Endosomal Markers Research Articles

Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassius carassius).

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

Pathogenic and Apathogenic Strains of Lymphocytic Choriomeningitis Virus Have Distinct Entry and Innate Immune Activation Pathways.

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.

Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes.

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32–0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Osiris17 is essential for stable integrin localization and function during insect wing epithelia remodeling

The dynamic adhesion between cells and their extracellular matrix is essential for the development and function of organs. During insect wing development, two epithelial sheets contact each other at their basal sites through the interaction of βPS integrins with the extracellular matrix. We report that Osiris17 contributes to the maintenance of βPS integrins localization and function in developing wing of Drosophila and locust. In flies with reduced Osiris17 expression the epithelia sheets fail to maintain the integrity of basal cytoplasmic junctional bridges and basal adhesion. In contrast to the continuous basal integrin localization in control wings, this localization is disrupted during late stages of wing development in Osiris17 depleted flies. In addition, the subcellular localization revealed that Osiris17 co-localizes with the endosomal markers Rab5 and Rab11. This observation suggests an involvement of Osiris17 in endosomal recycling of integrins. Indeed, Osiris17 depletion reduced the numbers of Rab5 and Rab11 positive endosomes. Moreover, overexpression of Osiris17 increased co-localization of Rab5 and βPS integrins and partially rescued the detachment phenotype in flies with reduced βPS integrins. Taken together, our data suggest that Osiris17 is an endosome related protein that contributes to epithelial remodeling and morphogenesis by assisting basal integrins localization in insects.

The Cl- transporter ClC-7 is essential for phagocytic clearance by microglia.

Microglia, professional phagocytic cells of the brain, rely upon the appropriate activation of lysosomes to execute their immune and clearance functions. Lysosomal activity is, in turn, modulated by a complex network of over 200 membrane and accessory proteins that relay extracellular cues to these key degradation centers. The ClC-7 chloride (Cl-)-proton (H+) antiporter (also known as CLCN7) is localized to the endolysosomal compartments and mutations in CLCN7 lead to osteopetrosis and neurodegeneration. Although the functions of ClC-7 have been extensively investigated in osteoclasts and neurons, its role in microglia in vivo remains largely unexamined. Here, we show that microglia and embryonic macrophages in zebrafish clcn7 mutants cannot effectively process extracellular debris in the form of apoptotic cells and β-amyloid. Despite these functional defects, microglia develop normally in clcn7 mutants and display normal expression of endosomal and lysosomal markers. We also find that mutants for ostm1, which encodes the β-subunit of ClC-7, have a phenotype that is strikingly similar to that of clcn7 mutants. Together, our observations uncover a previously unappreciated role of ClC-7 in microglia and contribute to the understanding of the neurodegenerative phenotypes that accompany mutations in this channel.

ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Nervous necrosis virus induced vacuolization is a Rab5- and actin-dependent process

ABSTRACT Cytoplasmic vacuolization is commonly induced by bacteria and viruses, reflecting the complex interactions between pathogens and the host. However, their characteristics and formation remain unclear. Nervous necrosis virus (NNV) infects more than 100 global fish species, causing enormous economic losses. Vacuolization is a hallmark of NNV infection in host cells, but remains a mystery. In this study, we developed a simple aptamer labelling technique to identify red-spotted grouper NNV (RGNNV) particles in fixed and live cells to explore RGNNV-induced vacuolization. We observed that RGNNV-induced vacuolization was positively associated with the infection time and virus uptake. During infection, most RGNNV particles, as well as viral genes, colocalized with vacuoles, but not giant vacuoles > 3 μm in diameter. Although the capsid protein (CP) is the only structural protein of RGNNV, its overexpression did not induce vacuolization, suggesting that vacuole formation probably requires virus entry and replication. Given that small Rab proteins and the cytoskeleton are key factors in regulating cellular vesicles, we further investigated their roles in RGNNV-induced vacuolization. Using live cell imaging, Rab5, a marker of early endosomes, was continuously located in vacuoles bearing RGNNV during giant vacuole formation. Rab5 is required for vacuole formation and interacts with CP according to siRNA interference and Co-IP analysis. Furthermore, actin formed distinct rings around small vacuoles, while vacuoles were located near microtubules. Actin, but not microtubules, plays an important role in vacuole formation using chemical inhibitors. These results provide valuable insights into the pathogenesis and control of RGNNV infections.

