Tubular Endoplasmic Reticulum Research Articles

Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo.

Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigate the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KOs in young mice show no major phenotypes; however, combined Fam134b and Fam134c deletion (Fam134b/cdKO), but not the combination including Fam134a deletion, leads to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/cdKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/cdKO mice exhibit expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities are present in cortical ER. Our study unveils the critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.

An Automated Imaging-Based Screen for Genetic Modulators of ER Organisation in Cultured Human Cells.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.

Combinatorial selective ER-phagy remodels the ER during neurogenesis

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

Accumulation of neutral lipids in dystrophic neurites surrounding amyloid plaques in Alzheimer's disease

Alzheimer's disease (AD) is characterized by the formation β-amyloid (Aβ) deposited neuritic plaques. Recent evidence suggests that abnormal lipid metabolism and accumulation could serve as biomarkers for neurodegenerative diseases, including AD. Tubular endoplasmic reticulum protein, reticulon 3 (RTN3), plays a crucial role in the development of neuritic plaque and lipid metabolism in AD brains. In present study, we sought to investigate a potential association between neutral lipid accumulation and AD pathology. BODIPY 500/510 dye was used to label neutral lipid surrounding Aβ plaques in APPNL-G-F mouse and AD postmortem brains samples. Immunofluorescent images were captured using confocal microscope and co-localization between lipid metabolism proteins and neutral lipids were evaluated. Lipid accumulation in Aβ plaque surrounding dystrophic neurites (DNs) was observed in the cortical region of AD mouse models and human AD brain samples. The neutral lipid staining was not co-localized with IBA1-labeled microglia or GFAP-labeled astrocytes, but it was co-labeled with VAMP2 and neurofilament. We further showed that neutral lipids were accumulated in RTN3 immunoreactive DNs. Both the neutral lipids accumulation and RIDNs formation showed age-dependent patterns in surrounding amyloid plaques. Mechanistic studies revealed that RTN3 likely contributes to the enrichment of neutral lipids near plaques by interacting with heat shock cognate protein 70 (HSC70) and diminishing its function in chaperone-mediated lipophagy. Our study provides immunohistochemical evidence of neutral lipids being enriched in DNs near amyloid plaques. Our findings shed light on RTN3-mediaed lipid accumulation in AD neuropathology and provide fresh insights into the role of RTN3 in neurodegenerative diseases.

Dynamic organelle changes and autophagic processes in lily pollen germination

Pollen germination is a crucial process in the life cycle of flowering plants, signifying the transition of quiescent pollen grains into active growth. This study delves into the dynamic changes within organelles and the pivotal role of autophagy during lily pollen germination. Initially, mature pollen grains harbor undifferentiated organelles, including amyloplasts, mitochondria, and the Golgi apparatus. However, germination unveils remarkable transformations, such as the redifferentiation of amyloplasts accompanied by starch granule accumulation. We investigate the self-sustained nature of amylogenesis during germination, shedding light on its association with osmotic pressure. Employing BODIPY 493/503 staining, we tracked lipid body distribution throughout pollen germination, both with or without autophagy inhibitors (3-MA, NEM). Typically, lipid bodies undergo polarized movement from pollen grains into elongating pollen tubes, a process crucial for directional growth. Inhibiting autophagy disrupted this essential lipid body redistribution, underscoring the interaction between autophagy and lipid body dynamics. Notably, the presence of tubular endoplasmic reticulum (ER)-like structures associated with developing amyloplasts and lipid bodies implies their participation in autophagy. Starch granules, lipid bodies, and membrane remnants observed within vacuoles further reinforce the involvement of autophagic processes. Among the autophagy inhibitors, particularly BFA, significantly impede germination and growth, thereby affecting Golgi morphology. Immunogold labeling substantiates the pivotal role of the ER in forming autophagosome-like compartments and protein localization. Our proposed speculative model of pollen germination encompasses proplastid differentiation and autophagosome formation. This study advances our understanding of organelle dynamics and autophagy during pollen germination, providing valuable insights into the realm of plant reproductive physiology.

Enrichment of ER tubule-derived microsomes by differential centrifugation and immunoprecipitation.

