SigmaR1 shapes rough endoplasmic reticulum membrane sheets.

Rough endoplasmic reticulum (ER) sheets are a fundamental domain of the ER and the gateway into the secretory pathway. Although reticulon proteins stabilize high-curvature ER tubules, it is unclear whether other proteins scaffold the flat membranes of rough ER sheets. Through a proteomics screen using ER sheet-localized RNA-binding proteins as bait, we identify the sigma-1 receptor (SigmaR1) as an ER sheet-shaping factor. High-resolution live cell imaging and electron tomography assign SigmaR1 as an ER sheet-localized factor whose levels determine the amount of rough ER sheets in cells. Structure-guided mutagenesis and in vitro reconstitution on giant unilamellar vesicles further support a mechanism whereby SigmaR1 oligomers use their extended arrays of amphipathic helices to bind and flatten the lumenal leaflet of ER membranes to oppose membrane curvature and stabilize rough ER sheets.

Endosome biogenesis is controlled by ER and the cytoskeleton at tripartite junctions.

The plasma membrane (PM) and its associated cargo are internalized into small vesicles via endocytosis funneling cargo into endosomes. The endosomal system must efficiently deliver cargos, as well as recycle cargo receptors and membrane to maintain homeostasis. In animal cells, endosome trafficking, maturation, and cargo recycling rely on the actin and microtubule cytoskeleton. Microtubules and their associated motor proteins provide the roads on which endosomes move and fuse during cargo sorting and delivery. In addition, highly dynamic assemblies of actin adjust the shape of the endosomal membrane to promote cargo segregation into budding domains allowing for receptor recycling. Recent work has revealed that the endoplasmic reticulum (ER) frequently acts as an intermediary between endosomes and their cytoskeletal regulators via membrane contact sites (MCSs). This review will discuss the factors which form these tripartite junction between the ER, endosomes, and the cytoskeleton as well as their function.

An ER phospholipid hydrolase drives ER-associated mitochondrial constriction for fission and fusion.

Mitochondria are dynamic organelles that undergo cycles of fission and fusion at a unified platform defined by endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCSs). These MCSs or nodes co-localize fission and fusion machinery. We set out to identify how ER-associated mitochondrial nodes can regulate both fission and fusion machinery assembly. We have used a promiscuous biotin ligase linked to the fusion machinery, Mfn1, and proteomics to identify an ER membrane protein, ABHD16A, as a major regulator of node formation. In the absence of ABHD16A, fission and fusion machineries fail to recruit to ER-associated mitochondrial nodes, and fission and fusion rates are significantly reduced. ABHD16A contains an acyltransferase motif and an α/ß hydrolase domain, and point mutations in critical residues of these regions fail to rescue the formation of ER-associated mitochondrial hot spots. These data suggest a mechanism whereby ABHD16A functions by altering phospholipid composition at ER-mitochondria MCSs. Our data present the first example of an ER membrane protein that regulates the recruitment of both fission and fusion machineries to mitochondria.

Coronin 1C restricts endosomal branched actin to organize ER contact and endosome fission.

ER contact sites define the position of endosome bud fission during actin-dependent cargo sorting. Disrupting endosomal actin structures prevents retrograde cargo movement; however, how actin affects ER contact site formation and endosome fission is not known. Here we show that in contrast with the WASH complex, actin, its nucleator ARP2/3, and COR1C form a contained structure at the bud neck that defines the site of bud fission. We found that actin confinement is facilitated by type I coronins. Depletion of type I coronins allows actin to extend along the length of the bud in an ARP2/3-dependent manner. We demonstrate that extension of branched actin prevents ER recruitment and stalls buds before fission. Finally, our structure-function studies show that the COR1C's coiled-coil domain is sufficient to restore actin confinement, ER recruitment, and endosome fission. Together, our data reveal how the dynamics of endosomal actin and activity of actin regulators organize ER-associated bud fission.

The ER ladder is a unique morphological feature of developing mammalian axons.

The endoplasmic reticulum (ER) confronts a challenge to accommodate long, smooth ER tubules into the structural complexity of the axonal compartment. Here, we describe a morphological feature for the axonal ER network in developing neurons we termed the ER ladder. Axonal ER ladders are composed of rungs that wrap tightly around the microtubule bundle and dynamic rails, which slide across microtubules. We found that the ER-shaping protein Reticulon 2 determines the architecture and dynamics of the axonal ER ladder by modulating its interaction with microtubules. Moreover, we show that ER ladder depletion impairs the trafficking of associated vesicular axonal cargoes. Finally, we demonstrate that stromal interaction molecule 1 (Stim1) localizes to ER rungs and translocates to ER-plasma membrane contact sites upon depletion of luminal Ca2+. Our findings uncover fundamental insights into the structural and functional organization of the axonal ER network in developing mammalian neurons.

