Membrane-shaping Proteins Research Articles

Two plant membrane-shaping reticulon-like proteins play contrasting complex roles in turnip mosaic virus infection.

Positive-sense RNA viruses remodel cellular cytoplasmic membranes as the membranous sources for the formation of viral replication organelles (VROs) for viral genome replication. In plants, they traffic through plasmodesmata (PD), plasma membrane-lined pores enabling cytoplasmic connections between cells for intercellular movement and systemic infection. In this study, we employed turnip mosaic virus (TuMV), a plant RNA virus to investigate the involvement of RTNLB3 and RTNLB6, two ER (endoplasmic reticulum) membrane-bending, PD-located reticulon-like (RTNL) non-metazoan group B proteins (RTNLBs) in viral infection. We show that RTNLB3 interacts with TuMV 6K2 integral membrane protein and RTNLB6 binds to TuMV coat protein (CP). Knockdown of RTNLB3 promoted viral infection, whereas downregulation of RTNLB6 restricted viral infection, suggesting that these two RTNLs play contrasting roles in TuMV infection. We further demonstrate that RTNLB3 targets the α-helix motif 42LRKSM46 of 6K2 to interrupt 6K2 self-interactions and compromise 6K2-induced VRO formation. Moreover, overexpression of AtRTNLB3 apparently promoted the selective degradation of the ER and ER-associated protein calnexin, but not 6K2. Intriguingly, mutation of the α-helix motif of 6K2 that is required for induction of VROs severely affected 6K2 stability and abolished TuMV infection. Thus, RTNLB3 attenuates TuMV replication, probably through the suppression of 6K2 function. We also show that RTNLB6 promotes viral intercellular movement but does not affect viral replication. Therefore, the proviral role of RTNLB6 is probably by enhancing viral cell-to-cell trafficking. Taken together, our data demonstrate that RTNL family proteins may play diverse complex, even opposite, roles in viral infection in plants.

RTN2 deficiency results in an autosomal recessive distal motor neuropathy with lower limb spasticity.

Heterozygous RTN2 variants have been previously identified in a limited cohort of families affected by autosomal dominant spastic paraplegia (SPG12-OMIM:604805) with a variable age of onset. Nevertheless, the definitive validity of SPG12 remains to be confidently confirmed due to the scarcity of supporting evidence. In this study, we identified and validated seven novel or ultra-rare homozygous loss-of-function RTN2 variants in 14 individuals from seven consanguineous families with distal hereditary motor neuropathy (dHMN) using exome, genome and Sanger sequencing coupled with deep-phenotyping. All affected individuals (seven males and seven females, aged 9-50 years) exhibited weakness in the distal upper and lower limbs, lower limb spasticity and hyperreflexia, with onset in the first decade of life. Nerve conduction studies revealed axonal motor neuropathy with neurogenic changes in the electromyography. Despite a slowly progressive disease course, all patients remained ambulatory over a mean disease duration of 19.71 ± 13.70 years. Characterization of Caenorhabditis elegans RTN2 homologous loss-of-function variants demonstrated morphological and behavioural differences compared with the parental strain. Treatment of the mutant with an endoplasmic/sarcoplasmic reticulum Ca2+ reuptake inhibitor (2,5-di-tert-butylhydroquinone) rescued key phenotypic differences, suggesting a potential therapeutic benefit for RTN2-disorder. Despite RTN2 being an endoplasmic reticulum (ER)-resident membrane shaping protein, our analysis of patient fibroblast cells did not find significant alterations in ER structure or the response to ER stress. Our findings delineate a distinct form of autosomal recessive dHMN with pyramidal features associated with RTN2 deficiency. This phenotype shares similarities with SIGMAR1-related dHMN and Silver-like syndromes, providing valuable insights into the clinical spectrum and potential therapeutic strategies for RTN2-related dHMN.

Molecular mechanisms of membrane-shaping proteins on the endoplasmic reticulum

Molecular mechanisms of membrane-shaping proteins on the endoplasmic reticulum

EHBP1 Is Critically Involved in the Dendritic Arbor Formation and Is Coupled to Factors Promoting Actin Filament Formation.

