Aurélie Riddhi Atul Jani

Di Cicco

Tal Keren-Kaplan

Silvia Vale-Costa

Daniel Hamaoui

Ilse Hurbain

Feng-Ching Tsai

Mathilde Di Marco

Anne-Sophie Macé

Yueyao Zhu

Riddhi Atul

Aurélie Di Cicco

Maria João Amorim

Patricia Bassereau

Juan S Bonifacino

Agathe Subtil

Michael S Marks

Daniel Lévy
email: daniel.levy@curie.fr

Graça Raposo

Cédric Delevoye
email: cedric.delevoye@curie.fr

PI4P and BLOC-1 remodel endosomal membranes into tubules

Keywords: phosphoinositide, protein complex, membrane remodeling, recycling endosomes

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The membrane-bound organelles of eukaryotic cells perform specific functions that are collectively needed for physiology. As a common feature, their limiting membrane isolates their lumen from the cytosol, while being actively remodeled to facilitate the exchange of components needed for the identity and the function of organelles [START_REF] Jarsch | Membrane curvature in cell biology: An integration of molecular mechanisms[END_REF]. Such remodeling of the organellar membranes leads to the biogenesis of tubulo-vesicular carriers through the bending, invagination or evagination, and scission at precise membrane subdomains [START_REF] Mcmahon | Membrane curvature and mechanisms of dynamic cell membrane remodelling[END_REF].

Relative to vesicles, tubular transport intermediates display a high surface-to-volume ratio that is best suited for transporting lipids and membrane-associated proteins [START_REF] Maxfield | Endocytic recycling[END_REF]. Tubular intermediates consist of elongated curved membrane structures that extend from largely planar membrane bilayers of many donor organelles [e.g. plasma membrane (PM), trans-Golgi Network (TGN), early endosomes, lysosomes and lysosomerelated organelles (LROs)]. Their biogenesis and maintenance require membrane asymmetry imposed by extrinsic and/or intrinsic factors [START_REF] Stachowiak | A cost-benefit analysis of the physical mechanisms of membrane curvature[END_REF]. Extrinsic factors include proteins that sense, impose, and/or stabilize specific membrane curvatures [e.g., BAR (Binamphiphysin-Rvs)-domain containing proteins] and cytoskeletal elements that exert mechanical force onto membranes. Intrinsic factors include the accumulation of membrane components with a characteristic shape (e.g., conical lipids or proteins) that can form membrane domains with a particular geometry [START_REF] Jarsch | Membrane curvature in cell biology: An integration of molecular mechanisms[END_REF].

Among membrane lipid species, phosphatidylinositols (PtdIns or PI) are minor components of total cellular phospholipids but play crucial roles in membrane trafficking. Up to 7 different PI-Phosphate (PIXP) species can be generated, through reversible phosphorylation of their myoinositol ring at positions -3, -4 and/ or -5. PIXPs contribute to the identity and function of organelles by dynamically concentrating at membrane subdomains, where they aid in the recruitment and/or stabilization of specific proteins that modulate the membrane biophysical properties [START_REF] Balla | Phosphoinositides: Tiny lipids with giant impact on cell regulation[END_REF]. For instance, PI(4,5)P2 at the PM cooperates with clathrin adaptor proteins to form carriers during endocytosis [START_REF] Chang-Ileto | Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulated by membrane curvature and facilitates membrane fission[END_REF]. PI3P on early endosomes favors membrane binding of trafficking effectors [START_REF] Gaullier | Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes. Role of conserved residues[END_REF][START_REF] Xu | SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P[END_REF] that support the formation of tubulo-vesicular endosomal carriers for cargo recycling [START_REF] Wallroth | Phosphoinositide conversion in endocytosis and the endolysosomal system[END_REF][START_REF] Ketel | A phosphoinositide conversion mechanism for exit from endosomes[END_REF][START_REF] Mcnally | Endosomal Retrieval of Cargo: Retromer Is Not Alone[END_REF]. And, PI4P (a.k.a., PtdIns4P) cooperates with membrane-shaping proteins and actin-based motors to effect the formation of TGN-derived carriers [START_REF] Rahajeng | Efficient Golgi Forward Trafficking Requires GOLPH3-Driven, PI4P-Dependent Membrane Curvature[END_REF]. Thus, the local concentration of PIXP defines a membrane trafficking hotspot for membrane remodeling events during the biogenesis of transport carriers [START_REF] Di Paolo | Phosphoinositides in cell regulation and membrane dynamics[END_REF][START_REF] Saarikangas | Regulation of the actin cytoskeletonplasma membrane interplay by phosphoinositides[END_REF][START_REF] Lemmon | Signal-dependent membrane targeting by pleckstrin homology (PH) domains[END_REF][START_REF] Kutateladze | Translation of the phosphoinositide code by PI effectors[END_REF].

Recycling endosomes (RE) are organelles that comprise a network of membrane tubules arising from the vacuolar domain of early sorting endosomes (SE) [START_REF] Willingham | Morphologic characterization of the pathway of transferrin endocytosis and recycling in human KB cells[END_REF][START_REF] Yamashiro | Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway[END_REF] and facilitating the sorting and trafficking of multiple cargoes to the PM and TGN, or to LROs in specialized cells [START_REF] Delevoye | Lysosome-related organelles as functional adaptations of the endolysosomal system[END_REF][START_REF] Klumperman | The complex ultrastructure of the endolysosomal system[END_REF]. Cargo transport through RE regulate many physiological processes at the cellular level such as nutrient uptake, cell migration, polarity, division or signaling, and autophagy, and at the tissue level such as neuronal plasticity and skin pigmentation [START_REF] Grant | Pathways and mechanisms of endocytic recycling[END_REF][START_REF] Puri | Diverse autophagosome membrane sources coalesce in recycling endosomes[END_REF][START_REF] Delevoye | Lysosome-related organelles as functional adaptations of the endolysosomal system[END_REF]. As such, RE malfunction is associated with diseases such as neurological disorders (e.g., Huntington and Alzheimer diseases) [START_REF] Li | A function of huntingtin in guanine nucleotide exchange on Rab11[END_REF][START_REF] Zhang | Presenilin/gamma-secretase activity regulates protein clearance from the endocytic recycling compartment[END_REF] or genetic forms of oculocutaneous albinism [e.g. Hermansky-Pudlak Syndrome (HPS)] [START_REF] Bowman | The road to lysosome-related organelles: Insights from Hermansky-Pudlak syndrome and other rare diseases[END_REF]. In addition, RE are hijacked during certain viral and bacterial infections [START_REF] Allgood | The recycling endosome and bacterial pathogens[END_REF]Vale-Costa and Amorim, 2016). However, it remains to be determined how specific lipids and proteins coordinate to remodel endosomal membranes to generate and stabilize RE tubules.

While PI4P is enriched on Golgi membranes [START_REF] Graham | Coordination of Golgi functions by phosphatidylinositol 4-kinases[END_REF], a cohort localizes also to early endosomes [START_REF] Hammond | Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2)[END_REF] and endosome-derived vesicles [START_REF] Ketel | A phosphoinositide conversion mechanism for exit from endosomes[END_REF] or tubules [START_REF] Jović | Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling[END_REF]. Cargo trafficking through the early endosomal system requires the spatiotemporal control of PI4P metabolism by two endosome-associated kinases (PI4KIIa and -b; hereafter referred as PI4KII) [START_REF] Craige | Phosphatidylinositol-4-kinase type II alpha contains an AP-3-sorting motif and a kinase domain that are both required for endosome traffic[END_REF][START_REF] Wieffer | PI4K2β/AP-1-based TGN-endosomal sorting regulates Wnt signaling[END_REF][START_REF] Hammond | A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi[END_REF] and at least one phosphatase (Sac2) [START_REF] Nakatsu | Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway[END_REF][START_REF] Hsu | Spatiotemporal control of phosphatidylinositol 4phosphate by Sac2 regulates endocytic recycling[END_REF] to respectively synthesize and deplete PI4P on endosomal membranes. Loss of function or expression of either PI4KIIa or Sac2 results in missorting of the transferrin receptor (TfR) and the epidermal growth factor receptor (EGFR) [START_REF] Minogue | Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor[END_REF][START_REF] Henmi | PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes[END_REF][START_REF] Nakatsu | Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway[END_REF][START_REF] Hsu | Spatiotemporal control of phosphatidylinositol 4phosphate by Sac2 regulates endocytic recycling[END_REF], and PI4P contributes to stabilize some endosomal tubules [START_REF] Jović | Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling[END_REF]. Moreover, loss of function of the PI4KII orthologue in Drosophila melanogaster larvae impairs RE function in the maturation of salivary gland secretory granules [START_REF] Ma | An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation[END_REF]. However, how PI4P production and consumption regulate RE tubule biology is not understood.

The eight-subunit biogenesis of lysosome-related organelles complex 1 (BLOC-1) is required for the elongation and release of nascent RE tubular carriers from SE in coordination with the microtubule-based kinesin-3 motor KIF13A and actin-related machineries [START_REF] Ripoll | Illuminating the dark side of recycling endosomes[END_REF][START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF][START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF][START_REF] Shakya | Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes[END_REF][START_REF] Thankachan | KIF13A-A Key Regulator of Recycling Endosome Dynamics[END_REF]. Certain BLOC-1-dependent RE carriers deliver a subset of cargoes to LROs in specialized cell types, such as melanosomes in pigment cells, and thus loss of BLOC-1 function underlies several variants of HPS [START_REF] Bowman | The road to lysosome-related organelles: Insights from Hermansky-Pudlak syndrome and other rare diseases[END_REF]. The BLOC-1-dependent trafficking pathway in melanocytes requires KIF13A [START_REF] Delevoye | AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis[END_REF] or PI4KII [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF]. In addition, PI4KIIa physically associates with BLOC-1 in other cell systems [START_REF] Salazar | Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells[END_REF][START_REF] Larimore | The schizophrenia susceptibility factor dysbindin and its associated complex sort cargoes from cell bodies to the synapse[END_REF]. Thus, BLOC-1 may be integrated with PI4P functions at endosomal membranes.