Reticulon-3 Promotes Endosome Maturation at ER Membrane Contact Sites.

ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.

Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport.

Secretory cargo is recognized, concentrated and trafficked from endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo-loaded COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII-coated vesicle. Here, we describe a recently developed live-cell cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We found that components of the COPII coat remain associated with the ERES while cargo is extruded into COPII-uncoated, non-ER associated, Rab1 (herein referring to Rab1a or Rab1b)-dependent carriers. Our data suggest that, in animal cells, COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.This article has an associated First Person interview with the first author of the paper.

Fission and fusion machineries converge at ER contact sites to regulate mitochondrial morphology.

The steady-state morphology of the mitochondrial network is maintained by a balance of constitutive fission and fusion reactions. Disruption of this steady-state morphology results in either a fragmented or elongated network, both of which are associated with altered metabolic states and disease. How the processes of fission and fusion are balanced by the cell is unclear. Here we show that mitochondrial fission and fusion are spatially coordinated at ER membrane contact sites (MCSs). Multiple measures indicate that the mitochondrial fusion machinery, Mitofusins, accumulate at ER MCSs where fusion occurs. Furthermore, fission and fusion machineries colocalize to form hotspots for membrane dynamics at ER MCSs that can persist through sequential events. Because these hotspots can undergo fission and fusion, they have the potential to quickly respond to metabolic cues. Indeed, we discover that ER MCSs define the interface between polarized and depolarized segments of mitochondria and can rescue the membrane potential of damaged mitochondria by ER-associated fusion.

Endoplasmic reticulum contact sites regulate the dynamics of membraneless organelles.

Tethered interactions between the endoplasmic reticulum (ER) and other membrane-bound organelles allow for efficient transfer of ions and/or macromolecules and provide a platform for organelle fission. Here, we describe an unconventional interface between membraneless ribonucleoprotein granules, such as processing bodies (P-bodies, or PBs) and stress granules, and the ER membrane. We found that PBs are tethered at molecular distances to the ER in human cells in a tunable fashion. ER-PB contact and PB biogenesis were modulated by altering PB composition, ER shape, or ER translational capacity. Furthermore, ER contact sites defined the position where PB and stress granule fission occurs. We thus suggest that the ER plays a fundamental role in regulating the assembly and disassembly of membraneless organelles.

A Novel Class of ER Membrane Proteins Regulates ER-Associated Endosome Fission.

Endoplasmic reticulum (ER) membrane contact sites (MCSs) mark positions where endosomes undergo fission for cargo sorting. To define the role of ER at this unique MCS, we targeted a promiscuous biotin ligase to cargo-sorting domains on endosome buds. This strategy identified the ER membrane protein TMCC1, a member of a conserved protein family. TMCC1 concentrates at the ER-endosome MCSs that are spatially and temporally linked to endosome fission. When TMCC1 is depleted, endosome morphology is normal, buds still form, but ER-associated bud fission and subsequent cargo sorting to the Golgi are impaired. We find that the endosome-localized actin regulator Coronin 1C is required for ER-associated fission of actin-dependent cargo-sorting domains. Coronin 1C is recruited to endosome buds independently of TMCC1, while TMCC1/ER recruitment requires Coronin 1C. This link between TMCC1 and Coronin 1C suggests that the timing of TMCC1-dependent ER recruitment is tightly regulated to occur after cargo has been properly sequestered into the bud.

Here, there, and everywhere: The importance of ER membrane contact sites.

Our textbook image of organelles has changed. Instead of revealing isolated cellular compartments, the picture now emerging shows organelles as largely interdependent structures that can communicate through membrane contact sites (MCSs). MCSs are sites where opposing organelles are tethered but do not fuse. MCSs provide a hybrid location where the tool kits of two different organelles can work together to perform vital cellular functions, such as lipid and ion transfer, signaling, and organelle division. Here, we focus on MCSs involving the endoplasmic reticulum (ER), an organelle forming an extensive network of cisternae and tubules. We highlight how the dynamic ER network regulates a plethora of cellular processes through MCSs with various organelles and with the plasma membrane.

SnapShot: Functions of Endoplasmic Reticulum Membrane Contact Sites.

The endoplasmic reticulum (ER) consists of the nuclear envelope and a reticulated interconnected network of tubules and sheets. ER sheets are studded with ribosomes and provide the entryway for proteins into the secretory pathway. ER tubules move dynamically on microtubules and form membrane contact sites with other organelles, where membranes are tethered, but not fused. This Snapshot reviews key biological processes that take place at ER contact sites with the Golgi, endosomes, and mitochondria.

