Gábor Sipka
email: sipka.gabor@brc.mta.hu

Pavel Müller

Klaus Brettel

Melinda Magyar

László Kovács

Qingjun Zhu

Yanan Xiao

Guangye Han

Petar H Lambrev

Jian-Ren Shen

Győző Garab
email: garab.gyozo@brc.mta.hu

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated Photosystem II core complex of Thermosynechococcus vulcanus

  Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus

excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (~50%) and 167 ns (~30%), and a longer-lived component (~20%). This kinetics are attributed to rereduction of P680 •+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 μs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (~60%) and 10 ns (~30%), attributed to recombination of the primary radical pair P680 •+ Pheo •-, and a small longer-lived component (~10%). These data confirm that only the first STSF is capable of generating stable charge separation -leading to the reduction of Q A ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

Introduction

Photosystem II (PSII) is the redox-active pigment-protein complex embedded in the thylakoid membrane that catalyzes the oxidation of water and the reduction of plastoquinone. PSII is probably the most-studied light-inducible enzyme, not only for its relevance to biochemistry (it is the O 2producing enzyme) but also because it serves as basis of inspiration for artificial photocatalyzers for water oxidation. The PSII reaction center complex (RC) is surrounded by CP47 and CP43 core antenna proteins. This PSII core complex (PSII CC) may be regarded as the minimal fully functional PSII complex capable of charge separation and stabilization, plastoquinone reduction and oxygen evolution [START_REF] Ghanotakis | Photosystem-II and the Oxygen-Evolving Complex[END_REF]Yocum 1990, van Grondelle et al. 1994). The structure of PSII CC from Thermosynechococcus vulcanus has been determined at 1.9 Å resolution by synchrotron radiation Xray crystallography (Umena et al. 2011); femtosecond X-ray free electron laser (XFEL) has provided a radiation-damage-free structure at a resolution of 1.95 Å (Suga et al. 2015). Each PSII CC contains the reaction center incorporated in the D1/D2 proteins, the α and β subunits of cytochrome b559 and two integral antenna proteins, CP43 and CP47, which carry 14 and 17 chlorophyll-a molecules (Chl-a), respectively. The RC D1/D2 proteins are located approximately symmetrically with respect to the transmembrane region, which is very similar to the arrangement of the L/M subunits in bacterial RC (bRC). Four Chls (two accessory chlorophylls Chl D1 and Chl D2 and a special pair of the Chl-a molecules P D1 and P D2 , conventionally denoted as P680), two pheophytins (Pheo D1 and Pheo D2 ), and two plastoquinones (Q A and Q B ) are arranged in two symmetrical branches. (It is to be noted that the nature of the primary donor is still under debate, and either a monomer, dimer or multimer of Chl-a molecules have been proposed [START_REF] Durrant | A multimer model for P680, the primary electron donor of photosystem II[END_REF][START_REF] Boussaad | Chlorophyll a dimer: a possible primary electron donor for the photosystem II[END_REF][START_REF] Barber | P680, the primary electron donor of photosystem II[END_REF], Romero et al. 2017a).) The absorption of a photon or transfer of an exciton to the primary electron donor P680 forms an electronically excited state. As in the simpler purple bRC [START_REF] Michel | Relevance of the photosynthetic reaction center from purple bacteria to the structure of Photosystem II[END_REF][START_REF] Rutherford | Photosystem II, the water-splitting enzyme[END_REF][START_REF] Kálmán | Comparison of bacterial reaction centers and photosystem II[END_REF], the subsequent charge separation in PSII is known to proceed asymmetrically, only along the D1 branch with the formation of P680 This article is protected by copyright. All rights reserved.

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When Q A is pre-reduced, only the primary radical pair P680 •+ Pheo •-can be formed. This pair then decays by charge recombination within a few nanoseconds. In this case, the lifetime and yield of fluorescence (emitted mostly by the antenna chlorophylls) are substantially increased because the energy transfer from the antennae to P680 becomes slower and less efficient and because delayed luminescence occurs during the (prolonged) lifetime of the P680 •+ Pheo •-pair (Shuvalov et al. 1980, Nuijs et al. 1986, Schatz et al. 1987, Schlodder and Brettel 1988, van Mieghem et al. 1995).

Despite the detailed structural information and the wealth of functional data [START_REF] Cardona | Charge separation in photosystem II: A comparative and evolutionary overview[END_REF][START_REF] Nelson | Structure and energy transfer in photosystems of oxygenic photosynthesis[END_REF][START_REF] Duan | Primary Charge Separation in the Photosystem II Reaction Center Revealed by a Global Analysis of the Twodimensional Electronic Spectra[END_REF], Romero et al. 2017b) F m depends solely on the redox state of the primary quinone electron acceptor, Q A (Duysens andSweers 1963, Strasser et al. 2004) has been challenged by several authors [START_REF] Joliot | Comparative study of the fluorescence yield and of the C550 absorption change at room temperature[END_REF][START_REF] Schansker | Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise[END_REF], Vredenberg 2011). Recently, it has been shown that a substantial part of the gradual fluorescence rise of PSII elicited by multiple µs-STSFs in the presence of the PSII electron transport inhibitor diuron (DCMU, 3-(3′,4′ dichlorophenyl)-1,1′ dimethylurea) that permits only one stable charge separation, originates from light-induced processes that occur after the stabilization of the charge separated state, i.e. in already closed RC where Q A is reduced (Magyar et al. 2018, Joliot and[START_REF] Joliot | Comparative study of the fluorescence yield and of the C550 absorption change at room temperature[END_REF]. This phenomenon has been observed in a variety of samples, from PSII CC to whole cells and from intact to TRIS-washed thylakoid membranes and at temperatures between about 170 and 280 K. Further, experiments using double flashes with variable delays (Δt) between them, have revealed the involvement of a previously unknown rate-limiting step in the light-induced reactions that lead to the gradual increase of the fluorescence level from F 1 (after one STSF) to F m (reached after several flashes). These observations cannot be reconciled with the mainstream model according to which "in order to reach [F m ], it is necessary, and sufficient, to have Q A completely reduced in all the active PSII centers" (Stirbet and Govindjee 2012). However, the underlying physical mechanism (or mechanisms) of the fluorescence yield increments and of the rate-limiting step has (have) not been elucidated.

An analysis of the temperature dependence of the fluorescence transients strongly suggested the involvement of conformational changes/dielectric relaxation in the F 1 -to-F m fluorescence increments.

With respect to the origin of the rate-limiting step, two hypotheses have been put forward [START_REF] Magyar | Rate-limiting steps in the dark-to-light transition of Photosystem II -revealed by chlorophyll-a fluorescence induction[END_REF]): (1) It might originate from a rate limitation at the level of primary photochemistry (e.g. the turnover of P680 This article is protected by copyright. All rights reserved.

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a gating mechanism operating in the time interval shortly after the first flash cannot be ruled out a priori -given the fact that the increments between F 1 and F 2 , F 2 and F 3 etc. are light-induced events, which require waiting time.

(2) Alternatively, the light-induced reactions might change the fluorescence yield of the PSII CC without gating the primary photochemistry. The fluorescence of PSII CC has been proposed to originate from the two antenna-proteins, and to depend on the excitation energy transfer reactions between P680 * and the domains of CP47 and CP43 (Shibata et al. 2013).

In this work, in an attempt to elucidate the origin of Chl-a fluorescence yield increments and the physical mechanism underlying the rate-limiting step in PSII, we measured, at room temperature, Chla fluorescence kinetics induced by trains of µs-STSFs, and saturating laser-flash induced nanosecond 819 nm transient absorption changes (originating mainly from the oxidation and re-reduction of P680)

in DCMU-treated PSII CC isolated from T. vulcanus. Our data confirm that the F o -to-F 1 fluorescence rise is associated with stable charge separation (down to S 2 (+) Q A •-; Fig. 1).

The consecutive F 1 -to-F 2 (and to F m ) increment(s) cannot be quantitatively explained by stable charge separation. Instead, rapid recombination of P680 •+ Pheo •-prevails upon second and repetitive flashes. We also conclude that the rate limitation in PSII does not mainly originate from gating of the primary charge separation and must involve other light-dependent processes affecting the Chl-a fluorescence yield.

