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Molecular Biology of the Cell Mar 2024The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex...
The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These "HOPS bodies" were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.
Topics: Humans; HeLa Cells; Endosomes; Endocytosis; Intracellular Membranes; Lysosomes
PubMed: 38198575
DOI: 10.1091/mbc.E23-08-0328 -
Current Opinion in Cell Biology Feb 2024The nuclear-localized lamins have long been thought to be the only intermediate filaments (IFs) with an impact on the architecture, properties, and functions of the... (Review)
Review
The nuclear-localized lamins have long been thought to be the only intermediate filaments (IFs) with an impact on the architecture, properties, and functions of the nucleus. Recent studies, however, uncovered significant roles for IFs other than lamins (here referred to as "non-lamin IFs") in regulating key properties of the nucleus in various cell types and biological settings. In the cytoplasm, IFs often occur in the perinuclear space where they contribute to local stiffness and impact the shape and/or the integrity of the nucleus, particularly in cells under stress. In addition, selective non-lamin IF proteins can occur inside the nucleus where they partake in fundamental processes including nuclear architecture and chromatin organization, regulation of gene expression, cell cycle progression, and the repair of DNA damage. This text reviews the evidence supporting a role for non-lamin IF proteins in regulating various properties of the nucleus and highlights opportunities for further study.
Topics: Lamins; Intermediate Filament Proteins; Cell Nucleus; Intermediate Filaments; Nuclear Envelope
PubMed: 38113712
DOI: 10.1016/j.ceb.2023.102303 -
BioEssays : News and Reviews in... Feb 2024Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis-regulated, is associated with...
Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis-regulated, is associated with diseases, tumor formation/growth, and cancer progression. Nuclear Envelope (NE)-budding is a newly appreciated nuclear export pathway for large macromolecular machineries, including those assembled to allow co-regulation of functionally related components, that bypasses canonical nuclear export through nuclear pores. In this pathway, large macromolecular complexes are enveloped by the inner nuclear membrane, transverse the perinuclear space, and then exit through the outer nuclear membrane to release its contents into the cytoplasm. NE-budding is a conserved process and shares many features with nuclear egress mechanisms used by herpesviruses. Despite its biological importance and clinical relevance, little is yet known about the regulatory and structural machineries that allow NE-budding to occur in any system. Here we summarize what is currently known or proposed for this intriguing nuclear export process.
Topics: Nuclear Envelope; Active Transport, Cell Nucleus; Herpesviridae; Cytoplasm; Cell Nucleus
PubMed: 38044581
DOI: 10.1002/bies.202300182 -
Advanced Science (Weinheim,... Nov 2023Advancing the technologies for cellular reprogramming with high efficiency has significant impact on regenerative therapy, disease modeling, and drug discovery....
Advancing the technologies for cellular reprogramming with high efficiency has significant impact on regenerative therapy, disease modeling, and drug discovery. Biophysical cues can tune the cell fate, yet the precise role of external physical forces during reprogramming remains elusive. Here the authors show that temporal cyclic-stretching of fibroblasts significantly enhances the efficiency of induced pluripotent stem cell (iPSC) production. Generated iPSCs are proven to express pluripotency markers and exhibit in vivo functionality. Bulk RNA-sequencing reveales that cyclic-stretching enhances biological characteristics required for pluripotency acquisition, including increased cell division and mesenchymal-epithelial transition. Of note, cyclic-stretching activates key mechanosensitive molecules (integrins, perinuclear actins, nesprin-2, and YAP), across the cytoskeletal-to-nuclear space. Furthermore, stretch-mediated cytoskeletal-nuclear mechano-coupling leads to altered epigenetic modifications, mainly downregulation in H3K9 methylation, and its global gene occupancy change, as revealed by genome-wide ChIP-sequencing and pharmacological inhibition tests. Single cell RNA-sequencing further identifies subcluster of mechano-responsive iPSCs and key epigenetic modifier in stretched cells. Collectively, cyclic-stretching activates iPSC reprogramming through mechanotransduction process and epigenetic changes accompanied by altered occupancy of mechanosensitive genes. This study highlights the strong link between external physical forces with subsequent mechanotransduction process and the epigenetic changes with expression of related genes in cellular reprogramming, holding substantial implications in the field of cell biology, tissue engineering, and regenerative medicine.
Topics: Mechanotransduction, Cellular; Cellular Reprogramming; Induced Pluripotent Stem Cells; Epigenesis, Genetic; RNA
PubMed: 37727069
DOI: 10.1002/advs.202303395 -
Cellular and Molecular Neurobiology Nov 2023Isolated exposure to intermittent hypoxia and permissive hypercapnia activates signaling mechanisms that induce ultrastructural changes in mitochondria and endoplasmic...