The coordinated action of a plethora of factors is required for the organization and dynamics of membranous structures critically underlying the development and function of cells, organs, and organisms. The evolutionary acquisition of additional amino acid motifs allows for expansion and/or specification of protein functions. We identify a thus far unrecognized motif specific for chordata EHBP1 proteins and demonstrate that this motif is critically required for interaction with syndapin I, an F-BAR domain-containing, membrane-shaping protein predominantly expressed in neurons. Gain-of-function and loss-of-function studies in rat primary hippocampal neurons (of mixed sexes) unraveled that EHBP1 has an important role in neuromorphogenesis. Surprisingly, our analyses uncovered that this newly identified function of EHBP1 did not require the domain responsible for Rab GTPase binding but was strictly dependent on EHBP1's syndapin I binding interface and on the presence of syndapin I in the developing neurons. These findings were underscored by temporally and spatially remarkable overlapping dynamics of EHBP1 and syndapin I at nascent dendritic branch sites. In addition, rescue experiments demonstrated the necessity of two additional EHBP1 domains for dendritic arborization, the C2 and CH domains. Importantly, the additionally uncovered critical involvement of the actin nucleator Cobl in EHBP1 functions suggested that not only static association with F-actin via EHBP1's CH domain is important for dendritic arbor formation but also actin nucleation. Syndapin interactions organize ternary protein complexes composed of EHBP1, syndapin I, and Cobl, and our functional data show that only together these factors give rise to proper cell shape during neuronal development.

Membrane shapers from two distinct superfamilies cooperate in the development of neuronal morphology.

Membrane-shaping proteins are driving forces behind establishment of proper cell morphology and function. Yet, their reported structural and in vitro properties are noticeably inconsistent with many physiological membrane topology requirements. We demonstrate that dendritic arborization of neurons is powered by physically coordinated shaping mechanisms elicited by members of two distinct classes of membrane shapers: the F-BAR protein syndapin I and the N-Ank superfamily protein ankycorbin. Strikingly, membrane-tubulating activities by syndapin I, which would be detrimental during dendritic branching, were suppressed by ankycorbin. Ankycorbin's integration into syndapin I-decorated membrane surfaces instead promoted curvatures and topologies reflecting those observed physiologically. In line with the functional importance of this mechanism, ankycorbin- and syndapin I-mediated functions in dendritic arborization mutually depend on each other and on a surprisingly specific interface mediating complex formation of the two membrane shapers. These striking results uncovered cooperative and interdependent functions of members of two fundamentally different membrane shaper superfamilies as a previously unknown, pivotal principle in neuronal shape development.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

First person – Lu Xu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Lu Xu is first author on ‘ Molecular basis of Climp63-mediated ER lumen spacing’, published in JCS. Lu conducted the research described in this article while a PhD student in Junjie Hu's lab at Nankai University, Tianjin, China. She is now a postdoc in the lab of Xin Bian at Nankai University, where she performs functional studies of membrane-shaping proteins.

Spatiotemporal Live-Cell Analysis of Photoreceptor Outer Segment Membrane Ingestion by the Retinal Pigment Epithelium Reveals Actin-Regulated Scission.

