family is composed of several members, including IKKα, IKKβ, IKKγ, TBK1 and IKKε, the structure of these factors is very similar50. Moreover, Bakkar et al.51 demonstrated that IKKα regulates mitochondrial biogenesis in muscle tissue. These data underline the complexity of IKKα function, which seems to be highly related to inflammation, mitochondria dynamics and mitophagy.It is noteworthy that IKKα is also fundamental in mitochondrial dynamics regulation by favouring OPA1 activation and therefore mitochondrial fusionIn sum, we prove that previously unravelled mitophagy-involved proteins, HUWE1 E3 ubiquitin ligase and IKKα kinase, play pivotal roles in AMBRA1-mediated mitophagy. HUWE1 is crucial to trigger AMBRA1-mediated mitophagy by favouring MFN2 degradation. This event is necessary for AMBRA1 phosphorylation on its S1014-mediated by IKKα. This kinase, in turn, regulates the engulfment of damaged mitochondria into autophagosomes by phosphorylating AMBRA1–S1014, thus enabling AMBRA1–mATG8 interactions. Since either HUWE1 or IKKα depletion inhibit mitophagy mediated by AMBRA1, we propose that these two proteins strongly contribute to two different steps in the AMBRA1-mediated mitophagy control . Penta-knockout HeLa cells (Penta-KO) and mt-mKeima expressing wild-type or PINK1 knockout HeLa cells  were a gift of R. Youle5. HEK293T-HUWE1 cells were donated by T. Mak.Cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% foetal bovine serum  and 1% penicillin–streptomycin solution  at 37 °C under 5% COTransient transfections of expression plasmids into HeLa, Penta-KO, HEK293T-HUWE1 and SH-SY5Y cells were performed using TurboFect or Lipofectamine 2000 according to the supplier’s instructions .All the transfected constructs presented in this article are described in Supplementary Table Mutations listed in the Supplementary Table RNA-interference was performed using Lipofectamine 2000. A list of siRNA constructs is described in Supplementary Table 5. HeLa cells were treated either with the combined treatment O/A (2.5 and 0.8 μM) or FCCP 10 μM  or EBSS  for the indicated time. In order to block IKKα activity, cells were treated with the irreversible inhibitor BAY-117082 at 1 or 2 μM, for the indicated time. Autophagosome–lysosome fusion was blocked with NH4Cl 40 mM .After 6 h of transfection or 24 h after cell seeding, cells were treated as described below. Penta-KO were treated with the combined treatment O/A /4 μM antimycin A (Sigma)), supplemented with 20 μM QVD  in fresh growth medium for 24 h, as in ref. 4VO3 0.1 mM , NaF 1 mM  and β-Glycerophosphate 5 mM .Cells were rinsed in phosphate-buffered saline (PBS) on ice and lysed in RIPA buffer supplemented with a protease inhibitor cocktail , Nag for 10 min at 4 °C. Protein concentrations were determined with the Bio-Rad Protein Assay Kit . Cell extracts or immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyvinylidene difluoride  or Nitrocellulose  membranes were incubated with primary antibodies followed by horseradish peroxidase-conjugate secondary antibodies  and visualized with ECL (Merck-Millipore WBKLS0500). Uncropped scans of the most important blots are reported in the Supplementary Information, as Supplementary Figure Cell extracts were centrifuged at 15,000 × After cell lysis, equal amounts of protein were incubated with primary antibodies with end-over-end rotation for 24 h. Then, 30 µl of protein A agarose beads  were added for 60 min. The beads were collected by centrifugation and washed 3 times with the RIPA buffer. Immunocomplexes were eluted with 30 µl of SDS sample buffer and heated at 95 °C for 10 min.52. For Supplementary Figure z-confocal planes.Cells were washed in PBS and fixed with 4% paraformaldehyde in PBS for 10 min. After permeabilization with 0.4% Triton X-100  in PBS for 5 min, cells were incubated overnight at 4 °C with primary antibodies and 2% normal goat serum . Cells were then washed with PBS  and incubated for 1 h with labelled anti-mouse  or anti-rabbit  secondary