Isolated exposure to intermittent hypoxia and permissive hypercapnia activates signaling mechanisms that induce ultrastructural changes in mitochondria and endoplasmic reticulum, accompanied by the development of maximal ischemic tolerance in neurons under the combined influence of these factors. However, there are a lack of data on the combined impact of these factors on the ultrastructure of neuronal organelles. The present study aims to comparatively assess the ultrastructural changes in neurons following isolated and combined exposure to hypoxia and hypercapnia, as well as to correlate these changes with the neuroprotective potential previously observed for these factors. Following a 15-session course of 30-min exposures to permissive hypercapnia (P ≈ 50 mmHg) and/or normobaric hypoxia (P ≈ 150 mmHg), morphometric assessment was conducted to evaluate the extent of ultrastructural changes in hippocampal neurons (mitochondria, perinuclear space, and granular endoplasmic reticulum). It was found that in hippocampal neurons from the CA1 region, permissive hypercapnia resulted in increased mitochondrial size, expansion of membranous compartments of the granular endoplasmic reticulum, and perinuclear space. Normobaric hypoxia affected only mitochondrial size, while hypercapnic hypoxia specifically widened the perinuclear space. These ultrastructural changes objectively reflect varying degrees of the influence of hypoxia and hypercapnia on organelles responsible for energy metabolism, anti-apoptotic, and synthetic functions of neurons. This confirms the effect of potentiation of their neuroprotective effects under combined exposure and highlights the dominant role of the hypercapnic component in this mechanism.
Topics: Humans; Hypercapnia; Hypoxia; Neurons; Cerebral Cortex; Hippocampus
PubMed: 37716927
DOI: 10.1007/s10571-023-01407-8 -
Journal of Virology Sep 2023Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space...
Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-β to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the genus within the subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.
Topics: Humans; Heat Shock Transcription Factors; Heat-Shock Response; Herpesvirus 6, Human; Viral Matrix Proteins; HSP90 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Promoter Regions, Genetic; Virus Replication; Phosphorylation; Gene Expression Regulation, Viral; Signal Transduction
PubMed: 37671864
DOI: 10.1128/jvi.00718-23 -
Frontiers in Cellular and Infection... 2023Ring finger protein 213 (RNF213) is a large E3 ubiquitin ligase with a molecular weight of 591 kDa that is associated with moyamoya disease, a rare cerebrovascular... (Review)
Review
Ring finger protein 213 (RNF213) is a large E3 ubiquitin ligase with a molecular weight of 591 kDa that is associated with moyamoya disease, a rare cerebrovascular disease. It is located in the cytosol and perinuclear space. Missense mutations in this gene have been found to be more prevalent in patients with moyamoya disease compared with that in healthy individuals. Understanding the molecular function of RNF213 could provide insights into moyamoya disease. RNF213 contains a C3HC4-type RING finger domain with an E3 ubiquitin ligase domain and six AAA+ adenosine triphosphatase (ATPase) domains. It is the only known protein with both AAA+ ATPase and ubiquitin ligase activities. Recent studies have highlighted the role of RNF213 in fighting against microbial infections, including viruses, parasites, bacteria, and chlamydiae. This review aims to summarize the recent research progress on the mechanisms of RNF213 in pathogenic infections, which will aid researchers in understanding the antimicrobial role of RNF213.
Topics: Humans; Moyamoya Disease; Ubiquitin-Protein Ligases; Genes, Regulator; Transcription Factors; Anti-Infective Agents; Adenosine Triphosphatases
PubMed: 37655297
DOI: 10.3389/fcimb.2023.1205355 -
Journal of Morphology Sep 2023Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic...
Asymmetry in previtellogenic and early vitellogenic oocytes, ultrastructure of follicular cells and egg envelope in the pigmented sterlet, Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).
Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic oocytes are polarized. Homogeneous ooplasm (contains ribosomes) and granular ooplasm (comprises nuage aggregations of nuclear origin, rough endoplasmic reticulum (RER), Golgi complexes, ribosomes, and mitochondria) are distinguished. Granular ooplasm is initially located near the nucleus, contacts the plasma membrane of the oocyte (oolemma) and forms a thin layer underneath its entire perimeter. Next, a ring that surrounds the nucleus is formed and sends strands directed toward the oolemma. The lipid body composed of lipid droplets forms adjacent to this ring. Later, the granular ooplasm and strands enlarge toward the oolemma, lipid body disperses, and homogeneous ooplasm is no longer present. A thin cortical ooplasm is formed underneath the oolemma and does not contain any organelles. The oocyte nucleus moves to the center. The nucleoplasm contains lampbrush chromosomes, nuclear bodies, and multiple nucleoli. Early vitellogenic oocytes are polarized, too. Three regions in the ooplasm are distinguished: the perinuclear (contains lipid droplets near the nuclear envelope), the endoplasm (contains yolk platelets and lipid droplets), and the periplasm (contains yolk spheres, pigment granules, and microtubules). In all these regions the RER, Golgi complexes, nuage, and mitochondria are present. Micropinocytotic vesicles, Golgi vesicles and precursors of the internal layer of the egg envelope are in the cortical ooplasm. Some FCs delaminate from the follicular epithelium, degenerate and vesicles are released into the perioocytic space. They may contain precursors of egg envelope and may be involved in "cell-cell" communication. The egg envelope (zona radiata, zona pellucida) is made up of three layers: the vitelline envelope (inner layer), the middle layer, and the outer layer. In its deposition, both the oocyte and FCs are engaged.