The photoreceptor outer segment (OS) is the phototransductive organelle in the vertebrate retina. OS tips are regularly ingested and degraded by the adjacent retinal pigment epithelium (RPE), offsetting the addition of new disk membrane at the base of the OS. This catabolic role of the RPE is essential for photoreceptor health, with defects in ingestion or degradation underlying different forms of retinal degeneration and blindness. Although proteins required for OS tip ingestion have been identified, spatiotemporal analysis of the ingestion process in live RPE cells is lacking; hence, the literature reflects no common understanding of the cellular mechanisms that affect ingestion. We imaged live RPE cells from mice (both sexes) to elucidate the ingestion events in real time. Our imaging revealed roles for f-actin dynamics and specific dynamic localizations of two BAR (Bin-Amphiphysin-Rvs) proteins, FBP17 and AMPH1-BAR, in shaping the RPE apical membrane as it surrounds the OS tip. Completion of ingestion was observed to occur by scission of the OS tip from the remainder of the OS, with a transient concentration of f-actin forming around the site of imminent scission. Actin dynamics were also required for regulating the size of the ingested OS tip, and the time course of the overall ingestion process. The size of the ingested tip is consistent with the term "phagocytosis." However, phagocytosis usually refers to engulfment of an entire particle or cell, whereas our observations of OS tip scission indicate a process that is more specifically described as "trogocytosis," in which one cell "nibbles" another cell.SIGNIFICANCE STATEMENT The ingestion of the photoreceptor outer segment (OS) tips by the retinal pigment epithelium (RPE) is a dynamic cellular process that has fascinated scientists for 60 years. Yet its molecular mechanisms had not been addressed in living cells. We developed a live-cell imaging approach to investigate OS tip ingestion, and focused on the dynamic participation of actin filaments and membrane-shaping BAR proteins. We observed scission of OS tips for the first time, and were able to monitor local changes in protein concentration preceding, during, and following scission. Our approach revealed that actin filaments were concentrated at the site of OS scission and were required for regulating the size of the ingested OS tip and the time course of the ingestion process.

Intrinsically disordered domains essential for membrane-shaping proteins

Disordered domains, frequently ignored when studying membrane interactions, have a high impact on membrane shape and curvature-inducing protein behavior.

Exploring the eukaryotic Yip and REEP/Yop superfamily of membrane-shaping adapter proteins (MSAPs): A cacophony or harmony of structure and function?

Polytopic cargo proteins are synthesized and exported along the secretory pathway from the endoplasmic reticulum (ER), through the Golgi apparatus, with eventual insertion into the plasma membrane (PM). While searching for proteins that could enhance cell surface expression of olfactory receptors, a new family of proteins termed “receptor expression-enhancing proteins” or REEPs were identified. These membrane-shaping hairpin proteins serve as adapters, interacting with intracellular transport machinery, to regulate cargo protein trafficking. However, REEPs belong to a larger family of proteins, the Yip (Ypt-interacting protein) family, conserved in yeast and higher eukaryotes. To date, eighteen mammalian Yip family members, divided into four subfamilies (Yipf, REEP, Yif, and PRAF), have been identified. Yeast research has revealed many intriguing aspects of yeast Yip function, functions that have not completely been explored with mammalian Yip family members. This review and analysis will clarify the different Yip family nomenclature that have encumbered prior comparisons between yeast, plants, and eukaryotic family members, to provide a more complete understanding of their interacting proteins, membrane topology, organelle localization, and role as regulators of cargo trafficking and localization. In addition, the biological role of membrane shaping and sensing hairpin and amphipathic helical domains of various Yip proteins and their potential cellular functions will be described. Lastly, this review will discuss the concept of Yip proteins as members of a larger superfamily of membrane-shaping adapter proteins (MSAPs), proteins that both shape membranes via membrane-sensing and hairpin insertion, and well as act as adapters for protein-protein interactions. MSAPs are defined by their localization to specific membranes, ability to alter membrane structure, interactions with other proteins via specific domains, and specific interactions/effects on cargo proteins.

Membrane shaping proteins, lipids, and cytoskeleton: Recipe for nascent lipid droplet formation.

Lipid droplets (LDs) are ubiquitous, neutral lipid storage organelles that act as hubs of metabolic processes. LDs are structurally unique with a hydrophobic core that mainly consists of neutral lipids, sterol esters, and triglycerides, enclosed within a phospholipid monolayer. Nascent LD formation begins with the accumulation of neutral lipids in the endoplasmic reticulum (ER) bilayer. The ER membrane proteins such as seipin, LDAF1, FIT, and MCTPs are reported to play an important role in the formation of nascent LDs. As the LDs grow, they unmix from the highly charged ER membrane to form mature LDs. LD biogenesis is an exciting, emerging research area, and herein, we discuss the recent progress in our understanding of the formation of eukaryotic nascent LDs. We focus on the role of ER membrane shaping proteins such as reticulons and reticulon-like proteins, membrane lipids, and cytoskeleton proteins such as septin in the formation of nascent LDs.