Materials and methods

Photosystem II core complexes and treatments PSII core complex of T. vulcanus was isolated as described earlier [START_REF] Shen | The Structure of Photosystem II and the Mechanism of Water Oxidation in Photosynthesis[END_REF][START_REF] Shen | Binding and functional properties of two new extrinsic components, cytochrome c-550 and a 12-kDa protein, in cyanobacterial photosystem II[END_REF][START_REF] Shen | Crystallization and the crystal properties of the oxygen-evolving photosystem II from Synechococcus vulcanus[END_REF]. For Chl-a fluorescence measurements, the Chl content of the PSII CC was adjusted to ~25 μg ml -1 in 20 mM MES (pH 6.0), 20 mM NaCl, 3 mM CaCl 2 and 5% glycerol. DCMU was dissolved in dimethyl sulfoxide (DMSO) and added to all samples immediately before the fluorescence measurements at a final concentration of 40 μM (the final concentration of DMSO did not exceed 1%). Before the measurements, the samples were dark adapted for 5 min at room temperature. For transient absorption spectroscopy, the same protocol was used for the dilution of PSII CC to a Chl concentration of ~250 µM and 40 µM DCMU was added to the samples immediately before the measurements.

Chlorophyll a fluorescence

Relative fluorescence yields were measured using a customized PAM 101 fluorometer (Walz), as

Transient absorption spectroscopy

The transient absorption spectroscopy system is optimized for fast response, high fidelity, and low noise; the setup has been described in detail in [START_REF] Byrdin | Use of ruthenium dyes for subnanosecond detector fidelity testing in real time transient absorption[END_REF][START_REF] Müller | ATP binding turns plant cryptochrome into an efficient natural photoswitch[END_REF][START_REF] Müller | Sub-nanosecond tryptophan radical deprotonation mediated by a protein-bound water cluster in class II DNA photolyases[END_REF]. Two pulsed lasers were used in single-and double-flash experiments. In double flash excitation experiments, the first excitation flash (~5 ns, 532 nm) was provided by a Nd:YAG pumped optical parametric oscillator (OPO; Brilliant B/Rainbow) and the second flash by a Continuum Leopard SS-10

Nd:YAG laser (~100 ps, 532 nm). The delay between the laser flashes was controlled by a high precision digital pulse and delay generator (DG535 from Stanford Research Systems, Inc.). The kinetic traces were recorded upon the second flashes. For single-and multiple-flash excitation experiments, the 100 ps laser was used. The laser energies were set to ~5 mJ cm -2 for the 5 ns pulse and ~1 mJ cm -2 for the 100 ps pulse, as estimated from energy measurements behind a cell filled with H 2 O using an energy meter (Gentec QE25SP-H-MB-D0) and from transient absorption signals using

[Ru(bpy) 3 ] 2+ as an actinometer [START_REF] Müller | Ru(bpy) ] 2+ as a reference in transient absorption spectroscopy: differential absorption coefficients for formation of the long-lived 3MLCT excited state[END_REF]. A small fraction of the 100 ps pulse was used to trigger the detection system via a fast photodiode. Continuous measuring light was provided by a laser diode emitting at 819 nm (SDL-5411-G1, Spectra Diode Labs), passed through a cut-off filter RG780 (to suppress weak contaminations below 750 nm in the emission of the laser diode). The .

Thermoluminescence (TL)

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TL was measured using a custom-made apparatus similar to the one described by [START_REF] Ducruet | Thermoluminescence: experimental[END_REF]. Samples of PSII core complexes, equivalent to 12 µg Chl, on a 13-mm-diameter glass-fiber filter paper (GF-2, Macherey-Nagel GmbH) were placed on a copper sample holder, which was connected to a cold finger immersed in liquid nitrogen. A heater coil, placed under the sample holder, ensured the desired temperature and heating rate of the sample during the measurements. Darkadapted samples were excited by one STSF at 10°C, or by 1 and 20 STSFs at -60°C. The glow curves were measured while heating the sample to 70°C with a constant heating rate of 0.5°C s -1 . The TL emission was measured with a Hamamatsu end-window photomultiplier (Hamamatsu Photosensor Modules H1071-20). The detector current was amplified using a DC transimpedance amplifier with a 10 V/µA conversion ratio (Hamamatsu C7319).

Results

Upon the excitation of DCMU-treated dark-adapted PSII CC of T. vulcanus by double STSFs with variable waiting times (Δt) between the two flashes, the Chl-a fluorescence yield at room temperature (23°C) displayed a stepwise rise (Fig. 2A). A similar pattern was observed earlier using a train of STSFs at 5°C [START_REF] Magyar | Rate-limiting steps in the dark-to-light transition of Photosystem II -revealed by chlorophyll-a fluorescence induction[END_REF]. STSF excitation of PSII CC in the presence of PSII inhibitor DCMU leads to the formation of S 2 (+) Q A •-state via three intermediate states (Fig. 1). As shown in Figs 2A and S1, the first STSF raised the fluorescence yield from F o to F 1 , with F 1 being significantly smaller than F m , 59 ± 3% of F m at room temperature. This value at room temperature and at 5°C varied from batch to batch between about 45 and 60% and about 35 and 45 %, respectively; the F v /F m parameter under the same conditions was 0.845 ± 0.035 (n = 9, obtained from 5 different batches; data not shown). These observations are in good agreement with the data reported earlier on PSII CC by [START_REF] Magyar | Rate-limiting steps in the dark-to-light transition of Photosystem II -revealed by chlorophyll-a fluorescence induction[END_REF] and in reasonable accordance with observations on thylakoid membranes [START_REF] Joliot | Comparative study of the fluorescence yield and of the C550 absorption change at room temperature[END_REF]Joliot 1979, Prášil et al. 2018). By using a second STSF, the fluorescence level could be further raised, by about another 15%but only after a sufficiently long Δt waiting time between the two flashes, and further excitations were required to reach the maximum level F m ; here MTSF excitation was used (the second MTSF laser pulse was applied to confirm that F m was reached).

As reported earlier [START_REF] Magyar | Rate-limiting steps in the dark-to-light transition of Photosystem II -revealed by chlorophyll-a fluorescence induction[END_REF]), the fluorescence level at the second and later flashes depended on the dark waiting time Δt between the flashes (Fig. 2B). Compared to F 1 , no fluorescence increment could be induced with the second flash if two STSFs were fired simultaneously, or with a short waiting time (Δt < 100 μs); the F 1 -to-F 2 increment was maximal at waiting times longer than 10 ms. The half-rise time (∆t 1/2 ) was about 3 ms. The effect of waiting time is also demonstrated in Fig. S1: the F 1 -to-F 2 fluorescence yield increment, i.e. the increase between the fluorescence levels after the first and second STSF, was negligible with Δt ≤ 100 µs, and reached its maximum with Δt ≥ 10 ms.

In order to elucidate the physical mechanism underlying the waiting-time dependent stepwise fluorescence yield rise in DCMU-treated PSII CC, we performed laser-flash-induced transient

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absorption measurements at 819 nm to monitor the photooxidation and re-reduction kinetics of P680, and the fate of P680 •+ Pheo •-primary radical pair. Note that both P680 •+ and Pheo •-absorb at this wavelength with extinction coefficients, respectively, (P680 •+ ) 819 nm  7 mM -1 [START_REF] Mathis | Near Infra-Red Absorption Spectra of the Chlorophyll a Cations and Triplet State in vitro and in vivo[END_REF] and (Pheo •-) 819 nm  6 mM -1 [START_REF] Fujita | Anion radicals of pheophytin and chlorophyll a: their role in the primary charge separations of plant photosynthesis[END_REF]. We recorded single, double and repetitive flashinduced transient absorption changes in the DCMU-treated dark-adapted PSII CC, using variable waiting times (∆t) between consecutive laser flashes.

As shown in Fig. 3A (black trace), upon single-flash excitation, the transient absorption signal at 819 nm exhibits a fast rise and an initial sub-ns decay phase (which is not well-resolved in our experiment and which we tentatively attribute to the re-oxidation of Pheo •-by Q A in a few hundred picoseconds; Fig. 1). This ultrafast phase is followed by an essentially biphasic decay, which can be well fitted in the time window from 1 to 800 ns by a bi-exponential decay with time constants of 16.6 ns (~50%) and 167 ns (~30%) and a constant (~20%) that accounts for decay components on the microsecond time scale (Table 1), in good agreement with literature data for the reduction of P680 •+ by the donor side of PSII (van Best and Mathis 1978[START_REF] Brettel | Nanosecond reduction kinetics of photooxidized chlorophyll-aII (P-680) in single flashes as a probe for the electron pathway, H + -release and charge accumulation in the O 2 -evolving complex[END_REF][START_REF] Schlodder | Temperature dependence of the oxidation kinetics of TyrZ and TyrD in oxygen-evolving photosystem II complexes throughout the range from 320 K to 5 K[END_REF]. The fastest decay component (~20 ns) has been clearly attributed to proton-coupled electron transport from TyrZ in the dark-adapted state (S 1 ) of the OEC (Tommos and Babcock 2000, [START_REF] Schlodder | Temperature dependence of the oxidation kinetics of TyrZ and TyrD in oxygen-evolving photosystem II complexes throughout the range from 320 K to 5 K[END_REF].