antibodies. Nuclei were stained with 1 μg/ml DAPI  and examined under a Zeiss LSM 700 100× oil-immersion objective . We used ImageJ software for image analysis. We calculated the mito content as percentage of cytosolic area occupied by mitochondria with Mitophagy macrog for 10 min, were resuspended in homogenization buffer , 10 mM HEPES pH 7 , EDTA ) with protease and phosphatase inhibitors cocktail. Nuclei, mitochondria and cytosol purifications proceed by standard differential centrifugations. After homogenization of the solution with 30–70 pulses (one pulse corresponds to approximately 1 s), cells were collected at 600 × g at 4 °C for 10 min. The pellet containing the nuclear fraction was suspended in RIPA buffer, instead the supernatant was subjected to a further centrifugation at 11,000 × g for 15 min at 4 °C. The mitochondrial pellet fraction was suspended in RIPA buffer. The supernatant fraction corresponded to soluble cytosolic proteins.Cells, rinsed in ice-cold PBS and centrifuged at 800 × WT or Flag-IKKαWT or HA-IKKαK44M. After 24 h, equal amounts of proteins (500 µg) were immunoprecipitated using anti-Myc, anti-Flag and anti-HA antibodies, respectively, overnight. Protein A Agarose beads (30 µl of 50% slurry)  were added to the samples at 4 °C with end-over-end rotation for 1 h. The immunoprecipitates were collected by centrifugation and washed 3 times with RIPA buffer. Pellets were washed for 3 min with 500 µl 1× Kinase Buffer ). The IP-Flag- IKKαWT or IP-HA-IKKαK44M final pellets were resuspended in 50 µl of 1× Kinase Buffer, while IP-Myc-AMBRA1 was suspended in 50 µl of RIPA buffer. In order to test kinase activity with a non-radioactive method, 1 µl of 10 mM adenosine triphosphate (ATP) and 20 µl of IP-Myc-AMBRA1 substrate were added to the IP kinases for 30 min at 30 °C. The reaction was stopped by adding 30 µl of 4× SDS Sample Buffer and denaturizing at 95 °C for 5 min. For the kinase assay with recombinant AMBRA1, the wild-type AMBRA1 protein was transcribed and translated in vitro with standard procedures . At the end of IKKαWT or IKKαK44M immunoprecipitation (as described above), 1 µl of 10 mM ATP and 2 µl of kinase substrate was added to the IP-kinases for 30 min at 30 °C. The reaction was stopped by adding 30 µl of SDS Sample Buffer and denaturizing at 95 °C for 5 min. For both of these kinase assays, samples were analysed by western blot.HeLa cells were transiently transfected with a vector encoding for Myc-AMBRA1The ITC experiments were performed at 25 °C and 35 °C using a MicroCal VP-ITC microcalorimeter . The peptides at concentrations of 0.35 mM were titrated into 0.015 mM LC3 and GABARAP proteins in 21 steps. The ITC data were analysed with the ITC-Origin 7.0 software with a one-site binding model. The protein and peptide concentrations were calculated from the UV absorption at 280 nm through a Nanodrop spectrophotometer .15N-labelled LC3B and GABARAP protein samples to which the non-labelled AMBRA1-LIR peptides were added stepwise until 8 times excess to LC3B protein, or 4 times excess to the GABARAP protein. Chemical shift perturbations (CSP) analysis was done according to the most recent review53. CSP values were calculated for each individual backbone proton using the formula: Δδ = [ΔδH2 + (ΔδN/5)2]1/2. The dissociation constants, KD, were calculated by a least squares fit to the titration data under the assumptions of a fast exchange regime and a one binding site mode of the protein interaction. The obtained CSP values were mapped on LC3B (PDB ID 1UGM54) and GABARAP (PDB ID 1GNU55) structures.All NMR experiments were performed at 298 K on Bruker Avance spectrometer operating at a proton frequencies of 600 MHz. Titration experiments were performed with 0.17 mM 15N-labelled LC3 and GABARAP proteins were obtained based on the protocols described elsewhere57. AMBRA1-LIR-containing peptides with different states of S1014 phosphorylation  were purchased from GenScript Inc. . Before experiments, all proteins and peptides were