Regulation Of Membrane Trafficking Research Articles

Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11-WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44-Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events.

Sphingosine kinase 2 regulates insulin receptor trafficking in hepatocytes.

Disturbed insulin receptor (InsR) trafficking is associated with impaired insulin signaling and the development of diabetes. Sphingosine kinase (SphK), including SphK1 and SphK2, is a key enzyme of sphingolipid metabolism, which has been implicated in the regulation of membrane trafficking. More recently, we have reported that SphK2 is a key regulator of hepatic insulin signaling and glucose homeostasis. However, the role of SphK in InsR trafficking is still undefined. Huh7 cells were treated with specific SphK1 and SphK2 inhibitors or SphK1- and SphK2-specific small interfering RNA (siRNA) in the presence or absence of insulin. Flow cytometry and immunofluorescence assays were carried out to investigate the role of SphK in InsR trafficking. InsR endocytosis, recycling, and insulin signaling were analyzed. Inhibition of SphK2, but not SphK1, by either specific pharmaceutic inhibitors or siRNA, significantly suppressed InsR endocytosis and recycling following insulin stimulation. Consequently, the insulin-stimulated Akt activation was significantly attenuated by SphK2 inhibition in hepatocytes. Moreover, the effect of SphK2 on InsR trafficking was mediated via the clathrin-dependent mechanism. Thus, our results show that SphK2 is able to regulate InsR trafficking. These findings suggest that SphK2 may impinge on hepatic insulin signaling by regulating InsR trafficking, providing further mechanistic evidence that SphK2 could serve as a potential intervention target against insulin resistance and T2D (type 2 diabetes).

Neuropilin-1 Mediates SARS-CoV-2 Infection of Astrocytes in Brain Organoids, Inducing Inflammation Leading to Dysfunction and Death of Neurons.

Coronavirus disease 2019 (COVID-19) is frequently associated with neurological deficits, but how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces these effects remains unclear. Here, we show that astrocytes are readily infected by SARS-CoV-2, but surprisingly, neuropilin-1, not angiotensin-converting enzyme 2 (ACE2), serves as the principal receptor mediating cell entry. Infection is further positively modulated by the two-pore segment channel 2 (TPC2) protein that regulates membrane trafficking and endocytosis. Astrocyte infection produces a pathological response closely resembling reactive astrogliosis characterized by elevated type I interferon (IFN) production, increased inflammation, and the decreased expression of transporters of water, ions, choline, and neurotransmitters. These combined events initiated within astrocytes produce a hostile microenvironment that promotes the dysfunction and death of uninfected bystander neurons. IMPORTANCE SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection, but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Here, we used induced pluripotent stem cell (iPSC)-derived brain organoids and primary human astrocytes from the cerebral cortex to study direct SARS-CoV-2 infection. Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. The deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

Divergent regulation of basement membrane trafficking by human macrophages and cancer cells

Macrophages and cancer cells populations are posited to navigate basement membrane barriers by either mobilizing proteolytic enzymes or deploying mechanical forces. Nevertheless, the relative roles, or identity, of the proteinase -dependent or -independent mechanisms used by macrophages versus cancer cells to transmigrate basement membrane barriers harboring physiologically-relevant covalent crosslinks remains ill-defined. Herein, both macrophages and cancer cells are shown to mobilize membrane-anchored matrix metalloproteinases to proteolytically remodel native basement membranes isolated from murine tissues while infiltrating the underlying interstitial matrix ex vivo. In the absence of proteolytic activity, however, only macrophages deploy actomyosin-generated forces to transmigrate basement membrane pores, thereby providing the cells with proteinase-independent access to the interstitial matrix while simultaneously exerting global effects on the macrophage transcriptome. By contrast, cancer cell invasive activity is reliant on metalloproteinase activity and neither mechanical force nor changes in nuclear rigidity rescue basement membrane transmigration. These studies identify membrane-anchored matrix metalloproteinases as key proteolytic effectors of basement membrane remodeling by macrophages and cancer cells while also defining the divergent invasive strategies used by normal and neoplastic cells to traverse native tissue barriers.

ER as master regulator of membrane trafficking and organelle function.

