The Lateral Organization and Mobility of Plasma Membrane Components.

Over the last several decades, an impressive array of advanced microscopic and analytical tools, such as single-particle tracking and nanoscopic fluorescence correlation spectroscopy, has been applied to characterize the lateral organization and mobility of components in the plasma membrane. Such analysis can tell researchers about the local dynamic composition and structure of membranes and is important for predicting the outcome of membrane-based reactions. However, owing to the unresolved complexity of the membrane and the structures peripheral to it, identification of the detailed molecular origin of the interactions that regulate the organization and mobility of the membrane has not proceeded quickly. This Perspective presents an overview of how cell-surface structure may give rise to the types of lateral mobility that are observed and some potentially fruitful future directions to elucidate the architecture of these structures in more molecular detail.

CerS6-Derived Sphingolipids Interact with Mff and Promote Mitochondrial Fragmentation in Obesity.

Ectopic lipid deposition and altered mitochondrial dynamics contribute to the development of obesity and insulin resistance. However, the mechanistic link between these processes remained unclear. Here we demonstrate that the C16:0 sphingolipid synthesizing ceramide synthases, CerS5 and CerS6, affect distinct sphingolipid pools and that abrogation of CerS6 but not of CerS5 protects from obesity and insulin resistance. We identify proteins that specifically interact with C16:0 sphingolipids derived from CerS5 or CerS6. Here, only CerS6-derived C16:0 sphingolipids bind the mitochondrial fission factor (Mff). CerS6 and Mff deficiency protect from fatty acid-induced mitochondrial fragmentation in vitro, and the two proteins genetically interact in vivo in obesity-induced mitochondrial fragmentation and development of insulin resistance. Our experiments reveal an unprecedented specificity of sphingolipid signaling depending on specific synthesizing enzymes, provide a mechanistic link between hepatic lipid deposition and mitochondrial fragmentation in obesity, and define the CerS6-derived sphingolipid/Mff interaction as a therapeutic target for metabolic diseases.

LAP2 Proteins Chaperone GLI1 Movement between the Lamina and Chromatin to Regulate Transcription.

Understanding transcription factor navigation through the nucleus remains critical for developing targeted therapeutics. The GLI1 transcription factor must maintain maximal Hedgehog pathway output in basal cell carcinomas (BCCs), and we have previously shown that resistant BCCs increase GLI1 deacetylation through atypical protein kinase Cι/λ (aPKC) and HDAC1. Here we identify a lamina-associated polypeptide 2 (LAP2) isoform-dependent nuclear chaperoning system that regulates GLI1 movement between the nuclear lamina and nucleoplasm to achieve maximal activation. LAP2ß forms a two-site interaction with the GLI1 zinc-finger domain and acetylation site, stabilizing an acetylation-dependent reserve on the inner nuclear membrane (INM). By contrast, the nucleoplasmic LAP2α competes with LAP2ß for GLI1 while scaffolding HDAC1 to deacetylate the secondary binding site. aPKC functions to promote GLI1 association with LAP2α, promoting egress off the INM. GLI1 intranuclear trafficking by LAP2 isoforms represents a powerful signal amplifier in BCCs with implications for zinc finger-based signal transduction and therapeutics.

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Molecular mechanisms and cellular functions of cGAS-STING signalling.