Slower decay components are thought to indicate relaxation processes involving hydrogen bond networks that shift the equilibrium P680 •+ TyrZ ↔ P680TyrZ • (H + ) to the right and reduction of TyrZ • (H + ) by the OEC [START_REF] Schilstra | Proton/Hydrogen Transfer Affects the S-State-Dependent Microsecond Phases of P680 + Reduction during Water Splitting[END_REF][START_REF] Christen | The role of hydrogen bonds for the multiphasic P680(+)* reduction by YZ in photosystem II with intact oxyen evolution capacity. Analysis of kinetic H/D isotope exchange effects[END_REF][START_REF] Schlodder | Temperature dependence of the oxidation kinetics of TyrZ and TyrD in oxygen-evolving photosystem II complexes throughout the range from 320 K to 5 K[END_REF].

Reduction of P680 •+ is also known to be slowed down in the presence of higher oxidation states (S 2 and S 3 ) of the OEC [START_REF] Brettel | Nanosecond reduction kinetics of photooxidized chlorophyll-aII (P-680) in single flashes as a probe for the electron pathway, H + -release and charge accumulation in the O 2 -evolving complex[END_REF][START_REF] Schlodder | Relation between microsecond reduction kinetics of photooxidized chlorophyll aII (P-680) and photosynthetic water oxidation[END_REF] and in PSII with inactive OEC [START_REF] Conjeaud | Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane[END_REF][START_REF] Conjeaud | The effect of pH on the reduction kinetics of P-680 in tris-treated chloroplasts[END_REF].

In contrast to the transient absorption changes upon a single laser-flash excitation, following one or several pre-flashes, the kinetics was dominated by a rapid decay in a few nanoseconds. This was most easily detected in an experiment with repetitive (2 s -1 ) excitation by 100 ps pulses. The red trace in Fig. 3A represents an average of 128 signals (which substantially improved the signal-to-noise ratio)

following 10 pre-flashes given to bring PSII to the state where F m is observed in the fluorescence yield measurements (Fig. 2A).

Even though the observed initial amplitudes of the signals of the single-flash and repetitive-flashes experiments were similar, note that this does not imply a similar yield of P680 •+ formationthe signal of the repetitive flash experiment contains a considerable contribution of Pheo •-, whereas Pheo •-is essentially absent at the beginning of our fit (1 ns) in the single-flash experiment. Considering the molar absorption coefficients given above, the P680 •+ Pheo •-pair yield in the repetitive flashes experiments amounts only to ~65% of the P680 •+ yield obtained in the single-flash experiment, close to the value (6015%) previously reported for PSII complexes from Synechococcus [START_REF] Schlodder | Primary charge separation in closed Photosystem II with a lifetime of 11 ns: Flash-absorption spectroscopy with O 2 -evolving Photosystem II complexes from Synechococcus[END_REF]).

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A bi-exponential fit of the repetitive flashes experiment in the interval between 360 ps (where the signal rise was completed) and 50 ns yielded τ 1 of 2 ns (~55%), τ 2 of 9 ns (~35%) and a minor longerlived component (10%). In line with previous works (Shuvalov et al. 1980[START_REF] Nuijs | Primary-charge separation and excitation of chlorophyll a in photosystem II particles from spinach as studied by picosecond absorbance-difference spectroscopy[END_REF][START_REF] Schlodder | Primary charge separation in closed Photosystem II with a lifetime of 11 ns: Flash-absorption spectroscopy with O 2 -evolving Photosystem II complexes from Synechococcus[END_REF], van Mieghem et al. 1995), we attribute the decay phases on the ns timescale to recombination of the primary radical pair P680

•+ Pheo •-in closed RC (containing Q A •-).
The time constants previously attributed to P680 •+ Pheo •-recombination vary considerably (between 1.6 ns [START_REF] Schatz | Picosecond kinetics of fluorescence and absorbance changes in photosystem II particles excited at low photon density[END_REF]) and ~25 ns (slower component of a biphasic decay; van Mieghem et al.

1995), which may reflect differences in origins and types of the PSII preparations (with (photo)chemically semi-or fully (pre-)reduced Q A ) and in the time window and resolution of the used techniques. It has been suggested that a biphasic nature of the decay is due to relaxation of the P680 •+ Pheo •-pair [START_REF] Schlodder | Primary charge separation in closed Photosystem II with a lifetime of 11 ns: Flash-absorption spectroscopy with O 2 -evolving Photosystem II complexes from Synechococcus[END_REF]. The small absorption change remaining after the ns decay may represent triplet states of Chl-a in the antenna and/or in the RC (van Mieghem et al. 1995).

We next studied the yield and kinetics of the P680 •+ Pheo •-pair upon the second flash in doubleflash experiments as a function of the delay between the two flashes (between 5 s and 100 ms).

Dependence of the initial amplitude on the delay would be a testament to gating of the primary charge separation, which could, in principle, be related to the lack of F 1 -to-F 2 fluorescence increment. In other terms, we wanted to clarify if the lack of F 1 -to-F 2 fluorescence increment upon the second STSF excitation with Δt < 100 μs, is caused, or not, by the lack (gating) of primary photochemistry.

Remarkably, already for the delay (t) of 5 s, the 'typical' P680 •+ Pheo •-recombination signal was observed with a relative amplitude of as much as ~65% of the signal in the repetitive-flash experiment (with 500 ms intervals). This relative amplitude increased with delay length to ~90% at 100 ms (Fig. 3B). The signals at all delays (averages of 5-8 experiments each) could be well fitted with biexponential decay functions with shared time constants τ 1 of 2 ns and τ 2 of 10 ns (i.e. very similar to the repetitive flashes experiment). The amplitudes of the two phases, of the longer-lived component, and their sum (corresponding to the initial amplitude) are plotted against the delay time (t) in the inset of Fig. 3B. Note that between t of 5 s and 5 ms, the growth of the initial amplitudes (A 1 + A 2 + y 0 in the inset of Fig. 3B) is essentially due to the growth of the amplitude of the 2 ns phase. Between t of 5 and 100 ms, there seems to be a significant increase in the longer-lived component y 0 .

Discussion

The goal of this work was to elucidate a potential connection between nanosecond redox transients of

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fluorescence yield F m could only be reached by providing additional excitations to the sample, e.g.

with a train of several flasheswhich then gradually raised the fluorescence yield, from F 1 to F 2 and so forth (Fig. 2). It has also been shown, in accordance with earlier literature data obtained on isolated thylakoid membranes [START_REF] Joliot | Comparative study of the fluorescence yield and of the C550 absorption change at room temperature[END_REF], that only the first STSF leads to stable charge separation (Q A reduction) and the F 1 -to- Analysis of the decay kinetics of P680 •+ (Fig. 3, Table 1) confirmed that, as expected, the photochemical events elicited by the first and second flash excitations differed substantially from each other. The kinetics of ΔA 819 following a single-flash excitation of the dark-adapted DCMU-treated PSII CC could be assigned to the formation of the radical pair P680 •+ Q A •-and re-reduction of P680 •+ by the donor side of PSIIas reflected by the multiphasic decay kinetics containing an ~20 and an ~200 ns component and a slow component on the µs timescale, with ~50, ~30 and ~20% contributions, respectively (see Results for more details and for references). In contrast, in the presence of a pre-flash or under repetitive flash excitations, ΔA 819 decayed much faster, dominated by components of ~2 ns (~60%) and ~10 ns (~30%),that can be assigned to charge recombination of P680 •+ Pheo •-; small longer-lived absorption changes (~10%) are essentially attributed to Chl-a triplet states (see Results for more details and for references). In good accordance with the conclusion that after the first STSF the additional flashes do not lead to stable charge separation, no additional TL band was present following multiple-STSF excitation of the sample (Fig. S3).