Fission Impossible (?)-New Insights into Disorders of Peroxisome Dynamics.

Peroxisomes are highly dynamic and responsive organelles, which can adjust their morphology, number, intracellular position, and metabolic functions according to cellular needs. Peroxisome multiplication in mammalian cells involves the concerted action of the membrane-shaping protein PEX11β and division proteins, such as the membrane adaptors FIS1 and MFF, which recruit the fission GTPase DRP1 to the peroxisomal membrane. The latter proteins are also involved in mitochondrial division. Patients with loss of DRP1, MFF or PEX11β function have been identified, showing abnormalities in peroxisomal (and, for the shared proteins, mitochondrial) dynamics as well as developmental and neurological defects, whereas the metabolic functions of the organelles are often unaffected. Here, we provide a timely update on peroxisomal membrane dynamics with a particular focus on peroxisome formation by membrane growth and division. We address the function of PEX11β in these processes, as well as the role of peroxisome–ER contacts in lipid transfer for peroxisomal membrane expansion. Furthermore, we summarize the clinical phenotypes and pathophysiology of patients with defects in the key division proteins DRP1, MFF, and PEX11β as well as in the peroxisome–ER tether ACBD5. Potential therapeutic strategies for these rare disorders with limited treatment options are discussed.

Morphological Heterogeneity of the Endoplasmic Reticulum within Neurons and Its Implications in Neurodegeneration.

The endoplasmic reticulum (ER) is a multipurpose organelle comprising dynamic structural subdomains, such as ER sheets and tubules, serving to maintain protein, calcium, and lipid homeostasis. In neurons, the single ER is compartmentalized with a careful segregation of the structural subdomains in somatic and neurite (axodendritic) regions. The distribution and arrangement of these ER subdomains varies between different neuronal types. Mutations in ER membrane shaping proteins and morphological changes in the ER are associated with various neurodegenerative diseases implying significance of ER morphology in maintaining neuronal integrity. Specific neurons, such as the highly arborized dopaminergic neurons, are prone to stress and neurodegeneration. Differences in morphology and functionality of ER between the neurons may account for their varied sensitivity to stress and neurodegenerative changes. In this review, we explore the neuronal ER and discuss its distinct morphological attributes and specific functions. We hypothesize that morphological heterogeneity of the ER in neurons is an important factor that accounts for their selective susceptibility to neurodegeneration.

Organelle homeostasis principles: How organelle quality control and inter-organelle crosstalk promote cell survival.

To survive, cells sense their surroundings and adapt to enable homeostasis. Studies dissecting this process reveal organizational principles, including quality-control pathways, changes to organelle shape, and inter-organelle communication, that facilitate metabolic or developmental remodeling. In this issue, several reviews discuss these organelle homeostasis principles and how they are altered in disease.

Key interplay between the co-opted sorting nexin-BAR proteins and PI3P phosphoinositide in the formation of the tombusvirus replicase.

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.

The roles of the diversity of amphipathic lipids in shaping membranes by membrane-shaping proteins.

Lipid compositions of cells differ according to cell types and intracellular organelles. Phospholipids are major cell membrane lipids and have hydrophilic head groups and hydrophobic fatty acid tails. The cellular lipid membrane without any protein adapts to spherical shapes, and protein binding to the membrane is thought to be required for shaping the membrane for various cellular events. Until recently, modulation of cellular lipid membranes was initially shown to be mediated by proteins recognizing lipid head groups, including the negatively charged ones of phosphatidylserine and phosphoinositides. Recent studies have shown that the abilities of membrane-deforming proteins are also regulated by the composition of fatty acid tails, which cause different degrees of packing defects. The binding of proteins to cellular lipid membranes is affected by the packing defects, presumably through modulation of their interactions with hydrophobic amino acid residues. Therefore, lipid composition can be characterized by both packing defects and charge density. The lipid composition regarding fatty acid tails affects membrane bending via the proteins with amphipathic helices, including those with the ArfGAP1 lipid packing sensor (ALPS) motif and via membrane-deforming proteins with structural folding, including those with the Bin-Amphiphysin-Rvs167 (BAR) domains. This review focuses on how the fatty acid tails, in combination with the head groups of phospholipids, affect protein-mediated membrane deformation.