The endoplasmic reticulum (ER), which occupies a large portion of the cytoplasm, is the cell's main site for the biosynthesis of lipids and carbohydrate conjugates, and it is essential for folding, assembly, and biosynthetic transport of secreted proteins and integral membrane proteins. The discovery of abundant membrane contact sites (MCSs) between the ER and other membrane compartments has revealed that, in addition to its biosynthetic and secretory functions, the ER plays key roles in the regulation of organelle dynamics and functions. In this review, we will discuss how the ER regulates endosomes, lysosomes, autophagosomes, mitochondria, peroxisomes, and the Golgi apparatus via MCSs. Such regulation occurs via lipid and Ca2+ transfer and also via control of in trans dephosphorylation reactions and organelle motility, positioning, fusion, and fission. The diverse controls of other organelles via MCSs manifest the ER as master regulator of organelle biology.

Sponge/DOCK-dependent regulation of F-actin networks directing cortical cap behaviors and syncytial furrow ingression

In the early syncytial Drosophila embryo, rapid changes in filamentous actin networks and membrane trafficking pathways drive the formation and remodeling of cortical and furrow morphologies. Interestingly, genomic integrity and the completion of mitoses during cell cycles 10–13 depends on the formation of transient membrane furrows that serve to separate and anchor individual spindles during division. While substantial work has led to a better understanding of the core network components that are responsible for the formation of these furrows, less is known about the regulation that controls cytoskeletal and trafficking function. The DOCK protein Sponge was one of the first proteins identified as being required for syncytial furrow formation, and disruption of Sponge deeply compromises F-actin populations in the early embryo, but how this occurs is less clear. Here, we perform quantitative analysis of the effects of Sponge disruption on cortical cap growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. We find that membrane trafficking is relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but that Sponge acts as a master regulator of a diverse cohort of Arp2/3 regulatory proteins. As DOCK family proteins have been implicated in regulating GTP exchange on small GTPases, we also suggest that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, we demonstrate the phasic requirements for branched F-actin and linear F-actin networks in potentiating furrow ingression. In total, these results provide quantitative insights into how a large DOCK scaffolding protein coordinates the activity of a variety of different actin regulatory proteins to direct the remodeling of the apical cortex into cytokinetic-like furrows.

Reduced bone morphogenic protein signaling along the gut-neuron axis by heat shock factor promotes longevity.

Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF‐1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF‐1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF‐1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF‐1 represses the expression of the neuron‐specific BMP ligand, DBL‐1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL‐1 in the periphery, the SMAD transcriptional coactivator, SMA‐3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB‐5), late (RAB‐7), and recycling (RAB‐11.1) endosomal dynamics and the BMP receptor binding protein, SMA‐10. This reduces cell surface residency and steady‐state levels of the type I BMP receptor, SMA‐6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF‐1 to coordinate BMP signaling along the gut–brain axis is an important determinate in age progression.

Normal oil body formation in Marchantia polymorpha requires functional coat protein complex I proteins.

Eukaryotic cells possess endomembrane organelles equipped with specific sets of proteins, lipids, and polysaccharides that are fundamental for realizing each organelle’s specific function and shape. A tightly regulated membrane trafficking system mediates the transportation and localization of these substances. Generally, the secretory/exocytic pathway is responsible for transporting cargo to the plasma membrane and/or the extracellular space. However, in the case of oil body cells in the liverwort Marchantia polymorpha, the oil body, a liverwort-unique organelle, is thought to be formed by secretory vesicle fusion through redirection of the secretory pathway inside the cell. Although their formation mechanism remains largely unclear, oil bodies exhibit a complex and bumpy surface structure. In this study, we isolated a mutant with spherical oil bodies through visual screening of mutants with abnormally shaped oil bodies. This mutant harbored a mutation in a coat protein complex I (COPI) subunit MpSEC28, and a similar effect on oil body morphology was also detected in knockdown mutants of other COPI subunits. Fluorescently tagged MpSEC28 was localized to the periphery of the Golgi apparatus together with other subunits, suggesting that it is involved in retrograde transport from and/or in the Golgi apparatus as a component of the COPI coat. The Mpsec28 mutants also exhibited weakened stiffness of the thalli, suggesting impaired cell–cell adhesion and cell wall integrity. These findings suggest that the mechanism of cell wall biosynthesis is also involved in shaping the oil body in M. polymorpha, supporting the redirection of the secretory pathway inward the cell during oil body formation.