Our data confirm that, in the presence of DCMU, stable charge separation in PSII can only be induced by the first saturating flash. Hence the gradual fluorescence yield increments from F 1 to F m upon additional flashes are not caused by stable charge separation and must be triggered by other events [START_REF] Joliot | Comparative study of the fluorescence yield and of the C550 absorption change at room temperature[END_REF]Joliot 1979, Magyar et al. 2018).

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The 819 nm transient absorption experiments using pre-flashes (Fig. 3) also answer the question whether the rate-limiting reaction in PSII CC, reported by Chl-a fluorescence (Fig. 2), can be assigned to gating of the primary photochemical event (formation of P680 •+ Pheo •-). If this were the case, the dependence of the initial amplitude of the ΔA 819 on the delay Δt between the first (pre-)flash and second flash (inducing the observed absorption changes), should directly correlate with the appearance of the F 1 -to-F 2 fluorescence yield increment. Inspection of our experimental data (compare Fig. 3B, Table 1 and Fig. 2B) reveals, however, that both the extents of the effects and the delay times of their appearance are clearly different: While the fluorescence yield increment was virtually zero for Δt up to 10 µs, the initial ΔA 819 at Δt = 5 µs was already ~65% of the value obtained with repetitive flashes at 500 ms interval. Furthermore, most of the (moderate) increase of the initial ΔA 819 occurred between Δt = 5 µs and 100 µs, while most of the fluorescence yield increment occurred between Δt = 1 ms and 10 ms. We hence conclude that the rate limitation observed in fluorescence experiments does not originate mainly from gating of the primary charge separation.

We would like to mention that an increase of the initial ΔA 819 with increasing Δt was expected because charge separation is only possible in PSII in which P680 •+ formed by the pre-flash is already re-reduced. The extent of the increase (from ~65% (of the value under repetitive excitation) at Δt = 5

µs to ~90% at Δt = 100 ms) is, however, somewhat more pronounced than expected from the rereduction kinetics of P680 •+ formed by the pre-flash: according to the first-flash signal in Fig. 3A, at least 80% of P680 •+ should already be re-reduced at Δt = 5 µs. As a possible explanation, we suggest that the yield of primary charge separation may be diminished during the lifetime (~50 µs; Summarizing the direct conclusions from our experimental data, the waiting-time related fluorescence yield increment cannot be explained at the level of primary photochemistry of P680.

Instead, we must assume that the waiting time is needed for allowing the relaxation (or completion) of a presently unidentified process that affects the fluorescence yield of Chl-a, which has been proposed to be determined by an interplay between the RC and the two antenna proteins of PSII CC (Shibata et al. 2013). It must be clearly seen that this process is induced by light and it does not occur without additional excitation of the sample. Thus, simple mechanisms, such as allowing a quencher (e.g. P680 •+ ) or triplet states to relax, do not explain our observations. On the other hand, conformational changes and/or dielectric relaxation might have cumulative nature, and the occurrence of consecutive

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events may depend on the completion of the previous steps. In this context it is interesting to note that using a pump-probe technique, [START_REF] Schreiber | Two fundamentally different types of variable chlorophyll fluorescence in vivo[END_REF] have shown that in isolated DCMUtreated intact chloroplasts the initial fluorescence yield rise (measured at 20 µs, limited by the time resolution of the instrument) upon a STSF excitation was followed by a slower rise in the dark, which was completed in ~1 ms, with t 1/2 ~ 50 µs (Note that our Chl-a fluorescence measurements, such as shown in Fig. 2, do not resolve this fast rise). These data, on the one hand, confirm that the reduction of Q A alone, occurring on the (sub-)ns timescale (Fig. 1), cannot be held responsible for F v . On the other hand, given the comparable delay times, there is a possibility that the rise time in the fluorescence, observed by [START_REF] Schreiber | Two fundamentally different types of variable chlorophyll fluorescence in vivo[END_REF], and Δt, the (dark) waiting time needed for the F 1 -to-F 2 and F 2 -to-F 3 etc. fluorescence yield increments [START_REF] Magyar | Rate-limiting steps in the dark-to-light transition of Photosystem II -revealed by chlorophyll-a fluorescence induction[END_REF]; Fig. 2), share the same physical mechanism. This hypothesis should be tested experimentallyby comparing the outcomes of both techniques applied to the same sample under comparable conditions. As to the putative common underlying physical mechanism, while we cannot rule out other explanation, the slow rise after a STSF excitation may also originate from conformational changes/dielectric relaxation due to the generation This article is protected by copyright. All rights reserved.

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Figure legends

Fig. 1. Energy scheme of the electron transfer steps (until Q A reduction) in PSII occurring upon excitation of P680 and assuming that all centers are in the dark-adapted stable state (S 1 ). Based on [START_REF] Cser | Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803[END_REF]. dependence of the fitted amplitudes on the delay between the first (pre-) flash and the actinic flash (Table 1).

  , our understanding of the processes associated with or accompanying the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by Chl-a fluorescence induction but the presently available models and observations are still controversial (Stirbet and Govindjee 2012, Schansker et al. 2014) and the origin of variable fluorescence (F v = F m -F o ) remains to be elucidated. F o and F m are the minimum and maximum fluorescence levels in dark-adapted samples, obtained before and after a sufficiently long and strong excitation, respectively. The canonical view that the rise of fluorescence yield from F o to

  described earlier[START_REF] Magyar | Rate-limiting steps in the dark-to-light transition of Photosystem II -revealed by chlorophyll-a fluorescence induction[END_REF]) -with minor modifications. Variable fluorescence was induced by single-turnover saturating Xe flashes (Excelitas LS-1130-3 Flashpac with FX-1163 Flashtube with reflector) of 1.5 µs duration at half-peak intensity. The frequency of the modulated measuring light (low intensity, virtually non-actinic) was 1.6 kHz. In order to determine F m a 100 ms long multipleturnover saturating flash (MTSF) was used after the first pair of STSFs. MTSFs were delivered from a This article is protected by copyright. All rights reserved.Accepted Articlethe sample holder of a thermoluminescence (TL) apparatus in order to control the temperature. The timing of the flashes was controlled by a programmable digital pulse generator (BNC 577, Berkeley Nucleonics Corp) via custom-designed LabVIEW software. The kinetics of each measurement was recorded using a USB Multifunction I/O Device (NI DAQ 6001) via custom-designed LabVIEW software, with 20 ms -1 sampling rates. However, the time resolution of the measurements was determined by the 1.6 kHz modulation frequency of the measuring beam of the PAM 101 fluorometer.

  measuring light was perpendicular to the excitation laser beams and passed through the sample along the 10 mm path of a 2  8  10 mm (widthheightlength) quartz cell with self-masking solid black walls (from Starna). Flash-induced changes of the transmission of the sample were monitored behind the sample and an 820 nm interference filter (to suppress stray light and the fluorescence induced by the excitation flash) by a Si photodiode (Alphalas UPD-200-UP; rise time, 200 ps; sensitive area, 0.1 mm 2 ) coupled to a digital oscilloscope (Agilent Infinium 81004B; sampling rate, 40 Gsamples s -1 ; rms noise at 40 mV full scale, 340 µV; bandwidth, dc-10 GHz). To estimate the decay parameters, Levenberg-Marquardt least-squares optimization algorithm was used in OriginLab2018 (OriginLab Corporation,) to fit trend lines according to the equation: ( ) ⁄ ⁄

  P680 and the stepwise, waiting-time-dependent rise of the Chl-a fluorescence yield in dark-adapted, DCMU-treated PSII CCs excited with a series of STSFs. Earlier,[START_REF] Magyar | Rate-limiting steps in the dark-to-light transition of Photosystem II -revealed by chlorophyll-a fluorescence induction[END_REF] have shown that in these samples, which are capable of only a single stable charge separation, the fluorescence yield maximum (F m ), cannot be reached by exciting the sample with only one STSF. One flash has been shown to lead to a fluorescence level, termed F 1 , significantly lower than F m . The maximal This article is protected by copyright. All rights reserved.

  photooxidation of P680 and its decay kinetics on the nanosecond timescale. Single, double, and multiple laser-flash experiments were performed.

Fig

  + ) due to electrostatic interaction with P680 •+ . It is also of note that the amplitude y 0 of the longlived component of ΔA 819 increased slightly (from 12 to 17% of the initial amplitude), but significantly, between Δt = 5 ms and 100 ms. This may indicate that, in part of PSII, Q A•-formed by the pre-flash became re-oxidized in this time window so that a stable charge separation (forming longlived P680 •+ ) was possible upon the second flash. Re-oxidation of Q A •-may occur by charge recombination with the oxidizing equivalent on the donor side of PSII or by electron transfer to the secondary quinone Q B in a fraction of PSII in which this electron transfer was not completely blocked by DCMU.