The endoplasmic reticulum (ER) is an essential component of the endomembrane system in eukaryotes and plays a crucial role in protein and lipid synthesis, as well as the maintenance of calcium homeostasis. Morphologically, the ER is composed primarily of sheets and tubules. The tubular ER is composed of a network of tubular membrane structures, each with diameters ranging from 30 to 50 nanometers. In recent years, there has been in-depth research on the molecular mechanisms of membrane shaping and membrane fusion of the tubular ER. However, there is still limited understanding of the specific physiological functions of the tubular ER. Here, we report a protocol that combines differential centrifugation and immunoprecipitation to specifically enrich microsomes originating from the tubular ER in yeast. The ER tubule-derived microsomes can be further used for proteomic and lipidomic studies or other biochemical analyses.

Reticulons bind sphingolipids to activate the endoplasmic reticulum cell cycle checkpoint, the ER surveillance pathway

The inheritance of a functional endoplasmic reticulum (ER) is ensured by the ER stress surveillance (ERSU) pathway. Here, we made the unexpected discovery that reticulon 1 (Rtn1) and Yop1, well-known ER-curvature-generating proteins, each possess two sphingolipid-binding motifs within their transmembrane domains and that these motifs recognize the ER-stress-induced sphingolipid phytosphingosine (PHS), resulting in an ER inheritance block. Upon binding PHS, Rtn1/Yop1 accumulate on the ER tubule, poised to enter the emerging daughter cell, and cause its misdirection to the bud scars (i.e., previous cell division sites). Amino acid changes in the conserved PHS-binding motifs preclude Rtn1 or Yop1 from binding PHS and diminish their enrichment on the tubular ER, ultimately preventing the ER-stress-induced inheritance block. Conservation of these sphingolipid-binding motifs in human reticulons suggests that sphingolipid binding to Rtn1 and Yop1 represents an evolutionarily conserved mechanism that enables cells to respond to ER stress.

RTN3 interacted with FABP5 and promoted lipid droplet biogenesis in cardiomyocytes

Abstract Background Lipid droplet (LD) accumulation is a notable feature of obesity-induced cardiomyopathy; however, detailed processes governing cardiac LD content remains poorly understood. Reticulon 3 (RTN3), a tubular endoplasmic reticulum (ER) localized protein, was recently identified as a key modulator of lipid metabolism in adipocytes. But its role in cardiac lipid metabolism and the specific mechanism of RTN3 regulating lipid content are currently unknown. Purpose To investigate the role of RTN3 in cardiac LD accumulation and the mechanism by which RTN3 affects LD metabolism. Methods We labelled LD, free fatty acids (FFA), and nucleus in cardiomyocytes with fluorescent markers and upregulated/downregulated RTN3 expression with adenovirus. The effect of RTN3 on cardiac LD content and biogenesis was detected with confocal microscopy. Then we investigated the interaction partner of RTN3 with co-immunoprecipitation (Co-IP), liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and immunofluorescence staining. The function of the complex which RTN3 formed with its interaction protein was then explored. Results RTN3 overexpression significantly increased LD content in cardiomyocytes, which phenocopied the effects of palmitate stress. In contrast, palmitate-induced LD accumulation was blocked by RTN3 knockdown. Tubular ER, where RTN3 residents, is the primary organelle regulating LD biogenesis. Based on that, we hypothesized that the effect of RTN3 on LD content could be attributed to its direct effect on LD biogenesis in the ER. Further FFA tracing results demonstrated that RTN3 overexpression and palmitate could markedly increase FFA-LD colocalization, suggesting the LD biogenesis was activated, but this increase in colocalization was absent in cardiomyocytes infected with Ad sh-RTN3. Live cell imaging also revealed that upregulation of RTN3 significantly accelerated cardiac LD biogenesis and downregulation of RTN3 suppressed palmitate-induce LD biogenesis. Then we investigated the interaction partner of RTN3 and found that RTN3 formed a complex with fatty acids binding protein 5 (FABP5), which is an intracellular carrier for long-chain FFAs. Mechanistically, RTN3 directly bound with FABP5, which facilitated the directed transport of FFA to ER and thus promoted LD biogenesis. FABP5 knockdown blocked RTN3-mediated LD biogenesis. Moreover, we discovered that the 1st-194th amino acids of RTN3 was responsible for interacting with FABP5 and promoting LD biogenesis. Conclusions Our study reveals a novel mechanism contributing to LD biogenesis in cardiomyocytes. Based on our results, manipulating LD biogenesis by modulating RTN3 may be a potential strategy for treating LD accumulation in obese patients.RTN3 promoted lipid droplet biogenesisRTN3 formed a protein complex with FABP5

Arabidopsis reticulons inhibit ROOT HAIR DEFECTIVE3 to form a stable tubular endoplasmic reticulum network.