The cGAS-STING signalling axis, comprising the synthase for the second messenger cyclic GMP-AMP (cGAS) and the cyclic GMP-AMP receptor stimulator of interferon genes (STING), detects pathogenic DNA to trigger an innate immune reaction involving a strong type I interferon response against microbial infections. Notably however, besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including extranuclear chromatin resulting from genotoxic stress and DNA released from mitochondria, placing cGAS-STING as an important axis in autoimmunity, sterile inflammatory responses and cellular senescence. Initial models assumed that co-localization of cGAS and DNA in the cytosol defines the specificity of the pathway for non-self, but recent work revealed that cGAS is also present in the nucleus and at the plasma membrane, and such subcellular compartmentalization was linked to signalling specificity of cGAS. Further confounding the simple view of cGAS-STING signalling as a response mechanism to infectious agents, both cGAS and STING were shown to have additional functions, independent of interferon response. These involve non-catalytic roles of cGAS in regulating DNA repair and signalling via STING to NF-κB and MAPK as well as STING-mediated induction of autophagy and lysosome-dependent cell death. We have also learnt that cGAS dimers can multimerize and undergo liquid-liquid phase separation to form biomolecular condensates that could importantly regulate cGAS activation. Here, we review the molecular mechanisms and cellular functions underlying cGAS-STING activation and signalling, particularly highlighting the newly emerging diversity of this signalling pathway and discussing how the specificity towards normal, damage-induced and infection-associated DNA could be achieved.

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation.

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.

The rhomboid-like superfamily: molecular mechanisms and biological roles.

The rhomboid proteases were first discovered as regulators of Drosophila EGF receptor signaling; soon after, it was recognized that they represented the founder members of a widespread family of intramembrane serine proteases conserved in all kingdoms. More recently still, the family was promoted to a superfamily, encompassing a wide variety of distantly related proteins. One of the surprises has been that many members of the rhomboid-like superfamily are not active proteases. Given the size of this clan, and its relatively recent discovery, there is still much to learn. Nevertheless, we already understand much about how rhomboid proteases perform their surprising function of cleaving transmembrane domains. We also already know that members of the rhomboid-like superfamily participate in biological functions as diverse as growth factor signaling, mitochondrial dynamics, inflammation, parasite invasion, and the machinery of protein quality control. Their potential medical significance is now becoming apparent in several areas.

Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

The ER-Localized Transmembrane Protein TMEM39A/SUSR2 Regulates Autophagy by Controlling the Trafficking of the PtdIns(4)P Phosphatase SAC1.

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1 from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.

Structure, Function, and Regulation of the Junctophilin Family.

In both excitable and nonexcitable cells, diverse physiological processes are linked to different calcium microdomains within nanoscale junctions that form between the plasma membrane and endo-sarcoplasmic reticula. It is now appreciated that the junctophilin protein family is responsible for establishing, maintaining, and modulating the structure and function of these junctions. We review foundational findings from more than two decades of research that have uncovered how junctophilin-organized ultrastructural domains regulate evolutionarily conserved biological processes. We discuss what is known about the junctophilin family of proteins. Our goal is to summarize the current knowledge of junctophilin domain structure, function, and regulation and to highlight emerging avenues of research that help our understanding of the transcriptional, translational, and post-translational regulation of this gene family and its roles in health and during disease.

MIGA2 Links Mitochondria, the ER, and Lipid Droplets and Promotes De Novo Lipogenesis in Adipocytes.

Physical contact between organelles is vital to the function of eukaryotic cells. Lipid droplets (LDs) are dynamic organelles specialized in lipid storage that interact physically with mitochondria in several cell types. The mechanisms coupling these organelles are, however, poorly understood, and the cell-biological function of their interaction remains largely unknown. Here, we discover in adipocytes that the outer mitochondrial membrane protein MIGA2 links mitochondria to LDs. We identify an amphipathic LD-targeting motif and reveal that MIGA2 binds to the membrane proteins VAP-A or VAP-B in the endoplasmic reticulum (ER). We find that in adipocytes MIGA2 is involved in promoting triglyceride (TAG) synthesis from non-lipid precursors. Our data indicate that MIGA2 links reactions of de novo lipogenesis in mitochondria to TAG production in the ER, thereby facilitating efficient lipid storage in LDs. Based on its presence in many tissues, MIGA2 is likely critical for lipid and energy homeostasis in a wide spectrum of cell types.

Endoplasmic Reticulum-Plasma Membrane Junctions as Sites of Depolarization-Induced Ca2+ Signaling in Excitable Cells.