AP1/2β-mediated exocytosis of tapetum-specific transporters is required for pollen development in Arabidopsis thaliana.

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.

Physiological and Pathological Roles of the Cytohesin Family in Neurons.

The cytohesin proteins, consisting of four closely related members (cytohesins-1, -2, -3, and -4), are a subfamily of the Sec7 domain-containing guanine nucleotide exchange factors for ADP ribosylation factors (Arfs), which are critical regulators of membrane trafficking and actin cytoskeleton remodeling. Recent advances in molecular biological techniques and the development of a specific pharmacological inhibitor for cytohesins, SecinH3, have revealed the functional involvement of the cytohesin–Arf pathway in diverse neuronal functions from the formation of axons and dendrites, axonal pathfinding, and synaptic vesicle recycling, to pathophysiological processes including chronic pain and neurotoxicity induced by proteins related to neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer’s disease. Here, we review the physiological and pathological roles of the cytohesin–Arf pathway in neurons and discuss the future directions of this research field.

Dopey proteins are essential but overlooked regulators of membrane trafficking.

Dopey family proteins play crucial roles in diverse processes from morphogenesis to neural function and are conserved from yeast to mammals. Understanding the mechanisms behind these critical functions could have major clinical significance, as dysregulation of Dopey proteins has been linked to the cognitive defects in Down syndrome, as well as neurological diseases. Dopey proteins form a complex with the non-essential GEF-like protein Mon2 and an essential lipid flippase from the P4-ATPase family. Different combinations of Dopey, Mon2 and flippases have been linked to regulating membrane remodeling, from endosomal recycling to extracellular vesicle formation, through their interactions with lipids and other membrane trafficking regulators, such as ARL1, SNX3 and the kinesin-1 light chain KLC2. Despite these important functions and their likely clinical significance, Dopey proteins remain understudied and their roles elusive. Here, we review the major scientific discoveries relating to Dopey proteins and detail key open questions regarding their function to draw attention to these fascinating enigmas.

The GTP-Bound form of Rab3D Promotes Lipid Droplet Growth in Adipocyte

Rab GTPases are major regulators of membrane trafficking. Proteome profiling reveals dozens of Rab proteins associated with lipid droplets (LDs), but few of them have been functionally validated. Cell death activator CIDE-3 protein mediates LD fusion and growth. It is highly enriched at LD-LD contact sites. Here, we investigated the role of Rab3D in lipid storage in adipocyte. We verified that the adipose levels of Rab3D transcript were higher than that of other Rab3 family members; the differences were most pronounced in white adipose tissue. Moreover, we showed that Rab3D promotes LD growth in 3T3-L1 preadipocytes in a dose dependent manner, independently of the process of CIDE-3-mediated LD fusion. Finally, we confirmed that the GTP-bound form of Rab3D is its LD promoting form; it translocates from other vesicles to LDs during adipocyte differentiation. By contrast, the Rab3D-GDP form is retained in the cytoplasm and has no effect on LD sizes. Presented results provide evidence for the role of Rab3D in controlling formation of large LDs in adipocytes. We conclude that Rab3D is a novel LD regulator with characteristics differing from these of previously identified LD associated Rab proteins, such as Rab18 and Rab8a.

Hypothalamic Irak4 is a genetically controlled regulator of hypoglycemia-induced glucagon secretion

ObjectivesGlucagon secretion to stimulate hepatic glucose production is the first line of defense against hypoglycemia. This response is triggered by so far incompletely characterized central hypoglycemia-sensing mechanisms, which control autonomous nervous activity and hormone secretion. The objective of this study was to identify novel hypothalamic genes controlling insulin-induced glucagon secretion. MethodsTo obtain new information on the mechanisms of hypothalamic hypoglycemia sensing, we combined genetic and transcriptomic analysis of glucagon response to insulin-induced hypoglycemia in a panel of BXD recombinant inbred mice. ResultsWe identified two QTLs on chromosome 8 and chromosome 15. We further investigated the role of Irak4 and Cpne8, both located in the QTL on chromosome 15, in C57BL/6J and DBA/2J mice, the BXD mouse parental strains. We found that the poor glucagon response of DBA/2J mice was associated with higher hypothalamic expression of Irak4, which encodes a kinase acting downstream of the interleukin-1 receptor (Il-1R), and of Il-ß when compared with C57BL/6J mice. We showed that intracerebroventricular administration of an Il-1R antagonist in DBA/2J mice restored insulin-induced glucagon secretion; this was associated with increased c-fos expression in the arcuate and paraventricular nuclei of the hypothalamus and with higher activation of both branches of the autonomous nervous system. Whole body inactivation of Cpne8, which encodes a Ca++-dependent regulator of membrane trafficking and exocytosis, however, had no impact on insulin-induced glucagon secretion. ConclusionsCollectively, our data identify Irak4 as a genetically controlled regulator of hypoglycemia-activated hypothalamic neurons and glucagon secretion.

