The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.

Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics.

BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T>C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A>C (p.Thr66Pro) and c.124T>C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system.

Architecture of the ESCPE-1 membrane coat.

Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.

Inhibition of endolysosome fusion increases exosome secretion.

Exosomes are small vesicles that are secreted from cells to dispose of undegraded materials and mediate intercellular communication. A major source of exosomes is intraluminal vesicles within multivesicular endosomes that undergo exocytic fusion with the plasma membrane. An alternative fate of multivesicular endosomes is fusion with lysosomes, resulting in degradation of the intraluminal vesicles. The factors that determine whether multivesicular endosomes fuse with the plasma membrane or with lysosomes are unknown. In this study, we show that impairment of endolysosomal fusion by disruption of a pathway involving the BLOC-one-related complex (BORC), the small GTPase ARL8, and the tethering factor HOPS increases exosome secretion by preventing the delivery of intraluminal vesicles to lysosomes. These findings demonstrate that endolysosomal fusion is a critical determinant of the amount of exosome secretion and suggest that suppression of the BORC-ARL8-HOPS pathway could be used to boost exosome yields in biotechnology applications.

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes.

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Measurement of Lysosome Positioning by Shell Analysis and Line Scan.

Lysosomes are membrane-bound organelles that degrade diverse biomolecules and regulate a multitude of other essential processes including cell growth and metabolism, signaling, plasma membrane repair and infection. Such diverse functions of lysosomes are highly coordinated in space and time and are therefore tightly coupled to the directional transport of the organelles within the cytoplasm. Thus, robust quantitative assessments of lysosome positioning within the cell provide a valuable tool for researchers interested in understanding these multifunctional organelles. Here, we present point-by-point methodology to measure lysosome positioning by two straight forward and widely used techniques: shell analysis and line scan.

RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.

The small GTPase ARL8 associates with endolysosomes, leading to the recruitment of several effectors that couple endolysosomes to kinesins for anterograde transport along microtubules, and to tethering factors for eventual fusion with other organelles. Herein we report the identification of the RUN- and FYVE-domain-containing proteins RUFY3 and RUFY4 as ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin for retrograde transport along microtubules. Using various methodologies, we find that RUFY3 and RUFY4 interact with both GTP-bound ARL8 and dynein-dynactin. In addition, we show that RUFY3 and RUFY4 promote concentration of endolysosomes in the juxtanuclear area of non-neuronal cells, and drive redistribution of endolysosomes from the axon to the soma in hippocampal neurons. The function of RUFY3 in retrograde transport contributes to the juxtanuclear redistribution of endolysosomes upon cytosol alkalinization. These studies thus identify RUFY3 and RUFY4 as ARL8-dependent, dynein-dynactin adaptors or regulators, and highlight the role of ARL8 in the control of both anterograde and retrograde endolysosome transport.

The autophagy protein ATG9A enables lipid mobilization from lipid droplets.

The multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes.

Pain Management and Opioid Therapy: Persistent Knowledge Gaps Among Primary Care Providers.

INTRODUCTION: Given the opioid epidemic in the US, it is vital that clinicians who prescribe opioids for pain management to do so in an evidence-based manner, eg considering all pharmacologic and non-pharmacologic options, assessing risk of opioid use disorder prior to initiating opioids. Continuing education regarding the evidence-based prescribing of opioids is now required for US healthcare providers who prescribe opioids. A "blueprint" of the content to be included in continuing education programs was developed by the US Food and Drug Administration and updated in 2018. METHODS: To understand the baseline knowledge and confidence of healthcare professionals in prescribing opioids for pain management, we posed 27 unique knowledge-based questions and 1 confidence question to clinician participants before or during 2 continuing educational programs that were based respectively on the 2016 and 2018 FDA Risk Evaluation and Mitigation Strategy (REMS) educational blueprints for pain management. RESULTS: Overall, 5571 clinicians completed these programs, including 1925 physicians (1516 [79%] identifying as primary care), 1181 physician assistants, 737 advanced practice nurses, 719 nurses, and 479 pharmacists. Responses to pretest questions in both programs indicated profound and persistent gaps in knowledge, particularly in definitions and mechanisms of pain, general principles of pharmacologic analgesic therapy, and specific aspects of opioid analgesic therapy and addiction. Participants in both programs also expressed limited confidence in their abilities to incorporate patient engagement techniques into pain management or develop a treatment plan for a patient with chronic pain. DISCUSSION: These data support an ongoing need for comprehensive clinician-based education as outlined in the FDA REMS educational blueprint, especially given recent data of escalating overdose deaths during the COVID-19 pandemic.

SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum.

The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin.

Oxidative Stress Mediates the Fetal Programming of Hypertension by Glucocorticoids.

The field of cardiovascular fetal programming has emphasized the importance of the uterine environment on postnatal cardiovascular health. Studies have linked increased fetal glucocorticoid exposure, either from exogenous sources (such as dexamethasone (Dex) injections), or from maternal stress, to the development of adult cardiovascular pathologies. Although the mechanisms are not fully understood, alterations in gene expression driven by altered oxidative stress and epigenetic pathways are implicated in glucocorticoid-mediated cardiovascular programming. Antioxidants, such as the naturally occurring polyphenol epigallocatechin gallate (EGCG), or the superoxide dismutase (SOD) 4-hydroxy-TEMPO (TEMPOL), have shown promise in the prevention of cardiovascular dysfunction and programming. This study investigated maternal antioxidant administration with EGCG or TEMPOL and their ability to attenuate the fetal programming of hypertension via Dex injections in WKY rats. Results from this study indicate that, while Dex-programming increased blood pressure in male and female adult offspring, administration of EGCG or TEMPOL via maternal drinking water attenuated Dex-programmed increases in blood pressure, as well as changes in adrenal mRNA and protein levels of catecholamine biosynthetic enzymes phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT), in a sex-specific manner. Furthermore, programmed male offspring displayed reduced antioxidant glutathione peroxidase 1 (Gpx1) expression, increased superoxide dismutase 1 (SOD1) and catalase (CAT) expression, and increased pro-oxidant NADPH oxidase activator 1 (Noxa1) expression in the adrenal glands. In addition, prenatal Dex exposure alters expression of epigenetic regulators histone deacetylase (HDAC) 1, 5, 6, 7, 11, in male and HDAC7 in female offspring. These results suggest that glucocorticoids may mediate the fetal programming of hypertension via alteration of epigenetic machinery and oxidative stress pathways.

LRRK2 mediates tubulation and vesicle sorting from lysosomes.

Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson's disease (PD). However, the biological functions of LRRK2 remain incompletely understood. Here, we report that LRRK2 is recruited to lysosomes after exposure of cells to the lysosome membrane-rupturing agent LLOME. Using an unbiased proteomic screen, we identified the motor adaptor protein JIP4 as an LRRK2 partner at the lysosomal membrane. LRRK2 can recruit JIP4 to lysosomes in a kinase-dependent manner via the phosphorylation of RAB35 and RAB10. Using super-resolution live-cell imaging microscopy and FIB-SEM, we demonstrate that JIP4 promotes the formation of LAMP1-negative tubules that release membranous content from lysosomes. Thus, we describe a new process orchestrated by LRRK2, which we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2), by which lysosomal tubulation is used to release vesicles from lysosomes. Given the central role of the lysosome in PD, LYTL is likely to be disease relevant.

The FTS-Hook-FHIP (FHF) complex interacts with AP-4 to mediate perinuclear distribution of AP-4 and its cargo ATG9A.

The heterotetrameric adaptor protein complex 4 (AP-4) is a component of a protein coat associated with the trans-Golgi network (TGN). Mutations in AP-4 subunits cause a complicated form of autosomal-recessive hereditary spastic paraplegia termed AP-4-deficiency syndrome. Recent studies showed that AP-4 mediates export of the transmembrane autophagy protein ATG9A from the TGN to preautophagosomal structures. To identify additional proteins that cooperate with AP-4 in ATG9A trafficking, we performed affinity purification-mass spectrometry followed by validation of the hits by biochemical and functional analyses. This approach resulted in the identification of the fused toes homolog-Hook-FHIP (FHF) complex as a novel AP-4 accessory factor. We found that the AP-4-FHF interaction is mediated by direct binding of the AP-4 µ4 subunit to coiled-coil domains in the Hook1 and Hook2 subunits of FHF. Knockdown of FHF subunits resulted in dispersal of AP-4 and ATG9A from the perinuclear region of the cell, consistent with the previously demonstrated role of the FHF complex in coupling organelles to the microtubule (MT) retrograde motor dynein-dynactin. These findings thus uncover an additional mechanism for the distribution of ATG9A within cells and provide further evidence for a role of protein coats in coupling transport vesicles to MT motors.