The endoplasmic reticulum (ER) is a network of interconnected tubules and sheets stretching throughout the cytoplasm of plant cells. In Arabidopsis (Arabidopsis thaliana), ROOT HAIR DEFECTIVE3 (RHD3) mediates ER tubule fusion, while reticulon proteins induce ER membrane curvature to produce ER tubules. However, it is unclear if and how RHD3-reticulon interplay during the formation of the interconnected tubular ER network. We discovered that RHD3 physically interacts with Arabidopsis reticulon proteins, including reticulon-like protein subfamily B3 (RTNLB3), on ER tubules and at 3-way junctions of the ER. The RTNLB3 protein is widely expressed in Arabidopsis seedlings and localizes to ER tubules. Although the growth of knockout rtnlb3 mutant plants was relatively normal, root hairs of rtnlb3 were shorter than those of wild type. The ER in mature mutant cells was also more sheeted than that in wild type. rhd3 is known to have short roots and root hairs and less branched ER tubules in cells. Interestingly, rtnlb3 genetically antagonizes rhd3 in plant root development and in ER interconnectivity. We show that reticulons including RTNLB3 inhibit the ER fusion activity of RHD3, partly by interfering with RHD3 dimerization. We conclude that reticulon proteins negatively regulate RHD3 to balance its ER fusion activity for the formation of a stable tubular ER network in plant cell growth.

Lunapark deficiency leads to an autosomal recessive neurodevelopmental phenotype with a degenerative course, epilepsy and distinct brain anomalies.

LNPK encodes a conserved membrane protein that stabilizes the junctions of the tubular endoplasmic reticulum network playing crucial roles in diverse biological functions. Recently, homozygous variants in LNPK were shown to cause a neurodevelopmental disorder (OMIM#618090) in four patients displaying developmental delay, epilepsy and nonspecific brain malformations including corpus callosum hypoplasia and variable impairment of cerebellum. We sought to delineate the molecular and phenotypic spectrum of LNPK-related disorder. Exome or genome sequencing was carried out in 11 families. Thorough clinical and neuroradiological evaluation was performed for all the affected individuals, including review of previously reported patients. We identified 12 distinct homozygous loss-of-function variants in 16 individuals presenting with moderate to profound developmental delay, cognitive impairment, regression, refractory epilepsy and a recognizable neuroimaging pattern consisting of corpus callosum hypoplasia and signal alterations of the forceps minor ('ear-of-the-lynx' sign), variably associated with substantia nigra signal alterations, mild brain atrophy, short midbrain and cerebellar hypoplasia/atrophy. In summary, we define the core phenotype of LNPK-related disorder and expand the list of neurological disorders presenting with the 'ear-of-the-lynx' sign suggesting a possible common underlying mechanism related to endoplasmic reticulum-phagy dysfunction.

Reticulons 1 and 3 are essential for axonal growth and synaptic maintenance associated with intellectual development.

Reticulon (RTN) proteins are a family of proteins biochemically identified for shaping tubular endoplasmic reticulum, a subcellular structure important for vesicular transport and cell-to-cell communication. In our recent study of mice with knockout of both reticulon 1 (Rtn1) and Rtn3, we discovered that Rtn1-/-;Rtn3-/- (brief as R1R3dKO) mice exhibited neonatal lethality, despite the fact that mice deficient in either RTN1 or RTN3 alone exhibit no discernible phenotypes. This has been the first case to find early lethality in animals with deletion of partial members of RTN proteins. The complete penetrance for neonatal lethality can be attributed to multiple defects including the impaired neuromuscular junction found in the diaphragm. We also observed significantly impaired axonal growth in a regional-specific manner, detected by immunohistochemical staining with antibodies to neurofilament light chain and neurofilament medium chain. Ultrastructural examination by electron microscopy revealed a significant reduction in synaptic active zone length in the hippocampus. Mechanistic exploration by unbiased proteomic assays revealed reduction of proteins such as FMR1, Staufen2, Cyfip1, Cullin-4B and PDE2a, which are known components in the fragile X mental retardation pathway. Together, our results reveal that RTN1 and RTN3 are required to orchestrate neurofilament organization and intact synaptic structure of the central nervous system.