Myr-Arf1 conformational flexibility at the membrane surface: insights for its interactions with ASAP1

ADP ribosylation factor-1 (Arf1) is a major regulator of cell membrane trafficking and organelle structure, and Arf1 misregulation contributes to invasion and metastasis of cancer cells. The basis for Arf1 biological function is a highly regulated GDP/GTP cycle coupled to a cytosolic/membrane-bound cycle. Cell membranes play a crucial role for all steps of the Arf1 signaling cycle: 1) membranes are mandatory for Arf1 GTPase to acquire the active state; 2) Arf1 interacts with numerous proteins on the membrane surface.

Deciphering the molecular organization of Get pathway chaperones through native top-down dissociation of multi-protein complexes

From regulating membrane trafficking to the release of neurotransmitters at the neuronal synapses, C-terminal tail-anchored membrane proteins (TAPs) play a central role in eukaryotic biology. Trafficking of TAPs to their target physiological membrane follows the usual anterograde trafficking route from the endoplasmic reticulum (ER), where they are first inserted. Nevertheless, unlike other membrane proteins, their insertion into the ER membrane is not mediated via the signal recognition particle (SRP) pathway.

Generation of Endogenously Tagged Membrane Trafficking Regulators Using CRISPR Genome Editing.

Vesicle trafficking entails packaging and transport of membrane-associated proteins to their target membranes, and recycling or degradation of endocytosed proteins. Biochemical and cell biological studies of vesicle trafficking often require the introduction of epitope tags or fluorescent protein markers for protein purification and tracking in cells. Previously, such tagging experiments in mammalian cells mainly used overexpression systems, which could lead to artifacts. Abnormally high expression levels also prevent us from studying individual vesicle trafficking events with precision. With the advent of CRISPR technologies, epitope tags and fluorescent proteins can now be introduced into endogenous proteins in almost any cell type that are proliferating in culture. This chapter describes approaches for inserting tags at the endogenous loci of genes, with the vesicle tethering protein complex, exocyst, as an example.

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system.

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. They have recently been found to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, making it extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed cell lines with conditional KO (CKO) of either Rab1 (Rab1A and Rab1B) or Rab5 (Rab5A, Rab5B and Rab5C) by using the auxin-inducible protein degradation system. Rab1 CKO and Rab5 CKO led to eventual cell death from 18 h and 48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and endoplasmic reticulum (ER)-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system.

Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions-however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

Role of Rab GTPases in Hepatocellular Carcinoma.

The Rab GTPase family contains almost 70 genes in the human genome and acts as the key regulator of intracellular membrane trafficking in human cells. The dysregulation of Rab GTPase has been shown to be associated with multiple human diseases, ranging from neurodegeneration, and infection to cancer. Rab GTPases not only play important roles in genome replication, morphogenesis and the release of hepatitis B virus (HBV) or hepatitis C virus (HCV), but also contribute to hepatitis-related hepatocarcinogenesis and hepatocellular carcinoma (HCC) progression. The alteration of Rab GTPase expression in HCC plays an important role in tumour cell proliferation, invasion and migration. Notably, the expression of Rab genes can be regulated by some noncoding RNAs, such as miRNAs and circRNAs. Thus, Rab GTPases can serve as promising rational and therapeutic targets for HCC treatments. In this review, we summarized recent advancements in this field focusing on Rab GTPases in HCC.

P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors.

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca2+ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H1 histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca2+] is exaggerated, as detected with a low-affinity targeted Ca2+ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca2+ release and the regulation of lysosomal membrane trafficking.