  Photosynthesis. Springer, Dordrecht, pp 463-495 Suga M, Akita F, Hirata K, Ueno G, Murakami H, Nakajima Y, Shimizu T, Yamashita K, Yamamoto M, Ago H, Shen JR (2015) Native structure of photosystem II at 1.95 A resolution viewed by femtosecond X-ray pulses. Nature 517: 99-103 Tommos C, Babcock GT (2000) Proton and hydrogen currents in photosynthetic water oxidation. Biochim Biophys Acta-Bioenergetics 1458: 199-219 Umena Y, Kawakami K, Shen JR, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 angstrom. Nature 473: 55-U65 van Best JA, Mathis P (1978) Kinetics of reduction of the oxidized primary electron donor of photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation. Biochim Biophys Acta 503: 178-188 van Grondelle R, Dekker JP, Gillbro T, Sundstrom V (1994) Energy-Transfer and Trapping in Photosynthesis. Biochim Biophys Acta-Bioenergetics 1187: 1-65 van Mieghem F, Brettel K, Hillmann B, Kamlowski A, Rutherford AW, Schlodder E (1995) Charge recombination reactions in photosystem II. I. Yields, recombination pathways, and kinetics of the primary pair. Biochemistry 34: 4798-4813 Vredenberg W (2011) Kinetic analyses and mathematical modeling of primary photochemical and photoelectrochemical processes in plant photosystems. Biosystems 103(2): 138-151

Fig. S3 .

 S3 Fig. S3. Thermoluminescence curves of DCMU-treated PSII core complexes following their excitation at -60°C with 1 and 20 STSFsshowing that no additional TL band is produced upon multiple-flash excitation while the fluorescence yield increases.

Fig. 2 .

 2 Fig. 2. (A) Two kinetic traces of chlorophyll-a fluorescence yield changes induced by trains of double-STSFs and two MTSFs in PSII core complexes from T. vulcanus at room temperature in the presence of 40 µM DCMU. Traces in black and blue, 1 µs and 100 ms waiting time (Δt) between the first and second STSF, as indicated; the MTSFs were 100 ms long. The traces are normalized to F m -F o ; the F v /F m was 0.84 ± 0.02. (B) Dependence of the F 1 -to-F 2 increment on the delay (Δt) between the first and second STSFs. The dashed line represents a single-exponential fit of the data points, with a half-rise time (∆t 1/2 ) of 2.9 ± 0.4 ms. Data points, mean values ± SD (n = 4); the curve is normalized to the maximal variable fluorescence (F m -F o ).

Fig. 3 .

 3 Fig. 3. (A) Transient absorption kinetics in DCMU-treated, dark adapted PSII core complex from T.vulcanus at 819 nm using single flash excitation (black trace) and multiple flash excitations at 2 s -1 (red trace). The traces are averages of 4 and 128 signals, respectively. To reach the stationary state with multiple flash excitations, 10 pre-flashes were applied at the same repetition rate. The inset shows the kinetics up to 800 ns upon a single-flash excitation. The smooth lines represent bi-exponential fits of the curves (from 1 to 800 ns for the black curve and from 360 ps to 50 ns for the red one). (B) Traces in green, black, blue and red were recorded upon a second flash excitation, which followed a saturating (pre-)flash after Δt = 5 µs, 100 µs, 5 ms and 100 ms, respectively. The plotted signals are averages of 5 to 8 experiments on fresh samples from the same stock; the smooth lines represent global biexponential fits of the curves (fitting interval: 360 ps -50 ns, shared time constants); inset:

  •+ Pheo •-and this primary charge separation is stabilized by a re-oxidation of Pheo

  •-by Q A, forming

  •+ Pheo •-). Although in closed RC the generation and fast recombination of

  •+ Pheo •-have been thoroughly demonstrated(van Mieghem et al. 1995[START_REF] Nadtochenko | Formation and decay of P680 (P(D1)-P(D2))( + )PheoD1( -) radical ion pair in photosystem II core complexes[END_REF]

Table 1 .

 1 Amplitude (A 1 and A 2 ) and lifetime (τ 1 and τ 2 ) parameters of the transient absorption kinetics fitted with two exponentials and the amplitude of the residual long component (y 0 ) (see Methods).Numbers marked with a * are results of a global fit with shared time constants.

	observed flash	τ 1 (ns)	τ 2 (ns)	A 1 (mOD)	A 2 (mOD)	y 0 (mOD)	A 1 + A 2 + y 0
	1 st flash	16.6	167	8.8	5.5	4.2	(18.5)
	repetitive flashes	1.96	8.59	12.7	7.6	2.1	22.4
	2 nd flash, Δt 5 µs	2.05*	10.1*	9.5	3.8	1.2	14.5
	2 nd flash, Δt 100 µs	2.05*	10.1*	11.9	4.6	1.6	18.1
	2 nd flash, Δt 5 ms	2.05*	10.1*	13.1	4.5	2.3	19.9
	2 nd flash, Δt 100 ms	2.05*	10.1*	12.1	4.7	3.6	20.4

Acknowledgements

-This work was supported by a grants from the National Research Development and Innovation Office of Hungary (NKFIH OTKA KH 124985 and K 128679, and TÉT_15-1-2016-0144 (G.G.), NKFIH OTKA NN 124904 and GINOP-2.3.2-15-2016-00001 (P.H.L.) and OTKA PD 121225 (M.M.)), by a STSM Grant from the EBSA (G.S.) and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05 (P.M. and K.B.), and by a National Key R&D Program of China (2017YFA0503700), a Strategic Priority Research Program of Chinese This article is protected by copyright. All rights reserved. Accepted Article Academy of Sciences (XDB17000000) and a National Natural Science Foundation of China (31470339) (J.R.S., Q.Z., G.H. and Y.X.). The set-up used in the TL measurements (SI) was constructed by Drs. Jean-Marc Ducruet and Szilvia Zita Tóth with the financial support of the Lendület Program of the Hungarian Academy of Sciences (LP2014/19).


Karen Bellec

Isabelle Gicquel

Roland Le Borgne
email: roland.leborgne@univ-rennes1.fr

The Clathrin adaptor AP-1 and the Rab-stabilizing chaperone Stratum act in two parallel pathways to control the activation of the Notch pathway in Drosophila

Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells whose identity relies on the differential activation of Notch during cytokinesis. While Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, only the basal pool of Notch is reported to contribute to Notch activation in the pIIa cell. Such proper intralineage signalling therefore requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We previously reported that AP-1 and Stratum regulate the intracellular trafficking of Notch and Sanpodo from the trans-Golgi network to basolateral membrane. Loss of AP-1 or of Stratum caused mild Notch phenotype. Here, we report that the concomitant loss of AP-1 and Stratum result in the stabilization of the apical pool of Notch, Delta and Spdo, the loss of the basal pool of Notch at the pIIa-pIIb interface, and is associated with activation of Notch in the two SOP daughters. We propose that AP-1 and Stratum control two parallel pathways towards plasma membrane and that Notch intralineage signalling could also occur at the apical pIIa-pIIb interface.

Results and Discussion

Cell-cell signalling by the evolutionarily conserved Notch receptor promotes cell fate acquisition in a large variety of developmental processes in metazoans [START_REF] Artavanis-Tsakonas | Notch signaling: cell fate control and signal integration in development[END_REF][START_REF] Bray | Notch signalling in Drosophila: three ways to use a pathway[END_REF][START_REF] Kopan | The canonical Notch signaling pathway: unfolding the activation mechanism[END_REF]. In most of cases, Notch receptor is activated by transmembrane ligands present at the plasma membrane of adjacent cells.