Here, we used a combination of biochemical approaches, light and electron microscopy (EM) on live and fixed cells, and in vitro cryo-EM imaging of model membranes to test whether BLOC-1 and PI4P orchestrate the biogenesis of RE tubules. We show in vitro that BLOC-1 binds to negatively charged membranes and tubulate those containing endosome-associated PI3P and PI4P. In cells, endosomal PI4P contributes to remodel and stabilize membranes into RE tubules. The PI4KII are required for recycling of endosomal cargoes, and exploited by influenza A virus, or, together with BLOC-1, by the bacterium Chlamydia during their intracellular cycle. Together with the contribution of PI4KII to the formation of BLOC-1dependent endosomal tubular carriers in pigment cells [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF], our studies define the PI4P/ BLOC-1 module as a minimal machinery to generate and stabilize functional RE tubules that can be hijacked by certain infectious agents.

7

RESULTS

PI4P associates with recycling endosomal tubules.

To detect RE tubules by fluorescence microscopy (FM), we performed live imaging of HeLa cells transiently transfected with plasmids encoding the kinesin-3 proteins KIF13A or KIF13B fused to fluorescent proteins [i.e., KIF13A-YFP or mCherry-KIF13B [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF][START_REF] Yamada | KIF13B regulates angiogenesis through Golgi to plasma membrane trafficking of VEGFR2[END_REF][START_REF] Delevoye | Rab GTPases and kinesin motors in endosomal trafficking[END_REF]]. KIF13A is an effector of the RAB11 subfamily of small GTPases [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF], and KIF13A-YFP expression in HeLa cells generated mCherry-RAB11A-labeled RE tubules extending towards the cell periphery (Fig. 1 A, top panels, arrowheads). Its close homologue, KIF13B, was shown to localize to RAB5-positive ( + ) early endosomes in mouse embryonic fibroblasts [START_REF] Kanai | KIF13B enhances the endocytosis of LRP1 by recruiting LRP1 to caveolae[END_REF] or to RAB6A + secretory vesicles in HeLa cells [START_REF] Serra-Marques | Concerted action of kinesins KIF5B and KIF13B promotes efficient secretory vesicle transport to microtubule plus ends[END_REF]. We found that expression of mCherry-KIF13B (pseudocolored in green) in HeLa cells also generated numerous long GFP-RAB11A + RE tubules (pseudocolored in red) (Fig. 1 for the PI4P sensor SidC-GFP [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF]. Together, these data indicated that KIF13 expression drives the generation of RE tubules with which PI4P is specifically and abundantly associated.

BLOC-1, in vitro, generate tubules from membranes

Previously, we showed that the biogenesis of KIF13A + RE tubules from the limiting membrane of early endosomes relied on the BLOC-1-dependent elongation of short nascent tubules [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF]. Given the distribution of KIF13 to PI4P + RE tubules in HeLa cells (Fig. 1, C and D), the contribution of BLOC-1 in PI4P-dependent RE tubules in pigment cells [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF], and the biochemical association of BLOC-1 with the PI4P-producing kinase PI4KIIa [START_REF] Salazar | Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells[END_REF][START_REF] Ryder | The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4kinase type IIα[END_REF], we hypothesized that PI4P and BLOC-1 cooperate to remodel the endosomal membrane during the formation of RE tubules. We first analyzed whether BLOC-1 interacted with membranes using a minimal system composed of purified BLOC-1 and lipid tubules and/or vesicles. We expressed and purified the eightsubunits of BLOC-1 tagged with GST and 6xHis using a single polycistronic plasmid in E. coli [Fig. S2 A; and see [START_REF] Lee | Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)[END_REF]]. By negative staining EM, BLOC-1 appeared as curvilinear rods of ~30 nm in length, and of variable curvatures (Fig. 2 A) as previously described [START_REF] Lee | Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)[END_REF].

We then analyzed the interaction of purified BLOC-1 with membranes containing different lipids. Lipid strip assays showed that BLOC-1-GST interacted with several mono-and di-phosphorylated PIXPs (Fig. S2 B). Binding of BLOC-1 to vesicles was then tested by lipid floatation assays using swelled liposomes doped with negatively charged [PI3P, PI4P, PI(4,5)P2, phosphatidylserine (PS)] or neutral [phosphatidylcholine (PC)] phospholipids. By SDS-PAGE analysis, BLOC-1 co-fractionated with vesicles containing negatively charged phospholipids (Fig. S2 C).

We then performed a more detailed analysis of the binding of BLOC-1 to membrane systems of different diameters and compositions. We first exploited lipid nanotubes with a narrow and constant diameter of 27-nm for lengths of several hundreds of nm. Consistently with lipid floatation assay (Fig. S2 C), BLOC-1 was uniquely bound to GalCer/ Egg PC (EPC) nanotubes doped with negatively charged phospholipids compared with control (GalCer/ EPC) nanotubes (Fig. S2 D). Subsequently, and given the role of BLOC-1 in endosomal tubulation [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF], we selected lipid nanotubes doped with the endosomal associated PI3P or PI4P for high-resolution cryo-EM imaging. Cryo-EM images of control GalCer/ EPC lipid nanotubes incubated with or without BLOC-1 (Fig. S2 E, bottom or top panels, respectively) were similar and showed the two lipid leaflets of the tubes, but no BLOC-1 bound to the tubes (black arrowheads). By contrast, cryo-EM images of GalCer/ EPC nanotubes doped with 5 % of either PI3P or PI4P and incubated with BLOC-1 showed densities of black dots (white arrowheads) decorating the surface of both PI3P + (Fig. S2 F We next tested if BLOC-1 tubulates membranes by exploiting giant unilamellar vesicles (GUVs), which consist of a larger reservoir of relatively flat and highly deformable membranes compared to swelled vesicles. We used fluorescent GUVs containing 5 % PI4P or PI3P (Fig. analyzed by cryo-EM the tubules derived from BLOC-1-treated PI4P + GUVs (Fig. 2, E-G

2, D-G and

PI4KII are required for the formation and stabilization of recycling tubules

The formation of extended KIF13A + RE tubules [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF] relies on BLOC-1 expression in HeLa cells [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF]. These RE tubules were specifically decorated with PI4P fluorescent sensors (Fig. 1 C), as in pigment cells where functional PI4KII kinase domains are required for BLOC-1-dependent endosomal trafficking [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF].

Finally, our in vitro data showed that BLOC-1 concentrated on PI4P + and curved membranes, and shaped them into tubules (Fig. 2 andS2). We therefore investigated in cells if PI4P and its metabolism were needed for the biogenesis of RE tubules. Given that the production of PI4P on endosomal membranes relies on the PI4KIIa and PI4KIIb (PI4KII) [START_REF] Hammond | A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi[END_REF][START_REF] Minogue | Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor[END_REF][START_REF] Balla | Phosphatidylinositol 4-kinases: old enzymes with emerging functions[END_REF], we examined the contribution of PI4KII to the formation of RE tubules by siRNA-mediated knock-down. HeLa cells treated with a combination of two individual siRNAs against PI4KIIa and PI4KIIb (siPI4KII; see materials and methods) showed ~90 % reduction in both PI4KIIa and PI4KIIb compared to control cells treated with non-targeting control siRNA (siCTRL) (Fig. 3, A andB; siPI4KIIa : 10.9 ± 0.4 %, siPI4KIIb : 6.5 ± 3.5 %). By live-cell fluorescence imaging of siCTRL-treated cells transiently expressing KIF13A-YFP with or without mCherry-RAB11A, KIF13A and RAB11A largely decorated RE tubules extending towards the cell periphery [Fig. 3 C and Fig. S3 A, left panels, arrowheads; and [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF]]. In contrast, RE tubules were largely absent in siPI4KII-treated cells, in which KIF13A-YFP and mCherry-RAB11A were instead localized to enlarged vesicular structures (Fig. 3 C and Fig. S3 A, right panels, arrows). Relative to control cells, the percentage of PI4KII-depleted cells that exhibited at least one KIF13A-YFP + RE tubule was reduced by ~30 % (Fig. 3 D; see materials and methods), and the RE tubules that were detected were ~30 % shorter (Fig. 3 E; siCTRL: 2.2 ± 0.1 µm, siPI4KII: 1.5 ± 0.04 µm), revealing a defect in the generation and the elongation of RE tubules. As a control, the siRNAmediated depletion of the Golgi-associated PI4KIIIb [START_REF] Minogue | The Many Roles of Type II Phosphatidylinositol 4-Kinases in Membrane Trafficking: New Tricks for Old Dogs[END_REF] did not affect the overall formation of KIF13A + RE tubules or the percentage of cells with at least one RE tubule (Fig.

3 F, middle panel; and Fig. 3, G andH). Furthermore, the tubulation defect observed in PI4KIIdepleted cells reflected neither an altered microtubule network -similar in siCTRL-and siPI4KII-treated cells (Fig. S3 B) -nor an alteration in the overall association of the BLOC-1 subunit (pallidin) to cellular membranes as shown by immunoblotting of membrane fractions prepared from cells homogenates (Fig. S3 C). Therefore, and although PI4KII would not be required for the BLOC-1 association with membranes, this set of experiments demonstrated that PI4KII are specifically required for the formation and/or the elongation of KIF13 + recycling endosomal tubules.

