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The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH,RetentionUsingSelectiveHooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting Golgi ∼2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.

Biomolecules in the secretory pathway use membrane trafficking for reaching their final intracellular destination or for secretion outside the cell. This highly dynamic and multipartite process involves different organelles that communicate to one another while maintaining their identity, shape, and function. Recent studies unraveled new mechanisms of interorganelle communication that help organize the early secretory pathway. We highlight how the spatial proximity between endoplasmic reticulum (ER) exit sites and early Golgi elements provides novel means of ER-Golgi communication for ER export. We also review recent findings on how membrane contact sites between the ER and the trans-Golgi membranes can sustain anterograde traffic out of the Golgi complex.

Liquid-liquid phase separation (LLPS) is emerging as a key physical principle for biological organization inside living cells, forming condensates that play important regulatory roles. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify the transcriptional output of expressed genes. However, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single-molecule imaging, theory, and simulations to investigate the physical properties of TF condensates of the progesterone receptor (PR) in living cells. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable LLPS process. Using a machine learning architecture, we found that receptor diffusion within condensates follows fractional Brownian motion resulting from viscoelastic interactions with chromatin. Interestingly, condensate growth dynamics at shorter times is dominated by Brownian motion coalescence (BMC), followed by a growth plateau at longer timescales that result in nanoscale condensate sizes. To rationalize these observations, we extended on the BMC model by including the stochastic unbinding of particles within condensates. Our model reproduced the BMC behavior together with finite condensate sizes at the steady state, fully recapitulating our experimental data. Overall, our results are consistent with condensate growth dynamics being regulated by the escaping probability of PR molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other nuclear condensates and could also operate in multiple biological scenarios. The research leading to these results has received funding from BIST-Ignite funding (PHASE-CHROM) (to C.R.-A. and J.A.T.-P.); the European Commission H2020 Program under grant agreement ERC Adv788546 (NANO-MEMEC) (to M.F.G.-P.), ERC AdG NOQIA and EU Horizon 2020 FET-OPEN OPTOlogic (Grant No 899794) (to M.L.), and ERC Synergy Grant 609989 (to M.B.); the Government of Spain (Severo Ochoa CEX2019-000910-S (to M.L. and to M.F.G.P.), JdC-IJCI-2017-33160 (to J.A.T.-P.), (PGC2018-097027-B-I00/10.13039/501100011033, CEX2019-000910-S/10.13039/501100011033 and QUSPIN RTC2019-007196-7) (to M.L.), the State Research Agency (FIDEUA PID2019-106901GB-I00/10.13039/501100011033) (to M.L.), PID2020-113068RB-I00/10.13039/501100011033 (to M.F.G.-P.) and (RYC-2017–22227 and PID2019-106232RB-I00/10.13039/501100011033) (to F.C.); QuantumCAT_U16-011424 (to M.L.), co-funded by the ERDF Operational Program of Catalonia 2014-2020; (QUANTERA MAQS (funded by the State Research Agency under PCI2019-111828-2/321 10.13039/501100011033) and QUANTERA DYNAMITE PCI2022-132919) (to M.L.); Barcelona Supercomputing Center MareNostrum (FI-2022-1-0042) (to M.L.); Obra Social La Caixa (LCF-ICFO) and the Austrian Science Fund (FWF) through SFB BeyondC F7102 (to G.M.-G.); National Science Centre, Poland (Symfonia Grant No. 2016/20/W/ST4/00314) (to M.L.); Fundació CELLEX (Barcelona); Fundació Mir-Puig; and the Generalitat de Catalunya through the CERCA program and AGAUR (grant no. 2017 SGR 1341 to M.L. and no. 2017SGR1000 to M.F.G.-P.).