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, β2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of β2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

MiR-451a suppresses the proliferation and migration of high-grade serous ovarian cancer by targeting RAB5A through the Ras/Raf/MEK/ERK pathway.

Ovarian cancer is one of the most common cancers in women. Profiles changes of microRNAs (miRNAs) are closely linked to malignant tumors. In the present study, we investigated expression of miR-451a in high-grade serous ovarian cancer (HGSOC). We also investigated the potential pathological roles and the likely mechanism of miR-451a in the development of HGSOC using animal models and cell lines. Using bioinformatics techniques and a real-time PCR, we analyzed differently expressed miRNAs in HGSOC compared to normal tissue. MTT (i.e. 3-[4, 5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide), EDU (i.e. 5-ethynyl-2'-deoxyuridine) and transwell assays were performed to investigate the effect of miR-451a on the proliferation and migration of HGSOC SKOV-3 cells. A dual luciferase reporter assay was performed to verify the targeting relationship of miR-451 and RAB5A (one of the Rab GTPase proteins that regulates endocytosis and vesicle transport). Also, we analyzed levels of the RAB5A mRNA and protein by real-time PCR, western blotting and immunohistochemistry assays in HGSOC cells and tissues. Finally, we performed in vivo experiments using HGSOC mice. miR-451a was substantially upregulated in HGSOC and associated with favorable clinical characteristics. miR-451a knockdown significantly increased growth and metastasis of HGSOC cell line SKOV-3 through Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. In addition, RAB5A, an early endosome marker, was shown to be a direct target of miR-451a. Moreover, RAB5A is correlated with unfavorable clinical features and shows independent prognostic significance in HGSOC. We found that the miR-451a/RAB5A axis is associated with tumorigenesis and progression through the Ras/Raf/MEK/ERK pathway, providing prognostic indicators and therapeutic targets for patients with HGSOC.

Basal interaction of the orphan receptor GPR101 with arrestins leads to constitutive internalization

GPR101 is an orphan G protein-coupled receptor that promotes growth hormone secretion in the pituitary. The microduplication of the GPR101 gene has been linked with the X-linked acrogigantism, or X-LAG, syndrome. This disease is characterized by excessive growth hormone secretion and abnormal rapid growth beginning early in life. Mechanistically, GPR101 induces growth hormone secretion through constitutive activation of multiple heterotrimeric G proteins. However, the full scope of GPR101 signaling remains largely elusive. Herein, we investigated the association of GPR101 to multiple transducers and uncovered an important basal interaction with Arrestin 2 (β-arrestin 1) and Arrestin 3 (β-arrestin 2). By using a GPR101 mutant lacking the C-terminus and cell lines with an Arrestin 2/3 null background, we show that the arrestin association leads to constitutive clathrin- and dynamin-mediated GPR101 internalization. To further highlight GPR101 intracellular fate, we assessed the colocalization of GPR101 with Rab protein markers. Internalized GPR101 was mainly colocalized with the early endosome markers, Rab5 and EEA-1, and to a lesser degree with the late endosome marker Rab7. However, GPR101 was not colocalized with the recycling endosome marker Rab11. These findings show that the basal arrestin recruitment by GPR101 C-terminal tail drives the receptor constitutive clathrin-mediated internalization. Intracellularly, GPR101 concentrates in the endosomal compartment and is degraded through the lysosomal pathway. In conclusion, we uncovered a constitutive intracellular trafficking of GPR101 that potentially represents an important layer of regulation of its signaling and function.

