Novel Components of the Stress Assembly Sec Body Identified by Proximity Labeling.

Sec bodies are membraneless stress-induced assemblies that form by the coalescence of endoplasmic reticulum exit sites (ERES). Through APEX2 tagging of Sec24AB, we biotinylated and identified the full complement of Sec body proteins. In the presence of biotin-phenol and H2O2 (APEX on), APEX2 facilitates the transfer of a biotin moiety to nearby interactors of chimeric Sec24AB. Using this unbiased approach comparing APEX on and off (-H2O2) conditions, we identified 52 proteins specifically enriched in Sec bodies. These include a large proportion of ER and Golgi proteins, packaged without defined stoichiometry, which we could selectively verify by imaging. Interestingly, Sec body components are neither transcriptionally nor translationally regulated under the conditions that induce Sec body formation, suggesting that incorporation of these proteins into granules may be driven instead by the aggregation of nucleating proteins with a high content of intrinsically disordered regions. This reinforces the notion that Sec bodies may act as storage for ERES, ER and Golgi components during stress.

Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.

Phase separation of components of ER exit sites (ERES) into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon exposure to specific cellular stressors, namely, salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Finally, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo.

Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.

Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. Here, we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt. This article has an associated First Person interview with the first author of the paper.

Hherisomes, Hedgehog specialized recycling endosomes, are required for high level Hedgehog signaling and tissue growth.

In metazoans, tissue growth and patterning is partly controlled by the Hedgehog (Hh) morphogen. Using immuno-electron microscopy on Drosophila wing imaginal discs, we identified a cellular structure, the Hherisomes, which contain the majority of intracellular Hh. Hherisomes are recycling tubular endosomes, and their formation is specifically boosted by overexpression of Hh. Expression of Rab11, a small GTPase involved in recycling endosomes, boosts the size of Hherisomes and their Hh concentration. Conversely, increased expression of the transporter Dispatched, a regulator of Hh secretion, leads to their clearance. We show that increasing Hh density in Hherisomes through Rab11 overexpression enhances both the level of Hh signaling and disc pouch growth, whereas Dispatched overexpression decreases high-level Hh signaling and growth. We propose that, upon secretion, a pool of Hh triggers low-level signaling, whereas a second pool of Hh is endocytosed and recycled through Hherisomes to stimulate high-level signaling and disc pouch growth. Altogether, our data indicate that Hherisomes are required to sustain physiological Hh activity necessary for patterning and tissue growth in the wing disc.

The GTPase Rab8 differentially controls the long- and short-range activity of the Hedgehog morphogen gradient by regulating Hedgehog apico-basal distribution.

The Hedgehog (Hh) morphogen gradient is required for patterning during metazoan development, yet the mechanisms involved in Hh apical and basolateral release and how this influences short- and long-range target induction are poorly understood. We found that depletion of the GTPase Rab8 in Hh-producing cells induces an imbalance between the level of apically and laterally released Hh. This leads to non-cell-autonomous differential effects on the expression of Hh target genes, namely an increase in its short-range targets and a concomitant decrease in long-range targets. We further found that Rab8 regulates the endocytosis and apico-basal distribution of Ihog, a transmembrane protein known to bind to Hh and to be crucial for establishment of the Hh gradient. Our data provide new insights into morphogen gradient formation, whereby morphogen activity is functionally distributed between apically and basolaterally secreted pools.

Vps13 is required for timely removal of nurse cell corpses.

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.

The function of GORASPs in Golgi apparatus organization in vivo.

In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.

Membrane-Bound Meet Membraneless in Health and Disease.