Topics: Female; Animals; Oocytes; Ovarian Follicle; Fishes; Cytoplasm; Vitellogenesis
PubMed: 37585228
DOI: 10.1002/jmor.21631 -
BioRxiv : the Preprint Server For... Jul 2023TorsinA is an atypical ATPase that lacks intrinsic activity unless it is bound to its activators lamina-associated polypeptide 1 (LAP1) in the perinuclear space or...
TorsinA is an atypical ATPase that lacks intrinsic activity unless it is bound to its activators lamina-associated polypeptide 1 (LAP1) in the perinuclear space or luminal domain-like LAP1 (LULL1) throughout the endoplasmic reticulum. However, the interaction of torsinA with LAP1 and LULL1 has not yet been shown to modulate a defined physiological process in mammals . We previously demonstrated that depletion of torsinA from mouse hepatocytes leads to reduced liver triglyceride secretion and marked steatosis, whereas depletion of LAP1 had more modest similar effects. We now show that depletion of LULL1 alone does not significantly decrease liver triglyceride secretion or cause steatosis. However, simultaneous depletion of both LAP1 and LULL1 from hepatocytes leads to defective triglyceride secretion and marked steatosis similar to that observed with depletion of torsinA. Our results demonstrate that torsinA and its activators dynamically regulate a physiological process in mammals .
PubMed: 37547008
DOI: 10.1101/2023.06.21.545957 -
Medical Physics Sep 2023The introduction of Gold NanoParticles (GNPs) in radiotherapy treatments necessitates considerations such as GNP size, location, and quantity, as well as patient...
BACKGROUND
The introduction of Gold NanoParticles (GNPs) in radiotherapy treatments necessitates considerations such as GNP size, location, and quantity, as well as patient geometry and beam quality. Physics considerations span length scales across many orders of magnitude (nanometer-to-centimeter), presenting challenges that often limit the scope of dosimetric studies to either micro- or macroscopic scales.
PURPOSE
To investigate GNP dose-enhanced radiation Therapy (GNPT) through Monte Carlo (MC) simulations that bridge micro-to-macroscopic scales. The work is presented in two parts, with Part I (this work) investigating accurate and efficient MC modeling at the single cell level to calculate nucleus and cytoplasm Dose Enhancement Factors (n,cDEFs), considering a broad parameter space including GNP concentration, GNP intracellular distribution, cell size, and incident photon energy. Part II then evaluates cell dose enhancement factors across macroscopic (tumor) length scales.
METHODS
Different methods of modeling gold within cells are compared, from a contiguous volume of either pure gold or gold-tissue mixture to discrete GNPs in a hexagonal close-packed lattice. MC simulations with EGSnrc are performed to calculate n,cDEF for a cell with radius µm and nucleus µm considering 10 to 370 keV incident photons, gold concentrations from 4 to 24 mg /g , and three different GNP configurations within the cell: GNPs distributed around the surface of the nucleus (perinuclear) or GNPs packed into one (or four) endosome(s). Select simulations are extended to cells with different cell (and nucleus) sizes: 5 µm (2, 3, and 4 µm), 7.35 µm (4 and 6 µm), and 10 µm (7, 8, and 9 µm).
RESULTS
n,cDEFs are sensitive to the method of modeling gold in the cell, with differences of up to 17% observed; the hexagonal lattice of GNPs is chosen (as the most realistic model) for all subsequent simulations. Across cell/nucleus radii, source energies, and gold concentrations, both nDEF and cDEF are highest for GNPs in the perinuclear configuration, compared with GNPs in one (or four) endosome(s). Across all simulations of the (r , r ) = (7.35, 5) µm cell, nDEFs and cDEFs range from unity to 6.83 and 3.87, respectively. Including different cell sizes, nDEFs and cDEFs as high as 21.5 and 5.5, respectively, are observed. Both nDEF and cDEF are maximized at photon energies above the K- or L-edges of gold by 10 to 20 keV.
CONCLUSIONS
Considering 5000 unique simulation scenarios, this work comprehensively investigates many physics trends on DEFs at the cellular level, including demonstrating that cellular DEFs are sensitive to gold modeling approach, intracellular GNP configuration, cell/nucleus size, gold concentration, and incident source energy. These data should prove especially useful in research as well as treatment planning, allowing one to optimize or estimate DEF using not only GNP uptake, but also account for average tumor cell size, incident photon energy, and intracellular configuration of GNPs. Part II will expand the investigation, taking the Part I cell model and applying it in cm-scale phantoms.
Topics: Humans; Gold; Monte Carlo Method; Metal Nanoparticles; Photons; Radiometry
PubMed: 37211878
DOI: 10.1002/mp.16454