ERnet: a tool for the semantic segmentation and quantitative analysis of endoplasmic reticulum topology.

The ability to quantify structural changes of the endoplasmic reticulum (ER) is crucial for understanding the structure and function of this organelle. However, the rapid movement and complex topology of ER networks make this challenging. Here, we construct a state-of-the-art semantic segmentation method that we call ERnet for the automatic classification of sheet and tubular ER domains inside individual cells. Data are skeletonized and represented by connectivity graphs, enabling precise and efficient quantification of network connectivity. ERnet generates metrics on topology and integrity of ER structures and quantifies structural change in response to genetic or metabolic manipulation. We validate ERnet using data obtained by various ER-imaging methods from different cell types as well as ground truth images of synthetic ER structures. ERnet can be deployed in an automatic high-throughput and unbiased fashion and identifies subtle changes in ER phenotypes that may inform on disease progression and response to therapy.

Drosophila SPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics.

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+ defects in Rtnl1 mutants. Our results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP.

Insights into membrane association of the SMP domain of extended synaptotagmin

The Synaptotagmin-like Mitochondrial-lipid-binding Protein (SMP) domain is a newly identified lipid transfer module present in proteins that regulate lipid homeostasis at membrane contact sites (MCSs). However, how the SMP domain associates with the membrane to extract and unload lipids is unclear. Here, we performed in vitro DNA brick-assisted lipid transfer assays and in silico molecular dynamics simulations to investigate the molecular basis of the membrane association by the SMP domain of extended synaptotagmin (E-Syt), which tethers the tubular endoplasmic reticulum (ER) to the plasma membrane (PM). We demonstrate that the SMP domain uses its tip region to recognize the extremely curved subdomain of tubular ER and the acidic-lipid-enriched PM for highly efficient lipid transfer. Supporting these findings, disruption of these mechanisms results in a defect in autophagosome biogenesis contributed by E-Syt. Our results suggest a model that provides a coherent picture of the action of the SMP domain at MCSs.

The role of the tubular endoplasmic reticulum in the axonal degeneration associated with neurodegenerative disorders

Neurodegenerative disorders represent a group of aging-related diseases affecting the different parts of the central nervous system. Axonal degeneration is among the leading causes of morbidity and disease progression in Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and other neurogenerative disorders. The unique structures of axons may make them particularly vulnerable to internal homeostasis. The axonal endoplasmic reticulum (ER) has emerged as one of the most important hallmarks in those neurodegenerative disorders associated with dysfunction of axonal transport, lipid synthesis, calcium dynamics, and interactions with other organelles. In this review, we summarize the role of tubular ER and its resident proteins in axonal degeneration, which emerges as an early pathological event in the axonal degeneration process. We also discuss the potential relationship between autophagy and tubular ER. With this review, we can consolidate the recent research advances in the role of tubular ER in axonal degeneration associated with several major neurodegenerative disorders and improve our understanding of axon pathophysiology and potential target therapies.

Endosomal lipid signaling reshapes the endoplasmic reticulum to control mitochondrial function.

Cells respond to fluctuating nutrient supply by adaptive changes in organelle dynamics and in metabolism. How such changes are orchestrated on a cell-wide scale is unknown. We show that endosomal signaling lipid turnover by MTM1, a phosphatidylinositol 3-phosphate [PI(3)P] 3-phosphatase mutated in X-linked centronuclear myopathy in humans, controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes, resulting in the conversion of ER tubules into sheets, the inhibition of mitochondrial fission, and sustained oxidative metabolism. Our results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to fluctuating nutrient environments.

ATL1 inhibits the proliferation and invasion of trophoblast cells via inhibition of the mTOR signaling pathway.