Membraneless organelles (MLOs) are defined as cellular structures that are not sealed by a lipidic membrane and are shown to form by phase separation. They exist in both the nucleus and the cytoplasm that is also heavily populated by numerous membrane-bound organelles. Even though the name membraneless suggests that MLOs are free of membrane, both membrane and factors regulating membrane trafficking steps are emerging as important components of MLO formation and function. As a result, we name them biocondensates. In this review, we examine the relationships between biocondensates and membrane. First, inhibition of membrane trafficking in the early secretory pathway leads to the formation of biocondensates (P-bodies and Sec bodies). In the same vein, stress granules have a complex relationship with the cyto-nuclear transport machinery. Second, membrane contributes to the regulated formation of phase separation in the cells and we will present examples including clustering at the plasma membrane and at the synapse. Finally, the whole cell appears to transit from an interphase phase-separated state to a mitotic diffuse state in a DYRK3 dependent manner. This firmly establishes a crosstalk between the two types of cell organization that will need to be further explored.

Cellular stress leads to the formation of membraneless stress assemblies in eukaryotic cells.

In cells at steady state, two forms of cell compartmentalization coexist: membrane-bound organelles and phase-separated membraneless organelles that are present in both the nucleus and the cytoplasm. Strikingly, cellular stress is a strong inducer of the reversible membraneless compartments referred to as stress assemblies. Stress assemblies play key roles in survival during cell stress and in thriving of cells upon stress relief. The two best studied stress assemblies are the RNA-based processing-bodies (P-bodies) and stress granules that form in response to oxidative, endoplasmic reticulum (ER), osmotic and nutrient stress as well as many others. Interestingly, P-bodies and stress granules are heterogeneous with respect to both the pathways that lead to their formation and their protein and RNA content. Furthermore, in yeast and Drosophila, nutrient stress also leads to the formation of many other types of prosurvival cytoplasmic stress assemblies, such as metabolic enzymes foci, proteasome storage granules, EIF2B bodies, U-bodies and Sec bodies, some of which are not RNA-based. Nutrient stress leads to a drop in cytoplasmic pH, which combined with posttranslational modifications of granule contents, induces phase separation.

The Upper Hand of the Otu Amyloid Fibers: Increasing Enzymatic Activity and Prolonging Lifespan.

The formation of amyloid fibers is usually associated with aging and neurodegeneration. In this issue of Molecular Cell, Ji et al. (2019) demonstrate that the deubiquitinase Otu coalesces into amyloid-like fibers to enhance its activity and ensure its optimum biological function.

COPII vesicles and the expansion of the phagophore.

A new study has identified the proteins that adapt COPII vesicles to the needs of starving cells.

TMEM59 potentiates Wnt signaling by promoting signalosome formation.

Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.

Modulation of the secretory pathway by amino-acid starvation.

As a major anabolic pathway, the secretory pathway needs to adapt to the demands of the surrounding environment and responds to different exogenous signals and stimuli. In this context, the transport in the early secretory pathway from the endoplasmic reticulum (ER) to the Golgi apparatus appears particularly regulated. For instance, protein export from the ER is critically stimulated by growth factors. Conversely, nutrient starvation also modulates functions of the early secretory pathway in multiple ways. In this review, we focus on amino-acid starvation and how the function of the early secretory pathway is redirected to fuel autophagy, how the ER exit sites are remodeled into novel cytoprotective stress assemblies, and how secretion is modulated in vivo in starving organisms. With the increasingly exciting knowledge on mechanistic target of rapamycin complex 1 (mTORC1), the major nutrient sensor, it is also a good moment to establish how the modulation of the secretory pathway by amino-acid restriction intersects with this major signaling hub.

Detection of mRNA and Associated Molecules by ISH-IEM on Frozen Sections.

The use of tagged RNA probes to directly hybridize frozen sections of chemically fixed tissues, followed by the tag detection with specific antibodies and gold conjugates form the core of the in situ hybridization (ISH)-immunoelectron microscopy (IEM) method that we have developed and successfully used to detect endogenous gurken and bicoid mRNAs in Drosophila oocytes.

Retriever fetches integrins from endosomes.

Recycling from endosomes to the plasma membrane is an important step in cell homeostasis. The retromer/SNX27/WASH complex recycles numerous receptors, but key ones are still unaccounted for. Now a related conserved heterotrimer, called retriever, has been identified that, together with SNX17, the CCC complex and WASH, mediates the recycling of α5ß1 integrins.