SLC30A9encodes a ubiquitously zinc transporter (ZnT9) and has been consistently suggested as a candidate for positive selection in humans. However, no direct adaptive molecular phenotype has been demonstrated to date. Our results provide evidence for directional selection operating in two major complementary haplotypes in Africa and East Asia. These haplotypes are associated with differential gene expression but also differ in the Met50Val substitution (rs1047626) in ZnT9, which we show was adaptively introgressed from archaic humans. Although we found no significant differences in systemic zinc content between individuals with different rs1047626 genotypes, we demonstrate that the expression of the derived isoform (ZnT9 50Val) in HEK293 cells shows a gain of function when compared with the ancestral (ZnT9 50Met) variant. In particular, the ZnT9 50Val variant was found to avoid zinc overload in the endoplasmic reticulum and mitochondria, with an impact on mitochondrial metabolism. Overall, our results show that this adaptatively introgressed variant, which is prevalent in East Asians and present at intermediate-high frequencies in other non-African populations, is associated with functional differences in zinc handling by the mitochondria and endoplasmic reticulum. Given the essential role of the mitochondria in both skeletal muscle and brain, and that of zinc in glutamatergic neurotransmission, we propose that archaic adaptation to cold may have driven this selection event, while also impacting susceptibility to neuropsychiatric disorders in modern humans.

Lateral compartmentalization of the plasma membrane is a prominent feature present at multiple spatiotemporal scales that regulates key cellular functions. The extracellular glycocalyx matrix has recently emerged as an important player that modulates the organization of specific receptors and patterns the lipid bilayer itself. However, experimental limitations in investigating its impact on the membrane nanoscale dynamics have hampered detailed studies. Here, we used photonic nanoantenna arrays combined with fluorescence correlation spectroscopy to investigate the influence of hyaluronic acid (HA), a prominent glycosaminoglycan, on the nanoscale organization of mimetic lipid bilayers. Using atomic force microscopy and force spectroscopy, we further correlated our dynamic measurements with the morphology and mechanical properties of bilayers at the nanoscale. Overall, we find that HA has a profound effect on the dynamics, nanoscale organization, and mechanical properties of lipid bilayers that are enriched in sphingolipids and/or cholesterol, such as those present in living cells.

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DC) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the transmembrane protein TGN46 as a cargo receptor for the export of secretory proteins in CARTS – a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the TGN46 lumenal domain encodes for its cargo sorting function. Interestingly, this domain forms liquid droplets in vitro by means of liquid-liquid phase separation, a physical mechanism that may assist in TGN46-mediated cargo sorting. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN. These findings could open new therapeutic avenues for diseases that parallel secretory malfunction.

Liquid-liquid phase separation (LLPS) is emerging as key physical principle for biological organization inside living cells, forming condensates that play important roles in the regulation of multiple functions. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify transcriptional output of expressed genes. Yet, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single molecule imaging tools, theory and simulations to investigate the physical properties of TF condensates of the Progesterone Receptor (PR) in vivo. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable and regulated LLPS process. Using a machine learning architecture, we uncovered that diffusion within condensates follows fractional Brownian motion, reflecting viscoelastic interactions between PR and chromatin within condensates. High density single molecule localization maps further revealed that condensate growth dynamics is dominated by Brownian motion coalescence (BMC) at shorter times, but deviate at longer timescales reaching a growth plateau with nanoscale condensate sizes. To understand our observations we developed an extension of the BMC model by including stochastic unbinding of particles within condensates. The model reproduced the BMC behavior together with finite condensate sizes a steady-state, fully recapitulating our experimental data. Our results are thus consistent with droplet growth dynamics being regulated by the escaping probability of TFs molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other TF condensates and could also operate in multiple biological scenarios.