Following binding to Notch, endocytosis of the ligand induces pulling forces driving a change in the conformation of the Notch extracellular domain thereby unmasking the S2 cleavage site of Notch [START_REF] Gordon | Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch[END_REF][START_REF] Langridge | Epsin-Dependent Ligand Endocytosis Activates Notch by Force[END_REF][START_REF] Meloty-Kapella | Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin[END_REF][START_REF] Seo | A Mechanogenetic Toolkit for Interrogating Cell Signaling in Space and Time[END_REF][START_REF] Shergill | Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis[END_REF][START_REF] Wang | Defining single molecular forces required to activate integrin and notch signaling[END_REF]. This regulated cleavage is followed by a constitutive proteolytic cleavage of Notch by the gamma secretase complex [START_REF] Mumm | A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1[END_REF][START_REF] Struhl | Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins[END_REF] giving rise to the Notch intracellular domain, a polypeptide that is translocated into the nucleus and that acts as a transcriptional coactivator [START_REF] Artavanis-Tsakonas | Notch signaling: cell fate control and signal integration in development[END_REF][START_REF] Bray | Notch signalling in Drosophila: three ways to use a pathway[END_REF][START_REF] Kopan | The canonical Notch signaling pathway: unfolding the activation mechanism[END_REF]. Because proteolytic activation of the Notch receptor is by definition irreversible, Notch activation needs to be tightly controlled in time and in space. The model system of asymmetric cell division of the sensory organ precursors (SOPs) in the pupal notum of Drosophila has been instrumental to identify the site of Notch activation at the cell surface. SOPs are polarized epithelial cells that divide asymmetrically within the plane of the epithelium to generate two daughter cells whose fate depends on the differential activation of Notch signalling [START_REF] Schweisguth | Asymmetric cell division in the Drosophila bristle lineage: from the polarization of sensory organ precursor cells to Notch-mediated binary fate decision[END_REF]. It relies on the unequal partitioning of the two cell fate determinants Neuralized (Neur) and Numb in the anterior SOP daughter cell [START_REF] Le Borgne | Unequal segregation of Neuralized biases Notch activation during asymmetric cell division[END_REF][START_REF] Rhyu | Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells [see comments[END_REF]. Neur promotes the endocytosis of Delta (Le Borgne and [START_REF] Le Borgne | Unequal segregation of Neuralized biases Notch activation during asymmetric cell division[END_REF] while Numb inhibits the recycling of Notch and its cofactor Sanpodo (Spdo) towards the plasma membrane to instead promote their targeting towards late endosomal compartments [START_REF] Cotton | Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor[END_REF][START_REF] Couturier | Numb localizes at endosomes and controls the endosomal sorting of notch after asymmetric division in Drosophila[END_REF][START_REF] Johnson | Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells[END_REF][START_REF] Upadhyay | Sanpodo controls sensory organ precursor fate by directing Notch trafficking and binding gamma-secretase[END_REF].

Consequently, the anterior cell adopts the pIIb identity while Notch is selectively activated in the posterior cell that adopts the pIIa fate. Combination of live-imaging and FRAP experiments using GFP-tagged Notch revealed that proteolytic activation of Notch occurs during SOP cytokinesis and that a specific pool of Notch receptors located basal to the midbody is the main contributor to the signalling in the pIIa cell [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF]. These data imply a polarized trafficking of Notch, Delta and Spdo towards this specific subcellular location during cytokinesis. We previously reported that the clathrin adaptor complex AP-1 regulates the polarized sorting of Notch and Spdo from the trans-Golgi network (TGN) and the recycling endosomes towards the plasma membrane [START_REF] Benhra | AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors[END_REF]. Loss of AP-1 causes stabilization of Notch and Spdo at the adherens junctions following SOP division, a phenotype associated with a mild Notch gain-of-function phenotype (GOF). More recently, we reported that Stratum (Strat), a chaperone regulating Rab8 recruitment, controls the exit from the Golgi apparatus and the basolateral targeting of Notch, Delta and Spdo [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF]. As for AP-1, loss of Strat causes a mild Notch GOF phenotype. These data can be interpreted as AP-1 and Strat acting in the same transport pathway and therefore being simple fine-tune regulators of Notch-Delta trafficking and activation. However, as AP-1 and Strat/Rab8 both regulate basolateral trafficking, an alternative explanation could be that AP-1 and Strat function in two parallel pathways (Fig. 1A-A'). In this scenario, loss of one of the two components could be compensated by the other. Accordingly, the concomitant loss of AP-1 and Strat would exhibit a stronger phenotype.

Simultaneous loss of AP-1 and Strat causes a penetrant Notch GOF phenotype

To test this prediction, we induced clones of cells homozygote mutant for a null mutation of Strat (Strat KO CRISPR, [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF] in which the µ subunit of the AP-1 complex (also known as AP-47) was silenced using the previously described dsRNA [START_REF] Benhra | AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors[END_REF]. Silencing of AP-47 prevents the assembly of functional AP-1 complexes and gives similar Notch GOF phenotypes as two independent AP-47 null mutant alleles (AP-47 SHE11 and AP-47 deletion by CRISPR ( [START_REF] Benhra | AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors[END_REF] and data not shown)). Silencing of AP-47 is hereafter referred to as loss of AP-1. In wild-type sensory organ (SO), the pIIb cell divides two times to generate the internal cells among which is one neuron, labelled using an anti-Elav. The pIIa cell divides once to generate the external cells among which is one socket cell, labelled with Suppressor of Hairless (Su(H)). In agreement with previous studies, Notch GOF phenotypes corresponding to an excess of socket cells were observed in 6,7% and 7,5% of SO mutant for strat or depleted of AP-1, respectively [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF][START_REF] Benhra | AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors[END_REF], Fig. 1C). However simultaneous loss of AP-1 and Strat led to 49% of transformed SOs (Fig. 1B-C). Among them, 34% of SOs were transformed at the SOP division, i.e. SOP divided to generate two pIIa cells (n= 87/254, Fig. 1C and data not shown).

These lineage data indicate that AP-1 and Strat function in two distinct and complementary pathways to regulate Notch activation.

Numb is correctly partitioned upon loss of AP-1 and Strat

Because AP-1 interacts with Numb and loss of Numb exhibits a Notch GOF, we wondered if the phenotype observed upon the loss of AP-1 and Strat would result from the loss of Numb function. We previously reported that the unequal partitioning of Numb is unaffected by the loss of AP-1 [START_REF] Benhra | AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors[END_REF] or by the loss of Strat [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF]. Because unequal segregation of Numb is under the control of polarity genes, we first monitored the overall cell polarity [START_REF] Gho | Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila[END_REF][START_REF] Kraut | Role of inscuteable in orienting asymmetric cell divisions in Drosophila[END_REF][START_REF] Schober | Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts[END_REF].

We noticed that, as in AP-1 mutant, the cuticle looks thinner and less pigmented than in the control situation (Fig. S1A-B'). These phenotypes could be caused by a defective AP-1 dependent transport of the Menkes Copper transporter ATP7a that regulates cuticle pigmentation [START_REF] Holloway | Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2-, AP-1-, and Rab22-dependent steps[END_REF][START_REF] Norgate | Essential roles in development and pigmentation for the Drosophila copper transporter DmATP7[END_REF] and by a defective apical secretion of cuticle components that may rely on AP-1 function as does the apical glue granule secretion in salivary glands [START_REF] Burgess | AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila[END_REF]. Despite these cuticle defects, the localization of the junctional markers DE-Cadherin and Coracle, as well as that of the polarity determinant Par3, were unaffected by the loss of AP-1 and Strat indicating that the overall cell polarity is unaffected (Fig. S1C-D). We next monitored the localization of Numb during the division of strat mutant SOPs depleted of AP-1. Anti-Numb staining showed that Numb localizes asymmetrically during prometaphase in control and in absence of Strat and AP-1 (Fig. 2A-B) and live-imaging using a Numb::GFP crispr [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF] revealed that Numb is unequally partitioned in the anterior SOP daughter cell, as seen in the control situation (Fig. 2C-D). Thus, the Notch GOF phenotype cannot be explained by a defective Numb unequal partitioning during SOP division and this rather suggests that it could be due to a defect in the polarized trafficking of Notch signalling actors.

Notch is enriched at the apical pIIa-pIIb interface upon loss of AP-1 and Strat

Elegant work from F. Schweisguth laboratory identified two pools of Notch at the pIIa-pIIb interface, apical and basal to the midbody, and provides the compelling evidence that only the subset of receptors located basal to the midbody contributes to signalling in the pIIa cell.