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca2+ signaling as triggered by membrane depolarization. ER-PM junctions underlie crucial physiological processes such as excitation-contraction coupling, smooth muscle contraction and relaxation, and various forms of activity-dependent signaling and plasticity in neurons. In many cases the structure and molecular composition of ER-PM junctions in excitable cells comprise important regulatory feedback loops linking depolarization-induced Ca2+ signaling at these sites to the regulation of membrane potential. Here, we describe recent findings on physiological roles and molecular composition of native ER-PM junctions in excitable cells. We focus on recent studies that provide new insights into canonical forms of depolarization-induced Ca2+ signaling occurring at junctional triads and dyads of striated muscle, as well as the diversity of ER-PM junctions in these cells and in smooth muscle and neurons.

FMS-like Tyrosine Kinase 3/FLT3: From Basic Science to Clinical Implications.

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is expressed almost exclusively in the hematopoietic compartment. Its ligand, FLT3 ligand (FL), induces dimerization and activation of its intrinsic tyrosine kinase activity. Activation of FLT3 leads to its autophosphorylation and initiation of several signal transduction cascades. Signaling is initiated by the recruitment of signal transduction molecules to activated FLT3 through binding to specific phosphorylated tyrosine residues in the intracellular region of FLT3. Activation of FLT3 mediates cell survival, cell proliferation, and differentiation of hematopoietic progenitor cells. It acts in synergy with several other cytokines to promote its biological effects. Deregulated FLT3 activity has been implicated in several diseases, most prominently in acute myeloid leukemia where around one-third of patients carry an activating mutant of FLT3 which drives the disease and is correlated with poor prognosis. Overactivity of FLT3 has also been implicated in autoimmune diseases, such as rheumatoid arthritis. The observation that gain-of-function mutations of FLT3 can promote leukemogenesis has stimulated the development of inhibitors that target this receptor. Many of these are in clinical trials, and some have been approved for clinical use. However, problems with acquired resistance to these inhibitors are common and, furthermore, only a fraction of patients respond to these selective treatments. This review provides a summary of our current knowledge regarding structural and functional aspects of FLT3 signaling, both under normal and pathological conditions, and discusses challenges for the future regarding the use of targeted inhibition of these pathways for the treatment of patients.

Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic model.

The recent rapid accumulation of knowledge on the dynamics and structure of the plasma membrane has prompted major modifications of the textbook fluid-mosaic model. However, because the new data have been obtained in a variety of research contexts using various biological paradigms, the impact of the critical conceptual modifications on biomedical research and development has been limited. In this review, we try to synthesize our current biological, chemical, and physical knowledge about the plasma membrane to provide new fundamental organizing principles of this structure that underlie every molecular mechanism that realizes its functions. Special attention is paid to signal transduction function and the dynamic aspect of the organizing principles. We propose that the cooperative action of the hierarchical three-tiered mesoscale (2-300 nm) domains--actin-membrane-skeleton induced compartments (40-300 nm), raft domains (2-20 nm), and dynamic protein complex domains (3-10 nm)--is critical for membrane function and distinguishes the plasma membrane from a classical Singer-Nicolson-type model.

Structural basis of the unfolded protein response.

The unfolded protein response (UPR) is a network of intracellular signaling pathways that maintain the protein-folding capacity of the endoplasmic reticulum (ER) in eukaryotic cells. Dedicated molecular sensors embedded in the ER membrane detect incompletely folded or unfolded proteins in the ER lumen and activate a transcriptional program that increases the abundance of the ER according to need. In metazoans the UPR additionally regulates translation and thus relieves unfolded protein load by globally reducing protein synthesis. If homeostasis in the ER cannot be reestablished, the metazoan UPR switches from the prosurvival to the apoptotic mode. The UPR involves a complex, coordinated action of many genes that is controlled by one ER-embedded sensor, Ire1, in yeasts, and three sensors, Ire1, PERK, and ATF6, in higher eukaryotes, including human. We discuss the emerging molecular understanding of the UPR and focus on the structural biology of Ire1 and PERK, the two recently crystallized UPR sensors.