TANGO1 protein facilitates the endoplasmic reticulum (ER) export of large cargoes that cannot be accommodated in 60 nm transport vesicles. It assembles into a ring in the plane of the ER membrane to create a distinct domain. Its lumenal portion collects and sorts folded cargoes while its cytoplasmic domains collar COPII coats, recruit retrograde COPI-coated membranes that fuse within the TANGO1 ring, thus opening a tunnel for cargo transfer from the ER into a growing export conduit. This mode of cargo transfer bypasses the need for vesicular intermediates and is used to export the most abundant and bulky cargoes. The evolution of TANGO1 and its activities defines the difference between yeast and animal early secretory pathways. Vivek Malhotra thanks Felix Goñi for keeping him sane in Spain. We acknowledge the Spanish Ministry of Science and Innovation for its support of the European Molecular Biology Laboratory partnership, the Centro de Excelencia Severo Ochoa, and the Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya. We acknowledge financial support from the following sources: Ministerio de Economía y Competitividad (SEV-2012-0208, BFU2013-44188-P, and CSD2009- 00016 to V.M. and IJCI-2017-34751 to I.R.). FC acknowledges support from the Government of Spain (RYC-2017-22227, PID2019-106232RB-I00/10.13039/501100011033; Severo Ochoa CEX2019-000910-S), Fundació Cellex, Fundació Mir-Puig, and Generalitat de Catalunya (CERCA, AGAUR).

We introduce an innovative design of planar plasmonic nanogap antenna arrays and demonstrate its potential to study the spatiotemporal organization of mimetic biological membranes at the nanoscale. We exploit our novel nanogap antenna platform with different nanogap sizes (10-45 nm) combined with fluorescence correlation spectroscopy to reveal the existence of nanoscopic domains in mimetic biological membranes. Our approach takes advantage of the highly enhanced and confined excitation light provided by the antennas together with their extreme planarity to investigate membrane regions as small as 10 nm in size with microsecond temporal resolution. We first demonstrate the ultra-high confinement of photonic antennas on biological membranes. Moreover, we show that cholesterol slows down the diffusion of individual fluorescent molecules embedded in the lipid bilayer, consistent with the formation of nanoscopic domains enriched by cholesterol. Incorporation of hyaluronic acid (HA) to the ternary lipid mixture further slows down molecular diffusion, suggesting a synergistic effect of cholesterol and HA on the dynamic partitioning of mimetic biological membranes.

Wakana et al. describe a new role for SCAP, a key regulatory protein in cholesterol metabolism, in promoting Golgi PI4P turnover and the biogenesis of the TGN-derived transport carriers CARTS via interaction with the cholesterol/PI4P exchange machinery at ER–Golgi membrane contact sites., In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP–OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER–Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP–OSBP complex. As in the case of perturbation of lipid transfer complexes at ER–Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network–derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport–defective mutant, but not of cholesterol sensing–defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER–Golgi MCSs, depending on the ER cholesterol.

The endoplasmic reticulum (ER)-resident protein TANGO1 assembles into a ring around ER exit sites (ERES), and links procollagens in the ER lumen to COPII machinery, tethers, and ER-Golgi intermediate compartment (ERGIC) in the cytoplasm (Raote et al., 2018). Here, we present a theoretical approach to investigate the physical mechanisms of TANGO1 ring assembly and how COPII polymerization, membrane tension, and force facilitate the formation of a transport intermediate for procollagen export. Our results indicate that a TANGO1 ring, by acting as a linactant, stabilizes the open neck of a nascent COPII bud. Elongation of such a bud into a transport intermediate commensurate with bulky procollagens is then facilitated by two complementary mechanisms: (i) by relieving membrane tension, possibly by TANGO1-mediated fusion of retrograde ERGIC membranes and (ii) by force application. Altogether, our theoretical approach identifies key biophysical events in TANGO1-driven procollagen export. M Chabanon, MF Garcia-Parajo and F Campelo acknowledge support from the Government of Spain (FIS2015-63550-R, FIS2017-89560-R, BFU2015-73288-JIN, RYC-2017–22227, and PID2019-106232RB-I00/10.13039/501100011033; Severo Ochoa CEX2019-000910-S), Fundació Cellex, Fundació Mir-Puig, and Generalitat de Catalunya (CERCA, AGAUR), ERC Advanced Grant NANO-MEMEC (GA 788546) and LaserLab 4 Europe (GA 654148). I Raote and V Malhotra acknowledge funding by grants from the Ministerio de Economía, Industria y Competitividad Plan Nacional (BFU2013-44188-P) and Consolider (CSD2009-00016); support of the Spanish Ministry of Economy and Competitiveness, through the Programmes ‘Centro de Excelencia Severo Ochoa 2013–2017’ (SEV-2012–0208) and Maria de Maeztu Units of Excellence in R and D (MDM-2015–0502); and support of the CERCA Programme/Generalitat de Catalunya. I Raote, MF Garcia-Parajo, V Malhotra., and F Campelo acknowledge initial support by a BIST Ignite Grant (eTANGO). I Raote acknowledges support from the Spanish Ministry of Science, Innovation and Universities (IJCI-2017–34751). M Arroyo and N Walani acknowledge the support of the European Research Council (CoG-681434), and M Arroyo that of the Generalitat de Catalunya (2017-SGR-1278 and ICREA Academia prize for excellence in research) and of the Spanish Ministry of Economy and Competitiveness, through the Severo Ochoa Programme (CEX2018-000797- S)