To investigate whether AP-1 and Strat affect the apico-basal distribution of Notch receptors, we monitored the dynamics of Notch::GFP crispr [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF] throughout the asymmetric division of the SOP. First of all, in the control situation, we confirmed the presence of two pools of Notch along the apical-basal pIIa-pIIb interface, in agreement with previous studies ( [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF], Fig. 3A). Notch is transiently detected at the apical pIIa-pIIb interface at ~6-9 min after the anaphase onset with a signal intensity peaking at 15-20 min prior to progressively disappear at ~30 min (Fig. 3A-B and Movie S1). Basal to the midbody, and in agreement with previous studies [START_REF] Couturier | Endocytosis by Numb breaks Notch symmetry at cytokinesis[END_REF][START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF], we also detected punctate structures positive for Notch, hereafter called lateral clusters, that appear ~6-9 min following the anaphase onset and that persist longer than the apical pool of Notch (up to 45 min after the anaphase onset; Fig. 3A). In striking contrast to the control situation, upon loss of Strat and AP-1, the Notch lateral clusters were not detected (Fig. 3C). Instead, we found that Notch appears in higher amounts at the apical pIIa-pIIb interface cells ~6-9 minutes following the anaphase onset and persists there for longer periods of time compared to the control (at least 78 min post-anaphase versus ~30 min in the control; Fig. 3B-C and Movie S2). In addition to the apical accumulation, Notch is also detected in dotted structures restricted to the apical plane of the pIIa-pIIb daughter cells (white arrows, t= 39 min and t=78 min, Fig. 3C and Movie S2). Since lateral clusters from which Notch is activated are absent upon loss of Strat and AP-1, our results raise the possibility that the Notch GOF observed could be due to the enrichment of Notch at the apical pIIa-pIIb interface.

To investigate this possibility, we first tried to directly test the ability of the apical pool of Notch to be cleaved and to be translocated into the nucleus. To this aim, we inserted the green-to-red photoconvertible Dendra2 in the cytoplasmic domain of Notch (Notch::Dendra crispr ) with the purpose of photoconverting the apical pool of Notch::Dendra crispr and testing its ability to translocate into the nucleus following proteolytic activation. While Dendra2 is detected at the apical plasma membrane upon loss of Strat and AP-1 using an anti-Dendra on fixed specimen (Fig. S2A), Notch::Dendra crispr signal is not detectable at the plasma membrane in live specimens (Fig. S2B). In time-lapse imaging, Notch::Dendra crispr signal is restricted to intracellular dotted structures, likely endosomal compartments (Fig. S2B). This apparent discrepancy between fixed and living specimen could be due to a longer time of folding or maturation of the newly synthesized Dendra2 compared to the GFP probe, as already reported for other fluorescent probes [START_REF] Couturier | A fluorescent tagging approach in Drosophila reveals late endosomal trafficking of Notch and Sanpodo[END_REF]. This result is compatible with the idea that newly synthesized, e.g. not yet folded or matured, Notch::Dendra crispr receptors are targeted to the apical SOP daughter cells interface and are processed from this location upon loss of Strat and AP-1. Because the lack of detection of Notch::Dendra crispr at the plasma membrane prevented us to directly test our hypothesis of apical activation of Notch, we next investigated the localization of Spdo and Delta in SO mutant for Strat and depleted of AP-1.

Spdo and Delta are distributed with Notch at the apical pIIa-pIIb interface upon loss of

Strat and AP-1

The localization of Spdo and Delta was analysed on fixed specimens. As previously described, in the control situation, Spdo is faintly detected at the apical pole of SOP daughter cells, and localizes predominantly in endosomes in the pIIb cell, while in the pIIa cell, Spdo distributes not only in endosomes but also at the basolateral plasma membrane (Fig. 4A; [START_REF] Cotton | Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor[END_REF][START_REF] Couturier | Numb localizes at endosomes and controls the endosomal sorting of notch after asymmetric division in Drosophila[END_REF][START_REF] Hutterer | Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs[END_REF][START_REF] Langevin | Lethal giant larvae controls the localization of notch-signaling regulators numb, neuralized, and Sanpodo in Drosophila sensory-organ precursor cells[END_REF]. Upon loss of Strat and AP-1, while Spdo is still detected in dotted intracellular structures in SOP daughter cells, it is no longer detected at the basolateral pIIa-pIIb interface (Fig. 4B). Instead, Spdo is enriched at the apical plasma membrane, as observed in single AP-47 SHE11 [START_REF] Benhra | AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors[END_REF] or strat mutant background [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF]; Fig. 4B andC) as well as at the apical pIIa-pIIb interface, as observed in most of mutant cases (n=12/14, white arrows, Fig. 4B). Similarly to Spdo, and as described previously, Delta is detected into intracellular endocytic structures in pIIa and pIIb cells, at the pIIa-pIIb lateral interface in the control situation as well as the lateral plasma membrane of the pIIa cell. Delta is also weakly detected at the apical pIIa-pIIb interface (Fig. 4D). The loss of Strat and AP-1 leads to an increase in the localization of Delta at the apical pIIa-pIIb interface, together with Notch, in 60% of SOs against 44% in the control situation (Fig. 4E and data not shown). Together, our data confirm that, in the control situation, Notch, Delta and Spdo are present at the lateral pIIa-pIIb interface from which Notch activation was reported to take place [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF]. Upon loss of Strat and AP-1, sustained levels of Notch/Delta and Spdo at the apical pIIa-pIIb interface are observed, localization at the basolateral interface no longer detected, a phenotype associated with a Notch GOF.

Concluding remarks

Previous work showed that Strat controls the transport of Notch, Delta and Spdo from the TGN to the basolateral pole [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF] and AP-1, from the TGN to the basolateral membrane via recycling endosomes [START_REF] Benhra | AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors[END_REF][START_REF] Cotton | Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor[END_REF]. Here, we report that AP-1 and Strat act redundantly to control the polarized trafficking of Notch, Delta and Spdo in SOP daughter cells (Fig. 4F). Either of these two pathways can be used to ensure the proper localization of Notch, Delta and Spdo, explaining the compensation of one by the other. Upon concomitant loss of Strat and AP-1, Notch, Delta and Spdo are no longer detected in lateral clusters and are enriched at the apical pIIa-pIIb interface, a localization associated with a Notch GOF. How can Notch be activated in the absence of lateral clusters, the main contributors of intra-lineage Notch activation [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF])? Although we cannot exclude the possibility that a few, hard to detect lateral clusters could contribute to Notch activation upon loss of AP-1 and Strat, we favour the hypothesis according which Notch activation takes place at the apical pIIa-pIIb interface. The lack of AP-1 prevents Numb to repress the Notch-Spdo recycling normally driving endosomal degradation of Notch-Spdo [START_REF] Cotton | Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor[END_REF][START_REF] Couturier | Numb localizes at endosomes and controls the endosomal sorting of notch after asymmetric division in Drosophila[END_REF][START_REF] Johnson | Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells[END_REF]. Thus, despite its unequal partitioning, Numb is not able to repress Notch activation upon loss of AP-1 and Strat. Nonetheless, although the loss of both of them gives rise to a Notch GOF, it does not phenocopy the loss of Numb, where Notch is accumulated in lateral clusters at the pIIa-pIIb interface.

Proteolytic activation of Notch requires the binding of the ligand and its subsequent Neurmediated endocytosis. While Neur is unequally localized during SOP division (Le Borgne and [START_REF] Le Borgne | Unequal segregation of Neuralized biases Notch activation during asymmetric cell division[END_REF], Neur::GFP is detected in both SOP daughter cells, at the apical pIIa-pIIb interface following SOP division [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF]. As Delta localizes at the apical pIIa-pIIb interface upon loss of Strat and AP-1, we postulate that Neur can promote endocytosis of Delta in both daughter cells from the apical interface, thereby promoting bidirectional Notch proteolytic activation. If Notch-Delta signalling is primarily taking place at the lateral interface [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF], our study indicates that signalling can also occur apically. The fact that Sec15, a subunit of the exocyst complex, and Arp2/3-WASp (Actinrelated protein 2/3-Wiskott-Aldrich syndrome protein) are involved in apical trafficking of Spdo and Delta, respectively, and are required for Notch-Delta signalling is consistent with our proposal of apical activation of Notch [START_REF] Jafar-Nejad | Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors[END_REF][START_REF] Rajan | The Arp2/3 complex and WASp are required for apical trafficking of Delta into microvilli during cell fate specification of sensory organ precursors[END_REF].

However, the weak contribution of the apical pool of receptors to the Notch activation described in the control situation [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF] suggests that apical activation can only occur when a specific threshold of Notch, Delta and Spdo is reached, such as in strat SOs depleted of AP-1. In line with previous reports [START_REF] Trylinski | Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells[END_REF], our study further illustrates how the apico-basal targeting of Notch, Spdo and Delta along the pIIa-pIIb interface limits signalling between SOP daughter cells at the exit of mitosis. The question of whether this mechanism is conserved awaits further investigation, but given the key role played by Notch in the acquisition of cell fate in vertebrates, it is conceivable that the regulation described in this study may be general.