Arf6, JIP3, and dynein shape and mediate macropinocytosis.

Macropinocytosis is an actin-driven form of clathrin-independent endocytosis that generates an enlarged structure, the macropinosome. Although many studies focus on signaling molecules and phosphoinositides involved in initiating macropinocytosis, the commitment to forming a macropinosome and the handling of that membrane have not been studied in detail. Here we show in HT1080 cells, a human fibrosarcoma cell line, a requirement for microtubules, dynein, the JIP3 microtubule motor scaffold protein, and Arf6, a JIP3 interacting protein, for the formation and inward movement of the macropinosome. While actin and myosin II also play critical roles in the formation of ruffling membrane, microtubules provide an important tract for initiation, sealing, and transport of the macropinosome through the actin- and myosin-rich lamellar region.

Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL.

Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.

Comparative Analysis of Renin-Angiotensin System (RAS)-Related Gene Expression Between Hypertensive and Normotensive Rats.

BACKGROUND The renal renin-angiotensin system (RAS) is physiologically important for blood pressure regulation. Altered regulation of RAS-related genes has been observed in an animal model of hypertension (spontaneously hypertensive rats - SHRs). The current understanding of certain RAS-related gene expression differences between Wistar-Kyoto rats (WKYs) and SHRs is either limited or has not been compared. The purpose of this study was to compare the regulation of key RAS-related genes in the kidneys of adult WKYs and SHRs. MATERIAL AND METHODS Coronal sections were dissected through the hilus of kidneys from 16-week-old male WKYs and SHRs. RT-PCR analysis was performed for Ace, Ace2, Agt, Agtr1a, Agtr1b, Agtr2, Atp6ap2 (PRR), Mas1, Ren, Rnls, and Slc12a3 (NCC). RESULTS Increased mRNA expression was observed for Ace, Ace2, Agt, Agtr1a, Agtr1b, and Atp6ap2 in SHRs compared to WKYs. Mas1, Ren, Slc12a3, and Rnls showed no difference in expression between animal types. CONCLUSIONS This study shows that the upregulation of several key RAS-related genes in the kidney may account for the increased blood pressure of adult SHRs.

Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain.

Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B, and NM2C. The three NM2s have well established essential roles in cell motility, adhesion, and cytokinesis and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine-liposomes, but not to phosphatidylcholine-liposomes, has been reported, but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B, and NM2C and monomeric, non-filamentous heavy meromyosin bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol 4,5-diphosphate, and phosphatidylinositol 3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous heavy meromyosin; the addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin.

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.

Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

Educational outcomes in the era of the Affordable Care Act: impact of personalized education about non-small cell lung cancer.

INTRODUCTION: The Affordable Care Act is moving medical care-and medical education-toward a quality-driven environment. Quality medical education must be available when the health care provider is ready to learn, provide feedback, and maximize translation of knowledge from desk to clinic. To best accomplish these goals, medical education must be personalized to clinicians' needs. Research has defined multiple knowledge/performance gaps among oncologists who manage advanced non-small cell lung cancer (NSCLC). A study was conducted to determine the effectiveness with which a personalized learning approach for oncologists will diminish these gaps. METHODS: The authors undertook development, online distribution, and impact measurement of an NSCLC curriculum in which learners' responses to a preeducation self-assessment of knowledge, skills, and attitudes resulted in receipt of a personalized curriculum. Upon completion of the assessment, each learner received a personalized curriculum, which included up to 5 distinct activities selected to address identified knowledge and practice gaps. Feedback was provided at the completion of each activity. RESULTS: Ninety-two oncologists completed an individualized learning plan. Analysis shows that completion of the learning plan was associated with a high effect size (d = .70). Significant increases were seen in oncologists' ability to correctly identify the rationale for determining histological subtype (13% increase), prevalence of genetic abnormalities (21% increase, p = .04), appropriate use of maintenance therapy (31% increase, p = .01), and appropriate treatment regimens for squamous cell carcinoma (19% increase, p = .003) and of adenocarcinoma (44% increase, p < .001). DISCUSSION: This study demonstrates the feasibility and impact of a personalized targeted curriculum for improving the competence of oncologists treating patients with advanced NSCLC.