The Effect of APOE Genotype on Endolysosomal Dynamics in Neurons

AbstractBackgroundAPOE genotype is known as the most important genetic risk factor for developing AD. Understanding the cellular differences between the ApoE isoforms will be crucial for understanding the fundamental pathways that are disrupted in AD and potential pathways that can be therapeutically targeted. Accumulating evidence is suggesting that ApoE isoforms influence the endosomal system, one of the earliest sites affected in AD. However, our understanding of how the isoforms affect the endolysosomal pathway is still limited. We aim to understand how ApoE isoforms affect the endosomal pathway, but also understand the dynamics of the system in the context of ageing and activity.MethodTo study cellular changes in neurons we are using primary hippocampal/cortical brain cultures, harvested from ApoE KO mice and targeted replacement mice, expressing human ApoE3 or ApoE4. Neurons in culture have accelerated maturation and with time in culture gradually show signs of age‐like stress. To study ApoE in the context of ageing, mature cultures at 18 days in vitro (DIV18) and more aged cultures at DIV25 were used. To explore how ApoE isoforms adapt to elevated activity DIV18 primary cultures were treated for 48 H with the GABA receptor antagonist bicuculline.ResultOur preliminary data suggests that especially early endosomes are affected in ApoE4 neurons. Interestingly, the appearance of the early endosomes seems to differ depending on both the intracellular localization and time in culture. However, the protein level of the early endosomal marker EEA1 remained unaltered in ApoE4. In addition, preliminary data indicate that ApoE4 has reduced capacity to adjust the endosomal system following 48 hours bicuculline treatment. When studying the trafficking of fluorescently tagged EGF (which has an established endolysosomal route), preliminary results show potential differences between both DIV18 and DIV25 but also between ApoE‐isoforms, suggesting that both time in culture and ApoE isoforms influence endolysosomal trafficking.ConclusionThe endolysosomal pathway is a highly dynamic and important system for all cells, including neurons and is increasingly linked to AD. Our preliminary findings point toward an ApoE isoform effect on endosomal trafficking and that its dynamics might be disrupted by ApoE4.

Mechanisms of reduced Apolipoprotein E4 lipidation in iPSC‐derived astrocytes

AbstractBackgroundCarrying the apolipoprotein E ε4 (APOE4) allele is the highest known genetic risk factor for late‐onset Alzheimer’s disease (LOAD). Although how APOE4 leads to LOAD is not known, dysregulated endo‐lysosomal trafficking and lipid metabolism are key defects associated with APOE4. Cholesterol accumulation in glia instigates intracellular trafficking defects, increases cellular stress, and disrupts secretion. Additionally, enlarged endosomes were observed in the frontal lobes of APOE4‐carrying individuals, decades before the appearance of Aβ fibrilization. Therefore, we asked if cholesterol and endo‐lysosomal trafficking dysregulation in APOE4 carriers were due to differential lipidation of APOE3 and APOE4.MethodLocalization of ATP Binding Cassette Subfamily A Member 1 (ABCA1) in early, late, and recycling endosomal compartments were analyzed in human isogenic induced pluripotent stem cell (iPSC)‐derived neural cells and in cell lines treated with recombinant APOE in microscopic images. Membrane and cytoplasmic ABCA1 were compared using Western blots. Secreted high‐density lipoprotein (HDL) particles containing fluorescently tagged APOE3 or APOE4 were analyzed using ion‐mobility spectrometry. CS‐6253, an ABCA1 agonist peptide, was used to increase APOE lipidation.ResultThe protein levels of ABCA1, which mediates cholesterol and phospholipid efflux to the nascent apolipoprotein particles, were lower in APOE4/4 iPSC‐derived astrocytes compared to astrocytes derived from isogenic APOE3/3 iPSCs. Advanced image analysis revealed lower ABCA1 protein levels in Rab11+ recycling endosomes and less colocalization between ABCA1 and Rab7, a late endosome marker, in APOE4/4 astrocytes than APOE3/3 astrocytes. Consequently, less ABCA1 was detected at the cell membrane. Combined with previous data, these findings suggest APOE4 lipidation was reduced due to reduced ABCA1 activity. Additionally, BHK cells treated with recombinant APOE3 or APOE4 showed differences in ABCA1 localization that were altered by increasing APOE lipidation by activating ABCA1 via CS‐6253. Finally, fluorescently tagged APOE3 and APOE4 were incorporated in HDL particles and were secreted whose sizes were altered after CS‐6253 treatment, suggesting differences in lipidation levels.ConclusionWe demonstrated that diminished APOE4 lipidation is, at least in part, due to dysregulation of endo‐lysosomal trafficking of ABCA1 and that increasing APOE4 lipidation could alleviate cellular defects.

Adding hydrophobicity or positive charges to the cytosolic half of the α-synuclein 3-11 helix increases membrane association and S129 phosphorylation.

The neuronal protein α-synuclein is centrally involved in the neurodegeneration occurring in Parkinson's disease and related synucleinopathies. α-Synuclein's membrane-induced 3-11 helix conformation has a hydrophobic membrane-embedded half and a hydrophilic cytosolic half. Here, we studied the significance of(a) the surprising hydrophobicity of amino-acids at cytosol-exposed helix position 8; (b) the absence of positively charged lysine/arginine from all cytosol-exposed positions (1-5-8-9). We found that (a) further increasing hydrophobicity or adding lysine, but not glutamate, at position 8 augments both membrane interaction and S129 phosphorylation; (b) adding lysines at cytosol-exposed positions 1, 5, 8, or 9 has similar effects. Variants abundantly presentin membranes by biochemical fractionation markedly colocalized withtransferrin-receptor (an endosomal marker) in immunofluorescence-microscopy, indicating accumulation at vesicle membranes. Thus, we observed a striking correlation between membrane attraction and S129 phosphorylation, relevant for understanding α-synuclein biology in health and disease.

NDP52 SUMOylation contributes to low-dose X-rays-induced cardiac hypertrophy through PINK1/Parkin-mediated mitophagy via MUL1/SUMO2 signalling.

Radiation-induced heart damage caused by low-dose X-rays has a significant impact on tumour patients' prognosis, with cardiac hypertrophy being the most severe noncarcinogenic adverse effect. Our previous study demonstrated that mitophagy activation promoted cardiac hypertrophy, but the underlying mechanisms remained unclear. In the present study, PARL-IN-1 enhanced excessive hypertrophy of cardiomyocytes and exacerbated mitochondrial damage.Isobaric tags for relative and absolute quantification-based quantitative proteomics identified NDP52 as a crucial target mediating cardiac hypertrophy induced by low-dose X-rays. SUMOylation proteomics revealed that the SUMO E3 ligase MUL1 facilitated NDP52 SUMOylation through SUMO2. Co-IP coupled with LC-MS/MS identified a critical lysine residue at position 262 of NDP52 as the key site for SUMO2-mediated SUMOylation of NDP52. The point mutation plasmid NDP52K262R inhibited mitophagy under MUL1 overexpression, as evidenced by inhibition of LC3 interaction with NDP52, PINK1 and LAMP2A. A mitochondrial dissociation study revealed that NDP52K262R inhibited PINK1 targeting to endosomes early endosomal marker (EEA1), late/lysosome endosomal marker (LAMP2A) and recycling endosomal marker (RAB11), and laser confocal microscopy confirmed that NDP52K262R impaired the recruitment of mitochondria to the autophagic pathway through EEA1/RAB11 and ATG3, ATG5, ATG16L1and STX17, but did not affect mitochondrial delivery to lysosomes via LAMP2A for degradation. In conclusion, our findings suggest that MUL1-mediated SUMOylation of NDP52 plays a crucial role in regulating mitophagy in the context of low-dose X-ray-induced cardiac hypertrophy. Two hundred sixty-secondlysine of NDP52 is identified as a key SUMOylation site for low-dose X-ray promoting mitophagy activation and cardiac hypertrophy. Collectively, this study provides novel implications for the development of therapeutic strategies aimed at preventing the progression of cardiac hypertrophy induced by low-dose X-rays.

Timing of Phagosome Maturation Depends on Their Transport Switching from Actin to Microtubule Tracks.

Phagosomes, specialized membrane compartments responsible for digesting internalized pathogens, undergo sequential dynamic and biochemical changes as they mature from nascent phagosomes to degradative phagolysosomes. Maturation of phagosomes depends on their transport along actin filaments and microtubules. However, the specific quantitative relationship between the biochemical transformation and transport dynamics remains poorly characterized. The autonomous nature of phagosomes, moving and maturing at different rates, makes understanding this relationship challenging. Addressing this challenge, in this study we engineered particle sensors to image and quantify single phagosomes' maturation. We found that as phagosomes move from the actin cortex to microtubule tracks, the timing of their actin-to-microtubule transition governs the duration of the early phagosome stage before acquiring degradative capacities. Prolonged entrapment of phagosomes in the actin cortex extends the early phagosome stage by delaying the dissociation of early endosome markers and phagosome acidification. Conversely, a shortened transition from actin- to microtubule-based movements causes the opposite effect on phagosome maturation. These results suggest that the actin- and microtubule-based transport of phagosomes functions like a "clock" to coordinate the timing of biochemical events during phagosome maturation, which is crucial for effective pathogen degradation.

SifA SUMOylation governs Salmonella Typhimurium intracellular survival via modulation of lysosomal function.

One of the mechanisms shaping the pathophysiology during the infection of enteric pathogen Salmonella Typhimurium is host PTM machinery utilization by the pathogen encoded effectors. Salmonella Typhimurium (S. Tm) during infection in host cells thrives in a vacuolated compartment, Salmonella containing vacuole (SCV), which sequentially acquires host endosomal and lysosomal markers. Long tubular structures, called as Salmonella induced filaments (SIFs), are further generated by S. Tm, which are known to be required for SCV's nutrient acquisition, membrane maintenance and stability. A tightly coordinated interaction involving prominent effector SifA and various host adapters PLEKHM1, PLEKHM2 and Rab GTPases govern SCV integrity and SIF formation. Here, we report for the first time that the functional regulation of SifA is modulated by PTM SUMOylation at its 11th lysine. S. Tm expressing SUMOylation deficient lysine 11 mutants of SifA (SifAK11R) is defective in intracellular proliferation due to compromised SIF formation and enhanced lysosomal acidification. Furthermore, murine competitive index experiments reveal defective in vivo proliferation and weakened virulence of SifAK11R mutant. Concisely, our data reveal that SifAK11R mutant nearly behaves like a SifA knockout strain which impacts Rab9-MPR mediated lysosomal acidification pathway, the outcome of which culminates in reduced bacterial load in in vitro and in vivo infection model systems. Our results bring forth a novel pathogen-host crosstalk mechanism where the SUMOylation of effector SifA regulated S. Tm intracellular survival.

Accumulation and neuroprotective effects of lithium on hepatocellular carcinoma mice model

AimThe peripheral tumor growth is accompanied by the accumulation of inflammatory mediators in the blood that can negatively influence blood-brain barrier function and neuronal structure and develop the cancer-associated depression. The aim of the study was to evaluate the neurobiological effects of lithium on hepatocellular carcinoma mice model. MethodsIn this study we analyzed the locomotor activity of lithium-treated tumor-bearing mice using the Phenomaster instrument. Inductively coupled plasma mass-spectral analysis was used to determine lithium levels in blood, brain, liver, kidneys, tumors and muscle tissues. The prefrontal cortex neurons ultrastructure was assessed by transmission electron microscopy. Expression of BDNF, GRP78, EEA1, LAMP1, and LC3 beta in neurons was determined by immunohistochemical analysis. ResultsA decrease in locomotor activity was found in animals with tumors. At the same time, the low expression levels of the neurotrophic factor BDNF and early endosomal marker EEA1 were revealed, as well as the decreased amount of synaptic vesicles and synapses was shown. Signs of endoplasmic reticulum stress and autophagy development in neurons of animals with tumors were noted. Lithium carbonate administration had a corrective effect on animal’s behavior and the prefrontal cortex neurons structure. ConclusionsIn summary, lithium can restore the neuronal homeostasis in tumor-bearing mice.