Planar cell polarity and the developmental control of cell behavior in vertebrate embryos.

Planar cell polarity (PCP), the orientation and alignment of cells within a sheet, is a ubiquitous cellular property that is commonly governed by the conserved set of proteins encoded by so-called PCP genes. The PCP proteins coordinate developmental signaling cues with individual cell behaviors in a wildly diverse array of tissues. Consequently, disruptions of PCP protein functions are linked to defects in axis elongation, inner ear patterning, neural tube closure, directed ciliary beating, and left/right patterning, to name only a few. This review attempts to synthesize what is known about PCP and the PCP proteins in vertebrate animals, with a particular focus on the mechanisms by which individual cells respond to PCP cues in order to execute specific cellular behaviors.

The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Membrane Proteins.

Endoplasmic reticulum (ER)-associated degradation (ERAD) removes misfolded proteins from the ER membrane and lumen by the ubiquitin-proteasome pathway. Retrotranslocation of ubiquitinated substrates to the cytosol is a universal feature of ERAD that requires the Cdc48 AAA-ATPase. Despite intense efforts, the mechanism of ER exit, particularly for integral membrane (ERAD-M) substrates, has remained unclear. Using a self-ubiquitinating substrate (SUS), which undergoes normal retrotranslocation independently of known ERAD factors, and the new SPOCK (single plate orf compendium kit) micro-library to query all yeast genes, we found the rhomboid derlin Dfm1 was required for retrotranslocation of both HRD and DOA ERAD pathway integral membrane substrates. Dfm1 recruited Cdc48 to the ER membrane with its unique SHP motifs, and it catalyzed substrate extraction through its conserved rhomboid motifs. Surprisingly, dfm1Δ can undergo rapid suppression, restoring wild-type ERAD-M. This unexpected suppression explained earlier studies ruling out Dfm1, and it revealed an ancillary ERAD-M retrotranslocation pathway requiring Hrd1.

Cis-inhibition suppresses basal Notch signaling during sensory organ precursor selection.

The emergence of the sensory organ precursor (SOP) from an equivalence group in Drosophila is a paradigm for studying single-cell fate specification through Notch-mediated lateral inhibition. Yet, it remains unclear how only a single SOP is selected from a relatively large group of cells. We show here that a critical aspect of SOP selection is controlled by cis-inhibition (CI), whereby the Notch ligands, Delta (Dl), cis-inhibit Notch receptors in the same cell. Based on the observation that the mammalian ligand Dl-like 1 cannot cis-inhibit Notch in Drosophila, we probe the role of CI in vivo. We develop a mathematical model for SOP selection where Dl activity is independently regulated by the ubiquitin ligases Neuralized and Mindbomb1. We show theoretically and experimentally that Mindbomb1 induces basal Notch activity, which is suppressed by CI. Our results highlight the trade-off between basal Notch activity and CI as a mechanism for singling out a SOP from a large equivalence group.

Physiological Functions of CRAC Channels.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol 1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity.

From powerhouse to processing plant: conserved roles of mitochondrial outer membrane proteins in tRNA splicing.

The mitochondrial cytoplasmic surface serves as a processing site for numerous RNAs from budding yeast to metazoans. We report that budding yeast mitochondrial outer membrane (MOM) proteins that are subunits of the translocase of the outer mitochondrial membrane (Tom70 and Tom 22) and sorting and assembly machinery (Sam37) are required for efficient pretransfer RNA (pre-tRNA) splicing. Defective pre-tRNA splicing in MOM mutants is due not to loss of respiratory metabolism but instead inefficient targeting/tethering of tRNA splicing endonuclease (SEN) subunits to mitochondria. Schizosaccharomyces pombe SEN subunits also localize to mitochondria, and Tom70 is required for this localization and pre-tRNA splicing. Thus, the role of MOM protein in targeting/tethering SEN subunits to mitochondria has been conserved for >500 million years.