equilibrated with a buffer containing 50 mM Na2HPO4, 100 mM NaCl, pH 7.0; and supplemented with 5 mM protease inhibitor cocktail.For ITC and NMR experiments, the unlabelled or 58 to generate structural models of the AMBRA1-LIR motif (L1013NSGVEYYWDQLNET1026) in complex with LC3B to use as starting structures for the all-atom MD simulations. For the modelling, we used the X-ray structure of p62-LIR motif in complex with LC3B (PDB entry 2ZJD)59 as a template. We generated 1000 binding poses with Modeller and we then carried out structural clustering with the linkage algorithm based on the root mean square deviation (RMSD) of all the atoms of Ambra1-LIR peptide. We selected the average structures from each of the four more populated clusters  and we used each of them as a starting structure for all-atom explicit solvent molecular simulations using multiple replicates. For the phosphorylated variant of Ambra1-LIR, we generated a second set of models by in silico phosphorylation  on position S1014 in each of the four models previously selected using the web interface of Vienna-PTM60. We treated in our simulation the phosphoserine S1014 using the −2 state. Indeed, the pKa values of single protonated/un-protonated transition of phosphoserine suggest that both states are present at neutral pH, but the −2 state is predominant61.We used Modeller version 9.1562 with the protein force fields Charmm22/CMAP63 and Charmm22*64 in conjunction with the TIPS3P water model. Parameters for S2P were merged from ref. 65. All systems were solvated in dodecahedral boxes with a minimum distance between protein and box edge of 1.8 nm and charged ions were added to set a concentration of 0.15 M NaCl. The productive MD simulations were carried out at 298 K with simulation parameters and equilibration protocol66. The first 50 ns of each MD simulation required for equilibration of the system was discarded from downstream analyses.We carried out 250-ns all-atom MD simulations in explicit solvent for each of the selected four models in both unphosphorylated and phosphorylated states, as well as a simulation of the known LC3B-p62 complex as a reference. The MD simulations were performed with Gromacs version 5.1For each trajectory we calculated, as a measure of protein flexibility, the average RMSF for each Cα atom in the Ambra1-LIR or the p62-LIR peptides averaging the RMSF over 10-ns time windows and estimating the associated standard deviations.The four independent replicates of each system were merged into a concatenated trajectory for the analyses of electrostatic interactions.67. The interactions are defined as distances between the charged groups in the residue side chains of arginine, lysine, aspartate, glutamate and the phosphoserine. We employed a 5 Å cutoff in the analyses. To filter out interactions with low occurrence in the simulations, we included in the final networks only those interactions which are present in at least 20% of the simulation frames, as previously tested in other cases of study67. All the electrostatic interactions identified for each of the MD ensembles were visualized using the PyMOL plugin xPyder68.We estimated intra- and inter-molecular electrostatic interactions, as implemented in the PyInteraph suite of toolsl-glutamine, l-arginine and l-lysine, according to the supplier’s instructions. Accordingly, we obtained two different isotope amino-acid labelling media: Light Medium K0/R0 and SILAC Heavy medium (K6/R10). Cells were growth in these media for 2 weeks.SILAC-based strategy is used for comparative and quantitative AMBRA1-interacting proteins analysis in HeLa cells in mitophagy condition. Cells were growth in SILAC DMEM , supplemented with FBS 10%, 1% penicillin–streptomycin solution, ActA for 24 h. Equal amounts of harvested Light and Heavy lysates were immunoprecipitated with an anti-Myc antibody, as previously described in the immunoprecipitation protocol. Immunoprecipitated heavy proteins were then mixed 1:1 with light proteins and subjected to SDS–PAGE followed by band excision and trypsin-peptide digestion in order to allow LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analysis15.In order to analyse AMBRA1 interactors during mitophagy, Light and Heavy cells were transfected with a vector encoding for Myc-AMBRA1HEK293T cells stably expressing Flag-HUWE1 were lysed with Triton Buffer , supplemented with proteases and phosphatases inhibitors. Then, 3–5 mg of whole cell lysates were precleared with protein A/G-Sepharose beads  for 3 h and then incubated overnight at 4 °C with 5 mg of anti-Flag antibody  per sample. Finally, the immunocomplexes have been absorbed on protein A/G-Sepharose beads, washed six times in Triton lysis buffer and used for in vitro ubiquitylation assay or eluted by boiling in SDS loading buffer.2, 0.1 mM ubiquitin aldehyde) in a final volume of 30 μl. The assays have been carried out for 90 min at 30 °C, then the immunocomplexes were eluted by boiling in SDS loading buffer, loaded on SDS–PAGE, blotted onto PVDF membranes (Millipore) and then blocked with PBS-T  containing 5% non-fat dry milk for 1 h at room temperature (RT). The incubation with primary antibodies was performed for 2 h at RT, followed by appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. After extensive washing, detection was performed with the ECL Western Blot Chemiluminescence Reagent (Perkin Elmer).In vitro ubiquitylation assay was carried out using HUWE1 complex immunopurified from HEK293T cells stably expressing Flag-HUWE1 using the method described above. The beads containing the HUWE1 immunocomplexes were washed twice with equilibration buffer  and then added to the following reaction: 40 ng of E1, 0.5 μg of UbcH3, 0.5 μg of UbcH5, 0.3 μg of UbcH7, 1 μg of ubiquitin in the ubiquitylation buffer , 4 mM ATP, 10 mM MgCl69. In addition, cells were exposed to 1 h of OGD. Initially, OGD was performed in a medium previously satured with 95% N2 and 5% CO2 for 20 min and containing NaCl 116 mM, KCl 5.4 mM, MgSO4 0.8 mM, NaHCO3 26.2 mM, NaH2PO4 1 mM, CaCl2 1.8 mM, glycine 0.001 mM and 0.001 w/v phenol red70. Then, hypoxic conditions were maintained using a hypoxia chamber at 37 °C, 5% CO2 and 95% N2. At the end of OGD, the cells were incubated for 24 h in normoxic conditions using DMEM containing normal levels of O2 in a CO2 incubator at 37 °C RX) followed by lysis for western blot analysis. Whole cell lysate were prepared in RIPA buffer  Triton X-100, pH 8.0) containing protease and the phosphatase inhibitors.Preconditioning and ischaemia condition were recreated by exposing cells to 30 min and 3 h, respectively, of OGD7) were co-transfected with PcDNA3+GFP, Myc-AMBRA1WT+GFP, Myc-AMBRA1S1014A+GFP, Myc-AMBRA1S1014D+GFP, PcDNA3+GFP-ShCtr, Myc-Ambra1ActA+GFP-ShCtr or myc-Ambra1ActA+GFP-ShHUWE1, and treated as indicated in the text. GFP was used as a reporter for co-transfected cells. Live cells were photographed at 37 °C/5% CO2 through the live cell imaging microscope UltraView Vox (Perkin Elmer). mt-mKeima was excited both at 405 nm  and 561 nm  and detected through the same emission filter , while GFP was captured with standard procedures . A total of 100 randomized fields per condition were detected in each experiment with a 100× objective. Randomization was performed through an automated function of the miscroscope. mt-mKeima fluorescence intensity was quantified in single GFP-positive cells with the integrated function of the microscope software Volocity (Perkin Elmer). Fields with no GFP-positive cells were excluded from the analysis.Wild-type and PINK1-KO HeLa cells stably expressing mitochondrial-targeted mKeima  and Tukey’s post-test with a confidence interval of 95%. Significance is defined as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. No statistical methods were used to predetermine sample size.All statistical calculations were performed and graphed using GraphPad Prism 6. Comparisons between two groups were analysed using Student’s Supplementary InformationPeer Review File