To assess if the enzymatic activity of the PI4KII was required to generate RE tubules, we tested the effect of phenylarsine oxide (PAO), a PI4KII inhibitor that causes cellular PI4P depletion [START_REF] Yue | Inhibition of phosphatidylinositol 4-kinase results in a significant reduced respiratory burst in formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils[END_REF]. Similarly to PI4KII depletion, treatment of HeLa cells transiently expressing KIF13A-YFP for 30 min with PAO at low concentration (300 nM) reduced the percentage of cells harboring at least one KIF13 + tubule by ~70%, as compared to cells treated with DMSO alone (Fig. S3, D andE). Live imaging during the 30 min of PAO treatment showed that pre-existing KIF13A-YFP + RE tubules were gradually lost, concomitant with the appearance of vesicular structures (Fig. S3 F, top right panel), while the microtubule network remained intact (bottom panels). At this concentration, PAO could inhibit the enzymatic activity of the PI4KIIIa [START_REF] Balla | Characterization of type II phosphatidylinositol 4-kinase isoforms reveals association of the enzymes with endosomal vesicular compartments[END_REF], but cells similarly treated with the PI4KIIIa inhibitor GSK-A1 (100 nM) did not show defects associated with KIF13A-YFP + RE tubules (Fig. S3, D andE). In contrast, cells depleted of PI4KIIIb and similarly treated with PAO now displayed a loss of the KIF13A-YFP + RE tubules (Fig. 3 F, right panel), showing that the stability of pre-existing RE tubules specifically required PI4KII enzymatic activity. Finally, and because BLOC-1 concentrated in vitro on PI3P + membranes and tubulated them (Fig. S2,D, F and J), we sought to rule out the possible involvement of PI3P + endosomal membranes during the KIF13dependent tubulation process. First, HeLa cells expressing KIF13A-YFP and treated with a PI3K inhibitor (wortmannin, 10 µM) did not show defects associated with KIF13A + RE tubule formation (Fig. S3, D andE). Second, depletion of retromer components such as VPS35, or SNX1 and SNX2, which contribute to PI3P-dependent endosomal tubulation processes [START_REF] Cullen | Sorting nexins provide diversity for retromerdependent trafficking events[END_REF], did not affect the number of cells with at least one KIF13A-YFP + tubule [Fig. S3, G and H; and see [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF]]. Taken together, these data indicate that the formation of RE tubules and their stabilization rely on cellular PI4P synthesized by PI4KII.

Endosomal PI4P stabilizes the recycling endosomal tubule.

As PI4P is distributed among different organelles, including the Golgi apparatus and endosomes [START_REF] Hammond | A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi[END_REF], we asked which pool of PI4P contributes to generate the RE tubules. We exploited the rapamycin-inducible FRB-FKBP chemical dimerization system to trigger the recruitment of the FKBP-conjugated PI4P phosphatase enzymatic domain from Sac1 (FKBP-PJ-Sac) to early endosomal or Golgi membranes harboring FRB-conjugated RAB5 or -Giantin, respectively. Upon rapamycin addition, FKBP-PJ-Sac dimerizes rapidly with FRB-target proteins, allowing acute PI4P dephosphorylation on the cytosolic leaflet of the target organelle [Fig.

PI4KII are required for the budding and elongation of early endosomal membranes.

To form a RE tubule, the early endosomal membrane must be locally remodeled to generate a nascent tubule that is then elongated and ultimately released by membrane scission [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF][START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF]. We thus investigated if an imbalance of PI4P metabolism affects the remodeling of early endosomal membranes. We performed conventional EM in PI4KII-depleted cells immobilized by high-pressure freezing (HPF), which maintains the cellular membranes in a nearly native state [START_REF] Hurbain | Analyzing Lysosome-Related Organelles by Electron Microscopy[END_REF]. In siCTRL-treated cells, most early endosomes -defined as electron-lucent and round membrane-bound compartments containing a variable number of intra-luminal vesicles (ILVs) -harbored one or more nascent tubule profiles at their limiting membrane (Fig. 5 A, arrowheads; quantification in Fig. 5 C; nascent tubules/ endosome, siCTRL: 1.07 ± 0.17, siPI4KII: 0.36 ± 0.07). In PI4KII-depleted cells (Fig. 5 B), the limiting membranes of early endosomes harbored fewer deformations (arrowheads) as revealed by the ~70 % reduction of nascent tubules per endosomal structure (Fig. 5C). This reduction did not reflect a defect in early endosome biogenesis, as early endosomes were frequently observed in siPI4KII-treated cells -although many contained fewer ILVs (Fig. 5 B, arrows), suggesting a potential impairment in endosomal maturation.

This result shows that PI4KII expression is required for the initiation of nascent early endosomal tubules.

Though nascent tubules associated with endosomes were less numerous in PI4KII-depleted cells, three parameters were measured for those that were detected (Fig. 5, B andC, arrowheads): the maximum length, the maximum width, and the neck width (Fig. S4 A). In PI4KII-depleted cells, the nascent tubes were significantly shorter (Fig. 5 D; siCTRL: 98 ± 8 nm, siPI4KII: 76 ± 5 nm), consistent with a requirement for PI4KII and PI4P in tubule extension.

The average maximum width of the nascent tubules was similar (Fig. S4 B, siCTRL: 60 ± 3 nm, siPI4KII: 61 ± 3 nm). Hence, the average length-to-width ratio of the nascent endosomal tubules in PI4KII-depleted cells was significantly reduced as compared to the control (Fig. S4 C; siCTRL: 1.8 ± 0.2, siPI4KII: 1.4 ± 0.1). Tubular membrane carriers constrict their neck prior to their release [START_REF] Ripoll | Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers[END_REF]. The average neck width of the nascent tubules was marginally increased in PI4KII-depleted cells relative to control cells (Fig. S4 D; siCTRL: 49 ± 3 nm, siPI4KII: 58 ± 4 nm), although the increase did not reach statistical significance. These data indicate that PI4KII mainly controls the length of the nascent RE tubules.

Together, our data indicate that PI4KII and its PI4P product contribute to sequential stepsinitiation and elongation -in the remodeling of the early endosome limiting membrane into a RE tubule.

PI4KII control endosomal cargo recycling.

If early endosomal PI4P contributes to RE tubule initiation, elongation and stabilization, then PI4KII depletion should impair the endosomal recycling of conventional cargoes. To test this prediction, we analyzed the dynamics of internalized transferrin (Tf) as a readout for the model recycling cargo, Tf receptor. Control and PI4KII-depleted cells were pulsed for 10 min with fluorescent-Tf (Alexa Fluor 488 conjugated Tf) and then chased for different time points before processing by FM (Fig. S5 A) and quantification of signal intensity (Fig. S5, B andC). First, the overall Tf fluorescence intensity at time 0 of chase was ~20 % reduced in PI4KII-depleted cells as compared to controls (Fig. S5, A andB), showing that less Tf was internalized during the pulse. Second, the overall intracellular distribution of Tf + structures in PI4KII-depleted cells was distinct from the control cells (Fig. S5 A). At time 0, Tf + endosomes in control-and PI4KIIdepleted cells were distributed throughout the cell periphery (top panels). Whereas the general peripheral distribution tended to be maintained in control cells throughout the chase, Tf + endosomes in PI4KII-depleted cells were more prominent in the perinuclear area after both 20 and 40 min of chase (middle and bottom right panels, arrowheads). Third, whereas control cells showed a linear decay of the average normalized fluorescent intensity of Tf over the 40 min of chase, the decay of Tf intensity in PI4KII-depleted cells showed a significant plateau between the 20-and 40-min time points (Fig. S5 C), consistent with a recycling defect.

Together with observations showing that PI4KIIa or PI4KIIb-depleted pigment cells displayed defects in the trafficking of BLOC-1-dependent cargoes from endosomes to melanosomes [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF], the results proposed that PI4KII are required for the recycling of endosomal cargoes.

PI4KII and/or BLOC-1 are required by intracellular pathogens to enable their developmental cycle.

Several intracellular pathogens exploit RE to establish their niche and/or to replicate. Thus, we reasoned that such pathogens might require PI4KII or BLOC-1 for certain steps of their developmental cycle. Among pathogen model systems, we chose 1) the influenza A virus (IAV), which exploits the RE-associated KIF13A and RAB11 to transport and propagate viral genome segments (viral ribonucleoproteins; vRNPs) during infection (Vale-Costa and Amorim, 2016;[START_REF] Ramos-Nascimento | KIF13A mediates trafficking of influenza A virus ribonucleoproteins[END_REF][START_REF] Alenquer | Influenza A virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sites[END_REF], and 2) the obligate intracellular bacteria Chlamydia trachomatis (C. trachomatis), which develops in a membranebound vacuole (referred to as an inclusion) from which tubules decorated by the REassociated components RAB11, PI4KII and PI4P emerge [START_REF] Rzomp | Rab GTPases Are Recruited to Chlamydial Inclusions in Both a Species-Dependent and Species-Independent Manner[END_REF][START_REF] Moorhead | Multiple Host Proteins That Function in Phosphatidylinositol-4-Phosphate Metabolism Are Recruited to the Chlamydial Inclusion[END_REF], but whose function is unknown. The IAV model was used to define if PI4KII contribute to RE functions that are hijacked during viral infection, and the membrane tubules emanating from the C. trachomatis inclusion served as a proxy for PI4KII functions during membrane tubulation.

First, we explored the role of PI4KII in viral replication by IAV. In human A549 lung carcinoma cells, treatment with PI4KII siRNAs significantly reduced the expression of PI4KIIa and PI4KIIb mRNAs (Fig. S6, A andB). Cells treated with siCTRL or siPI4KII were mock-infected or infected with IAV/Puerto Rico/8/34 (hereafter referred as PR8 virus; see materials and methods), and viral production was assayed. Importantly, PI4KII depletion led to a modest but consistent ~10 % reduction of PR8 virus production at 8 hours post-infection (h.p.i.) relative to siCTRL-treated cells (Fig. 6 A). Given that the synthesis of viral matrix and capsid proteins was not affected by PI4KII depletion (Fig. S6, C-E), this finding suggested that the formation of IAV inclusions could be impaired. Viral inclusions are viewed as sites where complete genomes assemble before packaging into nascent virions to form fully infectious particles [START_REF] Amorim | A Comprehensive Review on the Interaction Between the Host GTPase Rab11 and Influenza A Virus[END_REF]. The packaging of influenza segmented genomes into productive virions requires RAB11, which redistributes during infection to intracellular sites of progeny RNA accumulation and is required to form liquid viral inclusions (i.e., not membrane bound), in which RAB11 + vesicles accumulate (Marta Alenquer et al., 2019). This is evident by IFM of PR8 infected cells, in which labeling for RAB11 and for IAV proteins such as NP overlap in punctate structures throughout the cell (Vale-Costa et al., 2016). Whereas both siCTRL-and siPI4KII-treated IAV-infected cells showed that RAB11 + puncta overlapped with NP labeling, these RAB11 and NP + structures in PI4KII-depleted cells were substantially larger (Fig. 6 siCTRL: 9.5 ± 1.2 µm, siPI4KII: 2.3 ± 0.5 µm). Similar results were observed in Ctr D infected cells that had been depleted of KIF13A or of BLOC-1 (Fig. S6, F-H). Compared to control cells, the IM tubes per cell were fewer in number (Fig. S6 G; siKIF13A: 0.8 ± 0.1, siBLOC-1: 0.8 ± 0.1) and shorter in length (Fig. S6 H; siKIF13A: 5.1 ± 1.0 µm, siBLOC-1: 5.8 ± 1.3 µm) after KIF13A or BLOC-1 depletion. These data revealed that components required to form RE tubules are also used to form tubules from the Chlamydia inclusion membrane.