Multiple dynamin family members collaborate to drive mitochondrial division.

Mitochondria cannot be generated de novo; they must grow, replicate their genome, and divide in order to be inherited by each daughter cell during mitosis. Mitochondrial division is a structural challenge that requires the substantial remodelling of membrane morphology. Although division factors differ across organisms, the need for multiple constriction steps and a dynamin-related protein (Drp1, Dnm1 in yeast) has been conserved. In mammalian cells, mitochondrial division has been shown to proceed with at least two sequential constriction steps: the endoplasmic reticulum and actin must first collaborate to generate constrictions suitable for Drp1 assembly on the mitochondrial outer membrane; Drp1 then further constricts membranes until mitochondrial fission occurs. In vitro experiments, however, indicate that Drp1 does not have the dynamic range to complete membrane fission. In contrast to Drp1, the neuron-specific classical dynamin dynamin-1 (Dyn1) has been shown to assemble on narrower lipid profiles and facilitate spontaneous membrane fission upon GTP hydrolysis. Here we report that the ubiquitously expressed classical dynamin-2 (Dyn2) is a fundamental component of the mitochondrial division machinery. A combination of live-cell and electron microscopy in three different mammalian cell lines reveals that Dyn2 works in concert with Drp1 to orchestrate sequential constriction events that build up to division. Our work underscores the biophysical limitations of Drp1 and positions Dyn2, which has intrinsic membrane fission properties, at the final step of mitochondrial division.

Structure and function of ER membrane contact sites with other organelles.

The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

ER contact sites define the position and timing of endosome fission.

Endocytic cargo and Rab GTPases are segregated to distinct domains of an endosome. These domains maintain their identity until they undergo fission to traffic cargo. It is not fully understood how segregation of cargo or Rab proteins is maintained along the continuous endosomal membrane or what machinery is required for fission. Endosomes form contact sites with the endoplasmic reticulum (ER) that are maintained during trafficking. Here, we show that stable contacts form between the ER and endosome at constricted sorting domains, and free diffusion of cargo is limited at these positions. We demonstrate that the site of constriction and fission for early and late endosomes is spatially and temporally linked to contact sites with the ER. Lastly, we show that altering ER structure and dynamics reduces the efficiency of endosome fission. Together, these data reveal a surprising role for ER contact in defining the timing and position of endosome fission.

ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast.

Mitochondrial division is important for mitochondrial distribution and function. Recent data have demonstrated that ER-mitochondria contacts mark mitochondrial division sites, but the molecular basis and functions of these contacts are not understood. Here we show that in yeast, the ER-mitochondria tethering complex, ERMES, and the highly conserved Miro GTPase, Gem1, are spatially and functionally linked to ER-associated mitochondrial division. Gem1 acts as a negative regulator of ER-mitochondria contacts, an activity required for the spatial resolution and distribution of newly generated mitochondrial tips following division. Previous data have demonstrated that ERMES localizes with a subset of actively replicating mitochondrial nucleoids. We show that mitochondrial division is spatially linked to nucleoids and that a majority of these nucleoids segregate prior to division, resulting in their distribution into newly generated tips in the mitochondrial network. Thus, we postulate that ER-associated division serves to link the distribution of mitochondria and mitochondrial nucleoids in cells. DOI:http://dx.doi.org/10.7554/eLife.00422.001.

Endoplasmic reticulum structure and interconnections with other organelles.

The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.

Endoplasmic reticulum-endosome contact increases as endosomes traffic and mature.

The endosomal pathway is responsible for plasma membrane cargo uptake, sorting, and, in many cases, lysosome targeting. Endosome maturation is complex, requiring proper spatiotemporal recruitment of factors that regulate the size, maturity, and positioning of endosomal compartments. In animal cells, it also requires trafficking of endosomes on microtubules. Recent work has revealed the presence of contact sites between some endosomes and the endoplasmic reticulum (ER). Although these contact sites are believed to have multiple functions, the frequency, dynamics, and physical attributes of these contacts are poorly understood. Here we use high-resolution three-dimensional electron microscopy to reveal that ER tubules wrap around endosomes and find that both organelles contact microtubules at or near membrane contact sites. As endosomes traffic, they remain bound to the ER, which causes the tubular ER to rearrange its structure around dynamic endosomes at contact sites. Finally, as endosomes transition through steps of maturation, they become more tightly associated with the ER. The major implication of these results is that endosomes mature and traffic while coupled to the ER membrane rather than in isolation.