The role of membrane-shaping BAR domain proteins in caveolar invagination: from mechanistic insights to pathophysiological consequences.

The formation of caveolae, bulb-shaped plasma membrane invaginations, requires the coordinated action of distinct lipid-interacting and -shaping proteins. The interdependence of caveolar structure and function has evoked substantial scientific interest given the association of human diseases with caveolar dysfunction. Model systems deficient of core components of caveolae, caveolins or cavins, did not allow for an explicit attribution of observed functional defects to the requirement of caveolar invagination as they lack both invaginated caveolae and caveolin proteins. Knockdown studies in cultured cells and recent knockout studies in mice identified an additional family of membrane-shaping proteins crucial for caveolar formation, syndapins (PACSINs) - BAR domain superfamily proteins characterized by crescent-shaped membrane binding interfaces recognizing and inducing distinct curved membrane topologies. Importantly, syndapin loss-of-function resulted exclusively in impairment of caveolar invagination without a reduction in caveolin or cavin at the plasma membrane, thereby allowing the specific role of the caveolar invagination to be unveiled. Muscle cells of syndapin III KO mice showed severe reductions of caveolae reminiscent of human caveolinopathies and were more vulnerable to membrane damage upon changes in membrane tensions. Consistent with the lack of syndapin III-dependent invaginated caveolae providing mechanoprotection by releasing membrane reservoirs through caveolar flattening, physical exercise of syndapin III KO mice resulted in pathological defects reminiscent of the clinical symptoms of human myopathies associated with caveolin 3 mutation suggesting that the ability of muscular caveolae to respond to mechanical forces is a key physiological process.

Phospholipid Scramblases Remodel the Shape of Asymmetric Membranes.

The cell membrane and many organellar membranes are asymmetric and highly curved. In experiments, it is challenging to reconstitute and characterize membranes that differ in the lipid composition of their leaflets. Here we use molecular dynamics simulations to study the large-scale membrane shape changes associated with lipid shuttling between asymmetric leaflets. We exploit leaflet asymmetry to create a stable, near-spherical vesicle bud connected to a flat bilayer under periodic boundary conditions. Then we demonstrate how the lipid scramblase nhTMEM16 relaxes the lipid-number asymmetry. By mediating the flipping of lipids, this transmembrane protein dissipates the mechanochemical gradient between the leaflets and drives a large-scale membrane reorganization, converting the vesicle bud into a flat membrane. Our procedure to exploit bilayer asymmetry for simulations of highly curved membranes can be used to study the function of other lipid transporters and membrane-shaping proteins.

Ankyrin repeat-containing N-Ank proteins shape cellular membranes.

Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from understood-mainly because knowledge of membrane-shaping proteins that can promote local membrane curvatures is still limited. Our analyses unveiled that several members of a large, previously unrecognised protein family, which we termed N-Ank proteins, use a combination of their ankyrin repeat array and an amino (N)-terminal amphipathic helix to bind and shape membranes. Consistently, functional analyses revealed that the N-Ank protein ankycorbin (NORPEG/RAI14), which was exemplarily characterised further, plays an important, ankyrin repeat-based and N-terminal amphipathic helix-dependent role in early morphogenesis of neurons. This function furthermore required coiled coil-mediated self-assembly and manifested as ankycorbin nanodomains marked by protrusive membrane topologies. In summary, here, we unveil a class of powerful membrane shapers and thereby assign mechanistic and cell biological functions to the N-Ank protein superfamily.

Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin's dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.