DISCUSSION

Recycling endosomal tubules are critical in the trafficking of essential physiological cargoes from early endosomes to other intracellular compartments. Here, by exploring the way lipids and proteins shape the early endosomal membrane to form RE tubules, we identified the phospholipid PI4P and the protein complex BLOC-1 as a unit that tubulates endosomal membranes. In vitro analysis with a minimal set of lipids shows that BLOC-1 binding to negatively charged membranes, such as those containing PI3P or PI4P, generates tubules.

As a working model (Fig. 6 G), we propose that the PI phosphatase Sac2 activity would maintain a low level of PI4P at the PI3P-rich early sorting endosomal membrane by counteracting the activity of PI4KII. While BLOC-1 could associate with early endosomes through binding to PI3P + membranes, the local elevation of PI4P by PI4KII would favor BLOC-1 concentration on endosomal membrane subdomains, which would elongate through the combination of curvature induction and microtubule-based motor engagement [e.g., KIF13A [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF]]. While the RE tubule would be stabilized at least in part by membraneassociated KIF13 bound to microtubules, BLOC-1 together with branched actin filaments would contribute to the constriction of the RE tubule neck prior to its release by membrane fission [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF]. Together, we conclude that BLOC-1 and PI4P play a central role in endosomal tubulogenesis during the initiation, elongation, and stabilization of RE tubules.

PI4P metabolism relies on specific kinases (PI4KII and -III, each comprising two members, a and b) and phosphatases (Sac1 and Sac2), which control PI4P abundance on the cytosolic leaflet of membranes of various organelles (e.g., Golgi apparatus, endosomes, and lysosomes) and transport carriers derived therefrom. Sac2 localization is restricted to early sorting vacuolar endosomal domains [START_REF] Nakatsu | Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway[END_REF][START_REF] Hsu | Spatiotemporal control of phosphatidylinositol 4phosphate by Sac2 regulates endocytic recycling[END_REF], whereas PI4KIIa can associate with both vacuolar sorting endosomes [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF] and/or endosomal derived tubules in Drosophila and mammalian cells [START_REF] Burgess | Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila[END_REF][START_REF] Henmi | PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes[END_REF][START_REF] Minogue | Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor[END_REF][START_REF] Ma | An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation[END_REF]. This indicates that PI4P production and consumption at early endosomal membranes are differently balanced between the vacuolar and tubular domains. Indeed, PI4P extensively decorates RE tubules in different cell types of different species such as human HeLa cells (this study) or mouse pigment cells [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF]. The relative PI4P abundance on RE tubules over RAB5 + early endosomes might thus reflect a prominent Sac2 activity at the sorting endosomal membrane that counteracts the PI4KII activity, together with a preferential sorting of PI4KII to RE tubules [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF]. Hence, a PI3P-to-PI4P gradient would be naturally implemented along the sorting-to-recycling pathway [START_REF] Ketel | A phosphoinositide conversion mechanism for exit from endosomes[END_REF]. We show that the PI4P + / KIF13 + RE tubules are apposed to PI3P + / RAB5 + sorting endosomes, and that the hydrolysis of endosomal PI4P (via endosomal targeting of PJ-Sac) destabilizes the RE tubules. Therefore, these results suggest that PI3P + sorting and PI4P + recycling endosomal membranes may form a continuum, as previously observed in pigment cells [START_REF] Dennis | BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery[END_REF][START_REF] Delevoye | AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis[END_REF].

Along such a continuum, local production of PI4P at the sorting endosomal membrane might be a signal to initiate the formation of RE tubules. Depleting PI4KII expression abrogates the biogenesis of RE tubules and leads to the accumulation of early vacuolar endosomes with fewer nascent tubules. This suggests that PI4P must first be produced at the sorting endosomal membrane to concentrate locally a curvature-inducing component, like BLOC-1, that contributes to initiate and/or to stabilize membrane curvature prior to elongation to form a RE tubular carrier. The PI4P contribution to the biogenesis of tubular transport carriers might not be restricted to endosomal membranes, as PI4P together with curvature-inducing proteins could aid their formation from various membrane sources, including the Golgi apparatus [START_REF] Dippold | GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch and shape the Golgi to promote budding[END_REF][START_REF] Rahajeng | Efficient Golgi Forward Trafficking Requires GOLPH3-Driven, PI4P-Dependent Membrane Curvature[END_REF], early endosomes in Drosophila [START_REF] Ma | An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation[END_REF]), autolysosomes (McGrath et al., 2021), or phagolysosomes [START_REF] Levin-Konigsberg | Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling[END_REF][START_REF] López-Haber | Phosphatidylinositol-4-kinase IIα licenses phagosomes for TLR4 signaling and MHC-II presentation in dendritic cells[END_REF].

We show that acute inhibition of PI4KII activity or rapid hydrolysis of endosomal PI4P destabilize pre-existing RE tubules. Moreover, the few tubules that still emerged from vacuolar sorting endosomes in cells depleted of PI4KII were unusually short, suggesting that PI4P contributes not only to the initiation of RE tubules, but also to their elongation and/or stabilization. The elongating/stabilizing function might not be limited to KIF13 + RE tubules, as PI4P decorates and maintains other extended endosomal tubules with cargo recycling or trafficking functions [START_REF] Jović | Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling[END_REF][START_REF] Ma | An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation[END_REF]. Given that PI4P, together with BLOC-1 [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF], contributes first to initiate the RE tubule and second to stabilize it, this implies that one or more PI4Ks must be required to supply PI4P along the tubules. This is consistent with data in melanocytes that both PI4KIIa and PI4KIIb are present on melanosome-bound RE tubules and that both are required for their stability [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF].

Because we show that the depletion of PI4KIIIb, which interacts with RAB11 [START_REF] Burke | Structures of PI4KIIIβ complexes show simultaneous recruitment of Rab11 and its effectors[END_REF], or the inhibition of PI4KIIIa enzymatic activity, does not affect the formation or stability of KIF13 + RE tubules, we propose that only PI4KII (a and/or b) produce PI4P (i) at the early sorting endosomal membrane to facilitate the biogenesis of RE tubules, and (ii) along the RE tubules to support their elongation and/or stabilization.

Here, BLOC-1 tubulates relatively curved (i.e., small liposomes) or flat (i.e., GUVs) membranes provided they are negatively charged, such as those containing PI4P or PI3P. As shown biochemically, the lack of PI4KII expression does not impact the ability of BLOC-1 to bind to cellular membranes [Fig. S3 C; and [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF]]. Thus, in cells, BLOC-1 could associate with PI3P-rich vacuolar domains of early endosomes, where local elevation of PI4P would allow its concentration and/or recruitment of PI4P-binding components contributing to RE tubulogenesis, such as the AP-1 complex [START_REF] Wang | Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi[END_REF]; a KIF13A partner [START_REF] Nakagawa | A novel motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane through direct interaction with AP-1 complex[END_REF][START_REF] Delevoye | AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis[END_REF][START_REF] Campagne | AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis[END_REF]. Therefore, the BLOC-1 concentration on the PI4P-rich endosomal domain would be sufficient to form and/or stabilize tubules. This is consistent with observations in cells, in which depleting BLOC-1 expression led to the accumulation of early sorting endosomes that harbored numerous buds that did not elongate [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF]. We therefore propose that BLOC-1 is a membrane shaping complex [START_REF] Johannes | Bending "on the rocks"--a cocktail of biophysical modules to build endocytic pathways[END_REF] that facilitates tubulation of PI4P + cellular membranes.

Several membrane remodeling components form supramolecular assemblies on membranes, such as dynamin [START_REF] Kong | Cryo-EM of the dynamin polymer assembled on lipid membrane[END_REF], the N-BAR protein Endophilin [START_REF] Mim | Structural basis of membrane bending by the N-BAR protein endophilin[END_REF], ESCRT-III complex proteins [START_REF] Nguyen | Membrane constriction and thinning by sequential ESCRT-III polymerization[END_REF], retromer [START_REF] Kovtun | Structure of the membrane-assembled retromer coat determined by cryo-electron tomography[END_REF], or COP-II complexes [START_REF] Hutchings | Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network[END_REF]. Interestingly, our data suggest that BLOC-1 can be densely packed on PI4P + or PI3P + nanotubes without forming a periodic arrangement. Given its 30 nm-length [START_REF] Lee | Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)[END_REF], several BLOC-1 molecules are likely needed to surround tubules. By organizing into various architectures, BLOC-1 assemblies could then stabilize tubules of different curvatures (and hence different diameters). Indeed, as shown by EM of purified BLOC-1, the linear chain made by the eight subunits can bend as much as 45° [START_REF] Lee | Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)[END_REF], suggesting together that BLOC-1 may tune its 3D architecture and/or assembly of molecules on curved membrane.

Since PI4P and BLOC-1 function as a module in endosomal membrane remodeling, loss of their functions should have similar consequences. As expected from previous studies [START_REF] Henmi | PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes[END_REF][START_REF] Jović | Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling[END_REF][START_REF] Hsu | Spatiotemporal control of phosphatidylinositol 4phosphate by Sac2 regulates endocytic recycling[END_REF] and as also observed in cells depleted of BLOC-1 or KIF13A [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF][START_REF] Setty | BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles[END_REF], cells depleted of PI4KII display slower Tf recycling associated with reduced Tf uptake, likely reflecting decreased Tf Receptor cell surface expression. BLOC-1-deficient cells also missort additional cargoes [START_REF] Monis | BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia[END_REF][START_REF] Setty | BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles[END_REF][START_REF] Di Pietro | BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes[END_REF][START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF], and consistently Zhu et al document missorting of similar cargoes in mouse melanocytes depleted of PI4KIIa or b [START_REF] Zhu | Two type II phosphatidylinositol 4-kinases function sequentially in tubule-mediated cargo delivery from early endosomes to melanosomes[END_REF]. Thus, by initiating RE tubulogenesis, BLOC-1 and PI4KII together maintain the trafficking and sorting of cargoes along the endocytic pathway.

The requirement for PI4KII in RE biogenesis was validated in our analyses of PI4KII function in the developmental cycle of two intracellular pathogens. We show that the bacterium Chlamydia trachomatis uses PI4KII (as well as BLOC-1 and KIF13A) to build the parasitophorous inclusion-derived tubules, while the virus influenza A used PI4KII to form adequately sized IAV liquid viral inclusions and to control viral progeny production. The role of PI4KII in Chlamydia inclusion maturation is consistent with reports showing the recruitment of RAB11 [START_REF] Rzomp | Rab GTPases Are Recruited to Chlamydial Inclusions in Both a Species-Dependent and Species-Independent Manner[END_REF] and of PI4KIIa, PI4P or PI4P-binding proteins [START_REF] Moorhead | Multiple Host Proteins That Function in Phosphatidylinositol-4-Phosphate Metabolism Are Recruited to the Chlamydial Inclusion[END_REF] to the inclusion. Such redistribution of components allows the internalized bacteria to manipulate intracellular pathways, like the slow recycling endosomal pathway [START_REF] Ouellette | A Functional Slow Recycling Pathway of Transferrin is Required for Growth of Chlamydia[END_REF], and to complete the Chlamydia infectious cycle [START_REF] Mölleken | Acquisition of Rab11 and Rab11-Fip2-A novel strategy for Chlamydia pneumoniae early survival[END_REF][START_REF] Leiva | Rab11-Family of Interacting Protein 2 associates with chlamydial inclusions through its Rab-binding domain and promotes bacterial multiplication[END_REF]. The role of PI4KII in IAV assembly is also consistent with reports of the exploitation of RE function by IAV. Cells infected by IAV redistribute RAB11 to vRNPs + liquid structures, which relocalize to the cell periphery upon KIF13A overexpression (Vale-Costa and Amorim, 2016;[START_REF] Ramos-Nascimento | KIF13A mediates trafficking of influenza A virus ribonucleoproteins[END_REF][START_REF] Amorim | A Rab11-and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNA[END_REF][START_REF] Vale-Costa | Clustering of Rab11 vesicles in influenza A virus infected cells creates hotspots containing the 8 viral ribonucleoproteins[END_REF][START_REF] Alenquer | Influenza A virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sites[END_REF]. Given the requirement of PI4P and BLOC-1 during the building of functional RE tubules, it is likely that many other intracellular pathogens exploit these components to hijack the host RE system to their own benefit [START_REF] Yong | Targeting Endosomal Recycling Pathways by Bacterial and Viral Pathogens[END_REF].

The dysregulation of PI4KII expression or genetic mutations of BLOC-1 subunits are associated with risks of development of cancers or neurological disorders [START_REF] Minogue | The Many Roles of Type II Phosphatidylinositol 4-Kinases in Membrane Trafficking: New Tricks for Old Dogs[END_REF][START_REF] Hartwig | Neurodevelopmental disease mechanisms, primary cilia, and endosomes converge on the BLOC-1 and BORC complexes[END_REF], and inactivating BLOC-1 subunit mutations are found in subtypes of HPS [START_REF] Bowman | The road to lysosome-related organelles: Insights from Hermansky-Pudlak syndrome and other rare diseases[END_REF], an inherited syndromic disorder characterized by albinism, excessive bleeding, and other defects. Given that an altered biogenesis and/or function of RE has been characterized in these different disorders [START_REF] Delevoye | Lysosome-related organelles as functional adaptations of the endolysosomal system[END_REF]; O'Sullivan and Lindsay, 2020), a more systematic investigation of whether and when PI4P and BLOC-1 act together as a universal and minimal machinery reshaping membranes into tubules will provide a better understanding of the mechanisms of membrane dynamics in physiology and of their malfunction in pathophysiology.

Materials and Methods

Cell culture, transfections, and infections

Cell Culture: Hela cells were grown in DMEM with GlutaMAX TM (Invitrogen), supplemented with 10% FBS at 37ºC under 5% CO2. For viral infection, the epithelial cells Madin-Darby Canine Kidney (MDCK) and A549 human alveolar basal carcinoma were kind gift of Pr. Paul Digard (Roslin Institute, UK), cultured as previously described [START_REF] Amorim | A Rab11-and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNA[END_REF], and regularly tested for mycoplasma contamination with the LookOut mycoplasma PCR detection kit (Sigma, MP0035), using JumpStart Taq DNA Polymerase (Sigma, D9307).

Transfection: For plasmid transfections in 6-well plate, cells [2.5 × 10 5 / well or fluorodish (World Precision Instrument)] were seeded at day 1, transfected on day 2 with respective plasmids (300 ng -1000 ng) using jetPRIME reagent (Polyplus-transfection), and processed on day 3 for IFM, live cell imaging or immunoblotting (IB). For rapamycin-inducible-sac system, live cell imaging was carried out by co-transfecting plasmids coding for mRFP-PJ-Sac-FKBP (600 ng) and KIF13A-YFP (1 µg) together with FRB-ECFP(W66A)-Giantin (400 ng) or iRFP-FRB-RAB5 (400 ng) and imaging was carried out before and after addition of rapamycin (1 mM, 20 min) [see [START_REF] Hammond | A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi[END_REF] for details]. For siRNA transfections (HeLa cells), cells were seeded in 6-well plate on day 1 (1.5 × 10 5 / well), transfected with respective siRNAs (200 pmol/ well) on day 2 and day 4 using oligofectamine (Invitrogen; 10 µl/ well), and processed on day 6 for IFM or biochemical analyses. For live cell imaging, siRNA-treated cells were transfected in fluorodish on day 5 with respective plasmids and imaged at day 6. For siRNA transfections (A549 cells), cells were grown in 6-well plates to ∼50 % confluency the day before transfection, and transfected (100 pmol/ well) using DharmaFECT (Dharmacon) for 48 h, and infected or mock-infected (i.e., non-infected; similar protocols as for infection, but without virus addition) with PR8 at MOI 3 for 8 h.

Infection with pathogens: For bacterial infection, 1.5 × 10 5 HeLa cells were seeded in 12-well plates before transfection with siRNA by Lipofectamine RNAimax (Invitrogen) for 24 h following the manufacturer's instructions. Transfected cells were infected with C. trachomatis serovar D stably transformed with the p2TK2-SW2 plasmid expressing mCherry [START_REF] Agaisse | A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing fluorescent proteins under the control of a C. trachomatis promoter[END_REF] in triplicates at a multiplicity of infection (MOI) of 0.2, with a 30 min centrifugation step at 270xg at 37 °C. For viral infection, reverse-genetics derived A/Puerto Rico/8/34 (PR8 WT; H1N1) was used as a model virus and titrated according to reference (Vale-Costa et al., 2016). Virus infections were performed at a MOI of 3 for 8 h. After 45 min, cells were overlaid with DMEM containing 10 % fetal bovine serum (Gibco, Life Technologies, 10500-064) and 1 % penicillin/ streptomycin mix (Biowest, L0022-100).

Plasmids

The KIF13A-YFP and mCherry-RAB11A were obtained by recombination by using the GATEWAY cloning system (Invitrogen) [START_REF] Delevoye | Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A[END_REF]; GFP-RAB11A was a kind gift from Dr. Bruno Goud (Institut Curie, Paris, France); mCherry-KIF13B plasmid was a kind gift from Pr. Kaori Horiguchi Yamada (University of Illinois, Chicago, USA); plasmid coding for the PI4P sensor (pEGFP-SidC 609-776 ) was previously described [START_REF] Domingues | The Salmonella effector SteA binds phosphatidylinositol 4phosphate for subcellular targeting within host cells[END_REF] Robin Irvine; [START_REF] Hammond | PI4P and PI(4,5)P2 Are Essential But Independent Lipid Determinants of Membrane Identity[END_REF]]; iRFP-FRB-RAB5 [# 51612, pEGFP-C1; Pr. Tamas Balla [START_REF] Hammond | A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi[END_REF]]. The BLOC-1 expression plasmid (pST39-BLOC-1) is described in [START_REF] Lee | Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)[END_REF]. The GST expression plasmid (pGST-parallel-1) is described in [START_REF] Sheffield | Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors[END_REF].

siRNAs

The sense strand of the following siRNAs was synthesized by Qiagen: 1) Control: 5'-AATTCTCCGAACGTGTCACGT-3'; 2) PI4KIIa: smartpool (Flexitube siRNA, GS55361); 3) PI4KIIb:

5'-CAGAGTACTGGCCTTGTTCAA-3' (PI4KIIb#7) and 5'-TCGGATTGTCCACCTGAGCAA-3' (PI4KIIb#9); 4) PI4KIIIb: smartpool (Flexitube siRNA, GS5298); 5) BLOC-1 (individual siRNAs against the following BLOC-1 subunits): 5'-AATGCTGGATTCGGGAATTTA-3' (snapin), 5'-ATGGTCCATGTTAAATGTAAA-3' (muted),

Drug treatments

Cells were incubated 30 min with PAO (300 -600 nM), or with wortmannin (10 mM), or with GSK-A1 (100 nM), or 20 min with rapamycin (1 mM) prior to analyses by live cell imaging.

Control cells were treated with the same volume of DMSO alone.

Preparation of purified BLOC-1

Preparation of BLOC-1 for in vitro experiments using membrane systems: BLOC-1 was prepared according to [START_REF] Lee | Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)[END_REF] with some modifications. The pST39-BLOC-1 plasmid encoding recombinant BLOC-1 was transformed into BL21gold(DE3)plysS cells (230134, Agilent). Several colonies from the plate were inoculated into a starter culture of 10 ml LB supplemented with 34 μg/ ml chloramphenicol and 100 μg/ ml ampicillin that was grown overnight at 37 °C with moderate shaking. Next day the starter culture was transferred to 1liter Terrific Broth medium supplemented with antibiotics and grown at 37 °C, 200 rpm for ~7 h. Cultures were induced with 0.8 mM IPTG and grown overnight at 16 °C, 200 rpm. Cells were centrifuged at 4 °C, 6000xg for 15 min and bacterial pellets were resuspended in buffer containing 50 mM Tris (pH 7.4), 300 mM NaCl, 5 mM β-mercaptoethanol supplemented with lysozyme, DNase I, and complete-EDTA free protease inhibitor tablets (1183617001, Roche)

and incubated at 4 °C with rotation for 30 min. The sample was further sonicated, and the soluble fraction was separated by centrifugation at 4 °C, 35,267xg for 45 minutes. The supernatant was loaded on a gravity chromatography column preloaded with glutathione-Sepharose 4B (17-0756-05, GE Healthcare) and incubated for 2 h at 4 °C with gentle rotation in batch mode. Following incubation, the column was extensively washed with buffer [50 mM Tris (pH 7.4), 300 mM NaCl, and 5 mM β-mercaptoethanol] and on-column cleavage of the GST and 6xHis tags using tobacco etch virus (His6-TEV) S219V protease (~0.5 mg/ L) was done overnight at 4 °C. The flow-through fraction containing the cleaved BLOC-1 was collected and loaded onto a Ni-NTA column. Flow-through material was collected, concentrated, and further purified by size exclusion chromatography on a Superose 6 10/300 column in buffer containing 50 mM Tris (pH 8.0), 0.4 M NaCl, 2 mM DTT, 5 % glycerol and 1 mM EDTA. Fractions containing BLOC-1 were pooled together, aliquoted, flash frozen in liquid nitrogen, and stored at -80 °C. The integrity of the complex was analyzed by SDS-PAGE.

Preparation of GST and BLOC-1-GST: Plasmids encoding GST and BLOC-1-GST were expressed and purified as described above except that after the binding of the protein to glutathione sepharose and following the washing step, the bound GST proteins were eluted from the beads with elution buffer containing 50 mM Tris (pH 8.0) and 10 mM glutathione. 21 nM of GST or BLOC-1-GST were used for the PIP array assays.

PIP array assay

PIP array (P-6100, Echelon Biosciences) binding assays were carried out according to the manufacturer's protocol. Briefly, PIP membranes were blocked in PBS-T (PBS supplemented with 0.1 % v/v Tween-20) supplemented with 3 % fatty acid free BSA for 1 h at room temperature (RT) with gentle shaking. Following blocking, membranes were incubated with the proteins that were diluted in blocking buffer and incubated for 1 h with gentle agitation.

Membranes were washed 3 times in 5 ml PBS-T for 5 min and then incubated with anti-GST antibody for 1 h at RT with gentle agitation. Following incubation, membranes were washed 3 times in 5 ml PBS-T for 5 min and incubated with HRP antibodies. Following incubation, membranes were washed 3 times in 5 ml PBS-T for 5 min and an additional final wash of PBS.

Membranes were blotted with Pierce™ ECL Western Blotting Substrate (cat# 185698) and signal was detected with Bio-Rad Chemidoc MP imaging system.

Liposome flotation assay

Dried films of EPC/ bPI4P (95/ 5 mol/mol), EPC/ 18:1 PI3P (95/ 5 mol/mol), EPC/ bPI(4,5)P2

(95/ 5 mol/mol), or EPC/ bPS (90/ 10 mol/mol), doped with 1% Texas Red™ DHPE, were made by evaporation of organic solution under vacuum for 1 h. The lipid film was resuspended in HK buffer [50 mM Hepes (pH 7.4), 120 mM potassium acetate] at 1 mM while vortexing.

Liposomes (0.2 mM) and BLOC-1 (0.5 μM) were incubated for 15 min in HK buffer at RT in a

Beckman polycarbonate centrifuge tube. A 150 μL mixture was mixed with HK buffer supplemented with 60 % sucrose (100 μL), overlaid with an intermediate sucrose solution (HK buffer with 24 % sucrose; 200 μL) and a layer of HK buffer (50 μL). After centrifugation (55,000 rpm, TLS 55 Beckman swing rotor) for 90 min at 4°C, three fractions (bottom, medium, top) of 250, 150 and 100 μL were collected. Top fractions that contain fluorescent vesicles were analyzed by SDS-PAGE.

In vitro experiments using membrane systems

Preparation of lipid nanotubes and binding assay with BLOC-1: GalCer/ EPC nanotubes doped or not with PIxP or PS were prepared according to [START_REF] Wilson-Kubalek | Lipid nanotubes as substrates for helical crystallization of macromolecules[END_REF]. Briefly, a mixture of GalCer, EPC, and bPI4P, 18:1 PI3P, bPI(4,5)P2 or bPS was dried under vacuum.

GalCer/ EPC (80/ 15 mol/mol), GalCer/ EPC/ bPI4P (80/ 15/ 5 or 10 mol/mol), GalCer/ EPC/ 18:1 PI3P (80/ 15/ 5 mol/mol), GalCer/ EPC/ bPI(4,5)P2 (80/ 15/ 5 mol/mol), or GalCer/ EPC/ bPS (80/ 15/ 10 mol/mol) tubes were formed at RT after resuspension of the dried film at 5 mg/ ml total lipid concentration in HK buffer followed by 5 cycles of 10 min vortex, 2 min at 40 °C. For experiments, tubes were aliquoted and stored at -20 °C. Tubes were diluted in HK buffer at 50 µM and BLOC-1 was added at a lipid/ BLOC-1 ratio of 300 mol/mol. After 5 min incubation, sample was used for EM analysis.

Preparation of lipid suspension and binding assay with BLOC-1: Dried films made of EPC/ bPS/ PI4P (85/ 10/ 5 mol/mol) was made by evaporation of organic solution under vacuum for 1 h. The lipid film was resuspended in HK buffer at 1 mM while vortexing. This led to the formation of various population of vesicles and tubules. For cryo-EM, the lipid suspension was diluted to 50 µM and BLOC-1 was added at lipid/ BLOC-1 ratio of 300 mol/mol and incubated 15 min at RT before grid preparation for cryo-EM.

Giant Unilamellar Vesicle (GUV) preparation: GUVs were generated using a lipid mixture composed of EPC/ DOPS/ bPi4P (85/ 10 /5 mol/mol); EPC/ DOPS/ bPI3P (85/ 10 /5 mol/mol);

or EPC only. 0.5 % mole of BODIPY TM TR ceramide was added to visualize GUVs by confocal fluorescence microscopy. The inner buffer used to generate GUVs is 50 mM NaCl, 20 mM sucrose and 20 mM Tris (pH 7.5). GUVs were prepared by using the polyvinyl alcohol (PVA) gel-assisted vesicle formation method as previously described in [START_REF] Weinberger | Gel-assisted formation of giant unilamellar vesicles[END_REF].

Briefly, a PVA gel solution (5 %, w/w, dissolved in 280 mM sucrose and 20 mM Tris, pH 7.5) warmed up to 50 °C was spread on clean coverslips (20 mm × 20 mm). Before use, the coverslips were washed once with ethanol and twice with ddH2O. For experiments, the PVAcoated coverslips were incubated at 50 °C for 30 min, and then approximately 5 µL of the lipid mixture was spread on the PVA-coated coverslips. The coverslips were then placed under vacuum for 30 min at RT, and then in a petri dish. Approximately 500 µL of the inner buffer was pipetted on top of the coverslips. The coverslips were kept at RT for at least 45 min, allowing GUVs to grow. Once done, we gently "ticked" the bottom of the petri dish to detach GUVs from the PVA gel. GUVs were collected using a 1 mL pipette tip with its tip cut to prevent breaking GUVs.

Electron Microscopy

Negatively stained images of BLOC-1: BLOC-1 (25 µg/ml) was deposited on a carbon-coated EM grid previously glow-discharged and negatively stained with 2 % uranyl acetate in water.

Negatively stained images of BLOC-1 were acquired with a QUEMESA CCD camera (Olympus, Germany) on a Tecnai Spirit (Thermo Fisher Scientific, USA) operating at 80kV.

Cryo-EM experiments, imaging, and analysis: Lacey carbon 300 mesh grids (Ted Pella, USA)

were used in all cryo-EM experiments. Blotting was carried out on the opposite side from the liquid drop and samples were plunge frozen in liquid ethane (EMGP, Leica, Germany). Cryo-EM images were acquired with a Tecnai G2 (Thermo Fisher Scientific, USA) Lab6 microscope operated at 200 kV and equipped with a 4k x 4k CMOS camera (F416, TVIPS). Image acquisition was performed under low dose conditions of 10 e -/Å 2 at a magnification of 50,000

or 6500 with a pixel size of 0.21 or 1.6 nm, respectively. The diameter of the tubes depicted in Fig. 2 F were measured from > 100 tubes in 17 images from 2 experiments. Diameters were measured every 60 nm along a tube leading to a total of 408 values.

High Pressure Freezing, sample preparation, and conventional EM imaging: HeLa cells were seeded on carbonated sapphire discs and grown for 2 days prior to transfection with either control or PI4KII siRNAs. After two consecutive siRNA transfections, cells were immobilized by HPF using either a HPM 100 (Leica Microssystems, Germany) or a HPM Live µ (CryoCapCell, France), and freeze substituted in anhydrous acetone containing 1 % OsO4/ 2 % H2O for 64 h using the Automatic Freeze Substitution unit (AFS; Leica Microsystems). The samples were included in Epon. Ultrathin sections were contrasted with uranylacetate/ H2O and Reynold's lead citrate solution. Electron micrographs were acquired using a Transmission Electron Microscope (Tecnai Spirit; Thermo Fisher Scientific, Eindhoven, The Netherlands) operated at 80kV, and equipped with a 4k CCD camera (Quemesa, EMSIS, Muenster, Germany).

Fluorescence Microscopy

Live-cell: Hela cells were grown on fluorodish and imaged in their respective media supplemented with 20 mM HEPES (GIBCO, Thermo Fisher Scientific).

Sample preparation of GUVs and fluorescent imaging: Experimental chambers were assembled by sandwiching two coverslips using parafilm®. Before use, the chambers were passivated with a b-casein solution at a concentration of 5 mg/ ml for at least 5 min at RT.

GUVs were incubated with 0.2 µM BLOC-1 in the experimental chambers for at least 15 min at RT before observation. To mix GUVs with BLOC-1, we first added 8 µL of the GUVs in 15 µL of an "outer buffer" (60 mM NaCl, 20 mM Tris, pH 7.5), following by adding 2 µl of BLOC-1 (2.5 µM in stock). Samples were observed using a spinning disk confocal microscope, Nikon eclipse Ti-E equipped with Yokogawa CSU-X1 confocal head, 100X CFI Plan Apo VC objective (Nikon) and a CMOS camera, Prime 95B (Photometrics). After formation of tubules, sample was pipetted off and flash frozen for cryo-EM experiments.

Immunofluorescence (IF): Hela cells grown on coverslips were first fixed with 4 % PFA in PBS for 15 min at RT and then subsequently incubated for 5 min each with PBS/ 2 % glycine and PBS/ 0.2 % BSA (blocking buffer) and PBS/ 0.2 % BSA/ 0.1 % Saponin (permeabilization buffer). The cells were washed three times with permeabilization buffer and incubated 45 min with primary antibodies, washed again and incubated 45 min with secondary antibodies diluted in permeabilization buffer before mounting with Prolong Gold Antifade (Invitrogen). For viral infection studies, A549 cells were similarly fixed with 4% PFA, permeabilized with 0.2%

TritonX and stained in PBS/ 1 % PBS and processed as described above. For bacterial inclusion studies, twenty hours after infection, HeLa cells were fixed and permeabilized for immunofluorescence using 0.05 % saponin, 5 mg/ ml BSA in PBS.

Fluorescence Imaging: HeLa cells were imaged on an inverted Eclipse Ti-E (Nikon) + Spinning disk CSU-X1 (Yokogawa) integrated in Metamorph software (Molecular devices, version 7.8.13) with an EMCCD camera (iXon 897; Andor Technology), a 100× 1.4 NA Plan-Apo objective lens and Z-images were taken at 0.2 mm with the piezoelectric motor (Nano z100, Mad City Lab). For imaging of virus-infected cells, single optical sections were imaged with a SP5 live confocal microscope (Leica). Images were post-processed using ImageJ/ FIJI (NIH).

Super-resolution (SR) microscopy: images were acquired on a spinning-disk system equipped with a super-resolution module (Live-SR; Gataca Systems, France) leading to superresolution spinning-disk confocal microscopy using optical photon reassignment [as described before in [START_REF] Azuma | Super-resolution spinning-disk confocal microscopy using optical photon reassignment[END_REF]]. This method allows combining the doubling resolution together with the physical optical sectioning of confocal microscopy. The maximum resolution is 128 nm with a pixel size in SR mode of 64 nm.

Transferrin recycling assay

Cells starved for 30 min in serum-free medium supplemented with Hepes (20 mM, pH 7.4;

Invitrogen) and 0.5 % (w/v) BSA medium were pulsed for 10 min with Tf-AF-488. Cells were then quickly incubated with cold acid buffer (0,5 M Glycine, pH 2.2) to dissociate surface bound Tf, extensively washed with PBS, and chased at 37 °C with complete media (containing 20 mM Hepes) for various time points prior to chemical fixation with 4 % PFA (Thermo Fisher Scientific) and processing by FM.

Cytosol-membrane cell fractionation

Two 10 cm-dishes of siRNA-treated Hela cells/ condition (~12. Bacterial inclusion analyses: 5 -10 pictures were taken randomly for each coverslips using the imageJ/Fiji software, the scale was set from pixels to micrometers and the length in µm of tubules was measured by using ImageJ/Fiji tracing tool and number of tubules were counted using the multi-point tool. We calculated the mean number of tubules and the average tubular length per cell for each condition. Each condition was analyzed in a blind fashion from three individual coverslips with a minimum of 50 inclusions per coverslips.

58

EM image of a suspension of EPC/ bPS/ bPI4P vesicles and tubes incubated with BLOC-1.

BLOC-1 bind to tubules (white arrowheads), but not to large vesicles (black arrowheads). Inset is the magnified boxed region. J) Representative live fluorescent images of EPC GUVs doped or not with PI3P and incubated 30 min with BLOC-1 (bottom or top, respectively). Membrane tubules (arrowheads) were observed in PI3P + GUVs. K) Cryo-EM image of slightly variable diameters tubules generated upon BLOC-1 (arrowheads) addition to PI4P + GUVs (see also

  A, bottom panels, arrowheads) that co-localized with KIF13A-YFP (Fig. 1 B, arrowheads). MCherry-KIF13B + RE tubules did not co-distribute with iRFP-RAB5 + early endosomes (pseudocolored in green), although they closely apposed (Fig. S1 A, arrows, and quantification). Thus, both KIF13A and KIF13B generate RE tubules. We next investigated the distribution of some PIXP species relative to RE tubules by coexpressing endosomal PIXP-specific fluorescent sensors with KIF13 proteins. Live-cell superresolution FM (SR-FM; spinning-disk confocal microscopy using optical photon reassignment leading to 1.4-fold increased resolution) revealed that the PI4P sensor SidC-GFP largely localized to peripheral punctate structures devoid of iRFP-RAB5 (pseudocolored in red) (Fig. S1 B, top panels, arrows), and to perinuclear structures that corresponded to the TGN labeled for TGN46 by immunofluorescence microscopy (IFM) (Fig. S1 C, top panels, arrowheads). The PI4P sensor also decorated to a lesser extent PM [here stained using fluorescentlyconjugated wheat germ agglutinin (WGA); Fig. S1 C, bottom panels, arrowheads]. To analyze if a specific PIXP is enriched at RE tubules, we co-transfected HeLa cells with plasmids encoding mCherry-KIF13B and sensors of PI4P (SidC-GFP) or PI3P (GFP-FYVE). Coexpression of mCherry-KIF13B led to the appearance of numerous PI4P + tubular structures (Fig. 1 C, top panels), many of which co-distributed with mCherry-KIF13B (arrowheads) as shown by linescan analyses of multiple tubules (Fig. 1 D, top panels). However, mCherry-KIF13B + tubules were negative for the related PI3P sensor GFP-FYVE (Fig. 1, C and D, bottom panels), which instead largely labeled iRFP-RAB5 + early endosomes (Fig. S1 B, bottom panels, arrowheads). Consistently, similar RE tubules in pigment cells were positive

  ) and PI4P + (Fig. S2, G and H) tubes, showing, together, that BLOC-1 binds to these PIXPs containing tubes. Because PI4P decorated BLOC-1-dependent KIF13 + RE tubules [Fig. 1 C; and (Delevoye et al., 2016)], we investigated if BLOC-1 bound to PI4P + membranes of larger curvature than the nanotubes by preparing lipid suspensions formed by swelling a dried lipid film containing EPC and 5 % PI4P. In the control condition without addition of BLOC-1 (Fig 2 B, top panel), tubules (white arrowheads) and vesicles (black arrowheads) of variable diameter were observed (8.5 tubules/ image, n = 20 images). But the addition of BLOC-1 (Fig 2 B, bottom panel) dramatically increased the number of tubules (white arrowheads; 18.6 tubules/ image, n = 23 images). At higher magnification by cryo-EM, BLOC-1 was present on tubules with diameters ranging from 25 nm to 80 nm (Fig. 2 C and Fig. S2 I, white arrowheads), but not on larger vesicles (Fig. S2 I, black arrowheads).

  Fig. S2, J and K). While control GUVs with BLOC-1 and without PIxP (Fig. S2 J, top panel), or with PI4P and without BLOC-1 (Fig. 2 D, top panel), appeared smooth and round by FM, only the addition of BLOC-1 to PI4P + or PI3P + GUVs induced the formation of tubules from their surface (Fig. 2 D, bottom panel, and Supplemental Video 1; and Fig. S2 J, bottom panel; arrowheads). Since PI4P associated with RE tubules in cells (Fig. 1 C), we

  Fig. S2 K), and showed that BLOC-1 decorated tubules with diameters centered around of

  Fig.3, we conclude that the pool of PI4P on early endosomal membranes functions in both

  , B and C). This difference in size could reflect aberrant viral inclusions with altered biophysical properties or a defect in transporting liquid viral inclusions. These data suggest that PI4KII contributes to the size and maintenance of liquid viral inclusions during IAV infection. Second, we investigated the impact of PI4KII on the formation of membrane tubules from the surface of bacterial inclusions formed during Chlamydia trachomatis serovar D (Ctr D) infection of HeLa cells. CTRL-or PI4KII-depleted cells were infected with Ctr D expressing mCherry for 12 hours before analysis by IFM. In both controls and cells depleted of PI4KIIa and PI4KIIb, Ctr D (Fig. 6 D, top panels) were internalized and developed within an inclusion (detected using an antibody to the Cap1 inclusion membrane protein) of comparable size and shape (Fig. 6 D, bottom panels). However, relative to control cells, PI4KII-depleted cells harbored ~60 % fewer elongated inclusion membrane (IM) tubes decorated by Cap1 (Fig. 6 D, arrowheads; and Fig. 6 E; IM tube/ cell, siCTRL: 1.3 ± 0.1; siPI4KII: 0.6 ± 0.1). Moreover, the remaining IM tubes in PI4KII-depleted cells were ~70 % shorter than in controls (Fig. 6 F;

  Drs. Bruno Antonny and Bruno Mesmin (Université Nice Sophia Antipolis, France) for insightful discussions. This work was supported by a grant from the LabEx Cell(n)Scale (ANR-11-LABX-0038, ANR-10-IDEX-0001-02). M.J.A. is funded by The European Research Council consolidator grant under the European Union's Horizon 2020 research and innovation program (No 101001521 -LOFlu) and by the Portuguese Fundação para a Ciência e a Tecnologia (CEECIND/02373/2020). S.V-C. is funded by a Junior Researcher working contract from Fundação para a Ciência e a Tecnologia (FCT) and Instituto Gulbenkian de Ciência (IGC, Portugal). M.D-M. is funded by a doctoral contract from the Ligue contre le cancer.

  ; plasmid coding for the PI3P sensor (pEGFP-2x-FYVE domain) was kind gift from Pr. Harald Stenmark (Department of Molecular Cell Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo, Norway). The plasmids coding for the following protein products were obtained from Addgene: non-fluorescent FRB-ECFP(W66A)-Giantin [# 67903, pEGFP-C1; Pr. Dorus Gadella (van Unen et al., 2015)]; mRFP-PJ-Sac -FKBP [Pseudojanin (PJ)-Sac is a mutated chimeric phosphatase which dephosphorylates PI4P to produce PI] [# 38000, pmRFP-C1; Dr.

  . For all PI4KII knockdown experiments, expressions of PI4KIIa and PI4KIIb were extinguished by using a mix of individual siRNAs targeting PI4KIIa or PI4KIIb.Antibodies and other reagentsPrimary antibodies: sheep polyclonal anti-TGN46 (Biorad; 1:200, IFM); mouse monoclonal anti-β-actin (Sigma A5316; 1:1000, IB); rabbit polyclonal anti-PI4KIIβ (1:1000, IB) and -PI4KIIα(1:1000, IB) were generous gifts from Pr. Pietro De Camilli (Yale School of Medicine, New Haven, USA); rabbit polyclonal anti-b-tubulin (Abcam ab6046; 1:1000, IB); rabbit polyclonal anti-Pallidin (Proteintech 10891-1-AP; 1:1000, IB); goat polyclonal anti-VPS35 (Abcam ab1099; 1:1000, IB); mouse monoclonal anti-SNX1 (BD Bioscience #611482; 1:1000, IB); mouse monoclonal anti-SNX2 (BD Bioscience #611308; 1:1000, IB); rabbit polyclonal anti-acalnexin (Enzo Life Sciences ADI-SPA-860D; 1:1000, IB); rabbit polyclonal anit-RAB11A (Proteintech #15903-1-AP; 1:200, IFM); mouse monoclonal anti-NP (Abcam ab20343; 1:1000, IF); rabbit polyclonal anti-NP (1:1000), -PB1 (v19/6; 1:500), -PB2 (2N580; 1:500), -PA (1:500) and -NS1 (v29; 1:500) for IB were kindly provided by Pr. Paul Digard (Roslin Institute, UK); mouse monoclonal anti-M2 (Abcam ab5416, clone 14C2; 1:500, IB); mouse monoclonal antiactin (Sigma #A5441; 1:1000, IB); goat anti-M1 (Abcam ab20910; 1:500, IB); goat polyclonal anti-GST (GE healthcare #27-4577-01; 1:2000, IB); rabbit polyclonal anti-Cap1 [1 µg/ml, prepared as described in[START_REF] Hamaoui | The Chlamydia effector CT622/TaiP targets a nonautophagy related function of ATG16L1[END_REF]].Secondary antibodies: HRP-conjugated goat anti-mouse, -rabbit, and -donkey (Abcamab6721 and ab6789; 1:10000, and Thermo Fisher Scientific A15999; 1:2000,IB), or donkey anti-goat (Thermo Fisher Scientific A15999; 1:2000, IB in PIP binding assay); fluorescent antibodies from IRDye range (LI-COR Biosciences; 1:10000, IB); Alexa Fluor (AF)-488, -555 or -647-conjugated anti-rabbit, anti-sheep, or anti-mouse (Invitrogen; 1:200, IFM). Other reagents: AF-488 (1:500) labelled-Tf and AF-647 labelled WGA (1:200) were from Invitrogen (Thermo Fisher Scientific); siR-Tubulin (1:1000) was from Spirochrome. PAO (PhenylArsineOxide), wortmannin, GSK-A1, and rapamycin were from Sigma Aldrich and stock solutions were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich). The following reagents for in vitro studies were from Avanti Polar Lipids: Egg phosphatidylcholine (EPC), brain phosphatidylserine (bPS), brain phosphatidylinositol-4-phosphate (bPI4P), 1,2-dioleoylsn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate) 18:1 (PI3P), brain phosphatidylinositol-4,5-bisphosphate [bPI(4,5)P2], C24:1 Galactosyl(b) Ceramide (GalCer), DOPS (1,2-dioleoylsn-glycero-3-phospho-L-serine). Texas Red™ 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine, triethylammonium salt (Texas Red™ DHPE) and BODIPY Texas Red ceramide (BODIPY™ TR ceramide) was from Thermo Fisher Scientific.

  10 6 cells) were washed, scrapped and pelleted in cold PBS. Pellets were incubated in cold hypotonic buffer[20 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM EDTA, 250 mM sucrose supplemented with protease inhibitor cocktail (Roche)] for15 min, and homogenized with a 27G syringe (30 x/ sample). Cell Measurement of fluorescence intensity: Fluorescence intensity of Tf labeling was measured using the "Measure" function in imageJ/Fiji on average z-projections from the raw images. For each condition, the intensities were normalized to their respective mean at 0 min of chase (Fig.S5, B and C).Line scan analysis: Hela cells co-expressing mCherry-KIF13B and SidC-EGFP or EGFP-FYVE were analyzed by live SR-FM. Using the image of the mCherry channel, a line of 30 pixels was drawn crossing the mCh-KIF13B tubule and the fluorescence intensities of the mCherry and GFP were measured per pixel using the "Plot Profile" function in imageJ/Fiji and normalized to the maximum value (Fig.1D).Distance analysis: Hela cells co-expressing mCherry-KIF13B and iRFP-RAB5, were analyzed by live SR-FM. Using the merged image of the two channels, the shortest line was drawn between the mCh-KIF13B + tubule and iRFP-RAB5 + vesicle, and the distance in pixels was measured by the "Line" function in imageJ/Fiji (Fig.S1 A). Image processing: Images were treated with Clarify.AI module (deep neural network trained to recognize and remove out-of-focus signal on each plane) proposed by Nikon NIS-Elements software in the NIS.AI (Fig. 1 A, top panel; and Fig. 6 D). Immunoblot quantification: Protein expression levels were quantified using imageJ/Fiji software and intensities were normalized to the respective bands of the loading control or by using the Image Lab software (version 5.2.1, Biorad). Quantifications were performed on 3 at least independent experiments (Fig. 3 B, and Fig. S3 C; bottom panel).

Figure 1 .

 1 Figure 1. PI4P associates with KIF13 + recycling endosomal tubules

Figure 2 .

 2 Figure 2. BLOC-1 in vitro generate tubules from PI4P containing membranes

Figure 5 :

 5 Figure 5: Depletion of PI4KII prevents the initiation and elongation of early endosomal tubules

Figure S1 .

 S1 Figure S1. PI4P and KIF13 do not associate with early sorting endosomes

Figure

  Figure S2. BLOC-1 binding to vesicles and formation of tubules

Figure 2 G

 2 Figure 2 G). Figures are representative of at least three independent experiments. * in E and I are carbon network of the grid. Bars: (D-I, and K) 25 nm; (J) 5 µm.

Figure S3 .

 S3 Figure S3. PI4KII enzymatic activity is required for the formation and stabilization of recycling endosomal tubules

Statistical analysis: Statistical data are represented as mean ± SEM. Graphpad (Prism 7 or 9) was used for all statistical analyses. Either unpaired two-tailed Student's t-test or ordinary Anova test was used, as indicated in the Figure legends. The significant differences noted are between the control and test condition, and only p< 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p< 0.001 ****p < 0.0001).

Acknowledgments

This work was supported by National Institutes of Health grants R01 EY015625 (to M.S.M. and G.R.), GENESPOIR (to C.D.), Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Curie, and the Centre National de la Recherche Scientifique (CNRS), and by the Intramural Program of NICHD, NIH (ZIA-HD001607, to J.S.B.). We thank the Cell and Tissue Imaging core facility (PICT IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). We thank CryoCapCell (France) for providing equipment (HPM Live µ) and expertise in high pressure freezing, as well as Alexis Canette from the IBPS (Institut de Biologie Paris-Seine) electron microscopy core facility with the support of Sorbonne-Université and CNRS. We thank J. Manzi (BMBC,

Author contributions

Competing Interest

The authors declare no competing interest. cDNA and 0.4 µM of each primer. The reaction was performed on an ABI QuantStudio-384 (Applied Biosciences) under the following PCR conditions: Cycle 1 (1 repeat): 95 °C for 2 min; Cycle 2 (40 repeats): 95 °C for 5 s and 60 °C for 30 s; Cycle 3: 95 °C for 5 s and melt curve 65 °C to 95 °C (increment 0.05 °C each 5 s). Data were analyzed using QuantStudioTM Real Time PCR software (v1.1.,Applied Biosciences). Primer sequences used for real-time RT-qPCR were the following: 1) GAPDH -Fw: 5'-CTCTGCTCCTCCTGTTCGAC-3', Rv: 5'-ACCAAATCCGTTGACTCCGAC-3'; 2) PI4KIIa -Fw: 5'-TCTTTCCCGAGCGCATCTAC-3', Rv:

5'-GCAGCCACTTGGTCCACTTA-3'; 3) PI4KIIb -Fw: 5'-AAGCGGGTGCCTATCTTGTG-3', Rv: 5'-TTTTGCACGGTCAATCGCAT-3'; 4) PI4KIIIb -Fw: 5'-TCAGCAGCAACCTGAAACGA-3', Rv: 5'-CAGTCGAACAGGGGAACTGA-3'.

Image analyses and quantifications

KIF13 Tubule quantification: Quantification of the average number of cells with at least one KIF13A + tubule was done manually (Fig. 3, D and H; and Fig. S3, E and G). Quantification of the average length of KIF13A + tubules was carried out using ImageJ/Fiji as described previously [START_REF] Delevoye | BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes[END_REF][START_REF] Dennis | BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery[END_REF]. In short, live cell fluorescent image was converted into binary and skeletonized (Fig. 3 C). The resulting skeletonized image was then analyzed using the integrated plugin 'AnalyzeSkeleton'. The skeletonized image was manually compared with the raw image to exclude false-positive tubules from the analysis. The length and number of tubules were determined after applying a threshold of 8 pixels (1.28 µm). Only tubules longer than 8 px were included in the analysis (Fig. 3 E BLOC-1 addition led to the diseapperance of the spherical shape of the GUV and to the appearance of multiple tubular structures (arrows). See also Figure 2. Bar: 5 µm.