Materials and Methods

Drosophila stocks and genetics

Drosophila melanogaster stocks were maintained and crossed at 25°C. Mitotic clones were induced using the FLP-FRT technique using the hs-FLP and by heat shocking (2×60 min at 37°C) at second and early third instar larvae. pnr-GAL4 was used to drive the expression of the AP-47 dsRNA . The following stocks were used in this study: y w hs-FLP; Ubi-GFP nls, FRT40A; pnr-GAL4/TM6 Tb (Fig. 1B,C;2B,D; Resource Center, w 1118 ; P(GD14206)v24017/TM3, Fig. 1B,C;2B;3B,C;4B,C,E; S2 and Movie S2); y, hs-FLP; Ubi-GFP nls, FRT40A (Fig. 1C), w; strat, FRT40A/CyO (Fig. 1C); Cad::GFP/+; pnr-GAL4/+ (Fig. 2A); +/CyO::CFP; pnr-GAL4/+ (Fig. 2A); y, hs-FLP; Ubi-RFP nls, FRT40A (Fig. 2C), w; Numb::GFP crispr , FRT40A/CyO (Fig. 2C), w hs-FLP neur-H2B::RFP;; Sb/TM6 Tb (Fig. 2D), w; strat, Numb::GFP crispr , FRT40A/CyO; AP-47 dsRNA /TM6 Tb (Fig. 2D), Notch::GFP crispr , neur-H2B::RFP; Dr/TM6 Tb (Fig. 3A-C; Movies S1 and S2), +/+ (Fig. S1A-A').

Immunofluorescence and antibodies

Pupae were aged for 16.5 to 18.5 h after puparium formation (APF) for SOPs and SOPs daughter cells analysis and were aged for 24 h to 28 h APF for lineage analysis. Pupae were dissected in 1x phosphate-buffered saline (1x PBS) and then fixed for 15 min in 4% paraformaldehyde at room temperature. Dissection and staining conditions were essentially as previously described [START_REF] Le Borgne | Unequal segregation of Neuralized biases Notch activation during asymmetric cell division[END_REF]. Primary antibodies used were rat anti-Elav (7E10, Developmental Studies Hybridoma Bank (DSHB), 1:200), goat anti-Su(H) (sc15813, Santa Cruz, 1:500), mouse anti-Cut (2B10, DSHB, 1:500), goat anti-Numb (SC23579, Santa Cruz, 1:200), rabbit anti-Spdo (a kind gift from J. Skeath, 1:2000) (O' Connor-Giles and Skeath, 2003), mouse anti-DECD (C594.9B, DSHB, 1:200), mouse anti-Cora (C615.16, DSHB, 1:500), rat anti-DE-Cad (DCAD2, DSHB, 1:500), rabbit anti-Par3 (a kind gift from A. Wodarz, 1:1000) [START_REF] Shahab | Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells[END_REF], mouse anti-HRS (27-4, DSHB, 1:100), rabbit anti-Syntaxin16 (ab32340, Abcam, 1:1000) and rabbit anti-Dendra (Antibodies-online.com, ABIN361314, 1:1000). Cy2-, Cy3-and Cy5-coupled secondary antibodies (1:400) were from Jackson's Laboratories.

Generation of Notch::Dendra crispr

The Notch::Dendra crispr construct was generated using the CRISPR/Cas9 method as previously described (Gratz et al., 2013a;[START_REF] Gratz | CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand[END_REF]. As for the Notch::GFP crispr construct, the following gRNAs were used: 5′-AACTTGAA|TGGATTGAACCC GGG-3′ and 5′-CGAACTGG|AGGGTTCTCCTGTTG-3′ to introduce Cas9 cuts in exon 6 [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF]. The Dendra and the 3xP3-DsRed cassette flanked by GVG linkers and by loxP, respectively, were introduced at the previously described position (NiYFP4) [START_REF] Couturier | Endocytosis by Numb breaks Notch symmetry at cytokinesis[END_REF]. Homology arms 1 and 2 were of 1064 bp and 1263 bp in length, respectively.

Injection was performed by Bestgene in the yw; attP40(nos-cas9)/Cyo stock. The correct position of the Dendra and the DsRed cassette was verified by PCR and sequencing. The DsRed was then removed by crossings with if/Cyo, Cre, w stock.

Imaging

Images of fixed nota were acquired with a Leica SPE confocal microscope and a Zeiss Airyscan microscope. The live imaging of Numb::GFP crispr and Notch::Dendra crispr was acquired with a Leica SPE confocal microscope. The live imaging of Notch::GFP crispr in wildtype SOP and in strat SOP expressing pnr-GAL4>AP-47 dsRNA was acquired with a Zeiss Airyscan microscope. All images were processed and assembled using ImageJ 1.48 and Adobe Illustrator.

Quantification of the enrichment of Notch at the apical interface

To quantify the signal of Notch::GFP at the apical interface, we measured the signal in a manually drawn area on sum slices of the two apical planes where the Notch signal at the apical interface is the strongest. We also measured the signal between two epithelial cells within an equivalent drawn area and on the same sum slices. Then, we calculated the following ratio: Average fluorescence intensity at the apical interface between SOP daughter cells/Average fluorescence intensity at the apical interface between epithelial cells. This ratio was calculated for each time point. In absence of AP-1 and Strat, the manually drawn area was minimized to avoid taking into account the apical punctate structures positive for Notch.

Quantification of the apical enrichment of Spdo

The fluorescence intensity was calculated with ImageJ, on two-cell stages, as previously described [START_REF] Bellec | Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo[END_REF]. Briefly, the average fluorescence intensity was measured in a manually drawn area on sum slices of the two most apical planes, where Spdo is enriched.

The background noise was measured in the same way and subtracted from the apical intensity value.

Statistical analysis

Statistical analyses were carried out using the GraphPad Prism 6.05 software. A two-way ANOVA was performed for the quantification of the enrichment of Notch at the apical pIIa-pIIb interface with a multiple comparison Bonferroni test. For the quantification of the apical enrichment of Spdo, because data do not follow a normal distribution, we performed a Wilcoxon test. Statistical significances are represented as follows: not significant (ns)≥0.05; *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001.

  w; strat, FRT40A/CyO; AP-47 dsRNA /TM6 Tb (stock #24017 from Vienna Drosophila

  w 1118 ; P(ry[+t7.2]=neoFRT}40A P(w[+mW.hs]=FRT(w[hs]))G13/CyO (stock #8217 from Bloomington, Fig.3A,B; 4D; and Movie S1), W1118 (Fig.4A,C), Notch::Dendra crispr , neur-H2B::RFP; Dr/TM6 Tb (Fig. 4D-E; S1B-B'; and S2) and Notch::Dendra crispr , neur-H2B::RFP;

Figure 1 :

 1 Figure 1: Loss of Strat and AP-1 causes Notch gain-of-function phenotype within SO lineage

Figure 2 :

 2 Figure 2: Loss of Strat and AP-1 does not affect the localization of Numb

Figure 3 :

 3 Figure 3: Notch is enriched at the apical pIIa-pIIb interface in the absence of Strat and AP-1

Figure 4 :

 4 Figure 4: Spdo and Delta are enriched at the apical pIIa-pIIb interface in absence of Strat and AP-1

Acknowledgements

We thank the Bloomington Stock Center, the Vienna Drosophila RNAi Center and the National Institute of Genetics Fly Stock Center for providing fly stocks. We also thank S. Dutertre and X. Pinson from the Microscopy Rennes Imaging Center-BIOSIT (France). The monoclonal antibodies against Elav, Cut, Cora and DE-Cad were obtained from the Developmental Studies Hybridoma Bank, generated under the auspices of the National Institute of Child Health and Human Development, and maintained by the University of Iowa Department of Biological Sciences. We thank members of R.L.B.'s laboratory for helpful discussions and critical reading of the manuscript.

Funding

This work was supported in part by the ARED programme from the Région Bretagne/Agence Nationale de la Recherche (ANR-16-CE13-004-01), the Fondation pour la Recherche Médicale (FDT20170436864 to K.B.), La Ligue contre le Cancer-Equipe Labellisée (R.L.B.) and the Association Nationale de la Recherche et de la Technologie programme PRC Vie, santé et bien-être CytoSIGN (ANR-16-CE13-004-01 to R.L.B.).

Competing interests

The authors declare no competing or financial interests.

Author contributions

Conceptualization