The Golgi complex is the membrane-bound organelle that lies at the center of the secretory pathway. Its main functions are to maintain cellular lipid homeostasis, to orchestrate protein processing and maturation, and to mediate protein sorting and export. These functions are not independent of one another, and they all require that the membranes of the Golgi complex have a well-defined biochemical composition. Importantly, a finely-regulated spatiotemporal organization of the Golgi membrane components is essential for the correct performance of the organelle. In here, we review our current mechanistic and molecular understanding of how Golgi membranes are spatially organized in the lateral and axial directions to fulfill their functions. In particular, we highlight the current evidence and proposed models of intra-Golgi transport, as well as the known mechanisms for the retention of Golgi residents and for the sorting and export of transmembrane cargo proteins. Despite the controversies, conflicting evidence, clashes between models, and technical limitations, the field has moved forward and we have gained extensive knowledge in this fascinating topic. However, there are still many important questions that remain to be completely answered. We hope that this review will help boost future investigations on these issues.

In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum (ER) to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here we show that in cholesterol-fed cells ER-localized SCAP interacts through Sac1 phosphoinositide 4-phosphate (PI4P) phosphatase with a VAP/OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi contact sites. SCAP knockdown inhibited the turnover of PI4P perhaps due to a cholesterol transport defect and altered the subcellular distribution of the VAP/OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi contact sites, SCAP knockdown inhibited the biogenesis of thetrans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP but not cholesterol sensing-defective mutants. Altogether, our findings reveal a new role of SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis at ER-Golgi contact sites, depending on the ER cholesterol.SummarySCAP is the key regulatory protein in cholesterol metabolism. Wakana et al. describe a new role of SCAP in controlling Golgi PI4P turnover and the biogenesis of the Golgi-derived transport carries CARTS via cholesterol/PI4P exchange machinery at ER-Golgi contact sites.

SummaryMembrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. ER-PM MCS are particularly abundant in S. cerevisiae, where approximately half of the PM surface is covered by cortical ER (cER). Several proteins, including Ist2, Scs2/22 and Tcb1/2/3 are implicated in cER formation, but the specific roles of these molecules are poorly understood. Here we use cryo-electron tomography to show that ER-PM tethers are key determinants of cER morphology. In particular, Tcb proteins form peaks of extreme curvature on the cER membrane facing the PM. Semi-quantitative modeling and functional assays suggest that Tcb-mediated cER peaks facilitate the transport of lipids from the cER to the PM, necessary to maintain PM integrity under stress conditions. ER peaks were also present at other MCS, implying that membrane curvature enforcement may be a widespread mechanism to expedite lipid transport at MCS.