Pre-eclampsia (PE) is a major cause of hypertension in maternal and fetal. Atlastin-1 (ATL1), one regulator of endoplasmic reticulum (ER) morphology, participates in tubular ER formation and protein synthesis. The objective of this study is to investigate the role and molecular mechanism of ATL1 in PE. GEO databases showed that ATL1 was upregulated in PE patients. Our data also found that ATL1 was highly expressed in PE placental tissues. The cell viability, proliferation, migration, and invasion of HTR-8/SVneo cells increased/decreased after the downregulation/upregulation of ATL1. The mTOR pathway is the downstream pathway of ATL1. The levels of p-p70S6K and p-mTOR were increased/decreased after the downregulation/upregulation of ATL1. Moreover, rapamycin, an inhibitor of mTOR pathway, reversed the promotive effect of siATL1 on proliferation, migration, and invasion in HTR-8/SVneo cells. In conclusion, ATL1 inhibits the proliferation and invasion of trophoblast cells via the inhibition of the mTOR signaling pathway in HTR-8/SVneo cells.

Lunapark ubiquitinates atlastin-2 for the tubular network formation of the endoplasmic reticulum.

Endoplasmic reticulum (ER) tubules are interconnected by three-way junctions, resulting in the formation of a tubular ER network. Lunapark (Lnp) localizes to and stabilizes the three-way junctions. The N-terminal cytoplasmic domain in Lnp has a ubiquitin ligase activity. However, the molecular mechanism of how the ubiquitin ligase activity of Lnp is involved in the formation of the tubular ER network remains unknown. In this study, we examined whether the ER membrane proteins responsible for the formation of the tubular ER network are ubiquitinated by Lnp. We found that atlastin-2 (ATL2), an isoform of the ATL family mediating the generation of the three-way junctions by connecting the ER tubules, is a novel substrate for ubiquitination by Lnp. The localization of Lnp at the three-way junctions is important for ubiquitination of ATL2. Lysine 56, 57, 282 and 302 are the potential ubiquitination sites by Lnp. Silencing ATL2 decreased the number of the three-way junctions, and the expression of the ATL2 mutant in which the lysine residues are substituted with arginine failed to rescue the decrease of the three-way junctions in the ATL2 knocked-down cells. These results suggest that Lnp ubiquitinates ATL2 at the three-way junctions for the proper tubular ER network formation.

TLR4 Promotes ER stress and Unfolded Protein Response in Hypertension‐Induced Kidney Injury

Hypertension is the second leading cause of chronic kidney injury in the world. Endoplasmic reticulum (ER) is an important cell organelle that is involved in the synthesis, folding, and modification of various proteins to maintain homeostasis. ER stress is reported in hypertension‐induced damage of kidney, heart and brain. Further, TLR4 activation and signaling is linked to the development and progression of hypertension induced damage in various organs; however, the interplay between TLR4 and ER stress in renal pathology remains unknown. In the present study, we investigated whether TLR4 activation contributes to renal ER stress and inflammation in Angiotensin‐II (Ang‐II) induced hypertension and whether TLR4 deficiency protects the kidney by suppressing ER stress. C3H/HeouJ (Normal TLR4, TLR4N) and C3H/HeJ (Dysfunctional TLR4, TLR4M) aged 12‐14 weeks were treated with Ang‐II (1000 ng/Kg/min. x 28 days) using osmotic pumps. We found renal function was impaired in TLR4N mice compared to TLR4M. The expression of ER stress markers (ATF6, p‐IRE1α and p‐PERK), renal inflammation (TNFα, IL‐1β, CCL2) and transcription factor p‐NFkB was upregulated in TLR4N kidneys compared to TLR4M. Further, ER stress in TLR4N mice was associated with mRNA changes in GRP78, sXBP1, ATF4, CHOP genes that are involved in unfolded protein response. In TLR4N mice, renal inflammation was predominant in the tubular area and ER stress was observed in both glomerular and tubular area. TLR4M mice showed reduced ER stress and inflammation in the kidney. Taken together, our results suggest significant interaction between TLR4 and ER stress that may act as an obligatory step in mediating renal inflammation and damage, and TLR4 deficiency attenuates injury by regulating ER stress and unfolded protein response in Ang‐II‐induced hypertension.

Formation of Nascent Lipid Droplets at Specialized ER subdomains

Lipid droplets (LDs) are neutral lipid containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain–containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain of these proteins is similar to a reticulon homology domain. Like reticulons, MCTPs tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the transmembrane domains (TMD) of MCTP promote LD biogenesis by increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER–LD contact sites. Together, our data indicate that MCTPs form specialized subdomains within ER tubules that regulate LD biogenesis, size, and ER–LD contacts. Interestingly, MCTP punctae are associated with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites.