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1.
J Physiol ; 602(5): 891-912, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38429930

ABSTRACT

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.


Subject(s)
NAD , Proteomics , Humans , Adult , Infant, Newborn , NAD/metabolism , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Mitochondria, Muscle/metabolism , Lipid Droplets/metabolism
2.
Cells ; 12(20)2023 10 18.
Article in English | MEDLINE | ID: mdl-37887318

ABSTRACT

A basic process in cancer is the breaching of basement-membrane barriers to permit tissue invasion. Cancer cells can use proteases and physical mechanisms to produce initial holes in basement membranes, but how cells squeeze through this barrier into matrix environments is not well understood. We used a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize the dynamic early steps in cancer-cell invasion across this barrier. We demonstrate that certain cancer cells extend exceptionally long (~30-100 µm) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. Concurrently, these long, organelle-rich protrusions pull surrounding collagen inward while propelling cancer cells outward through perforations in the basement-membrane barrier. These exceptionally long, contractile cellular protrusions can facilitate the breaching of the basement-membrane barrier as a first step in cancer metastasis.


Subject(s)
Actins , Collagen , Humans , Cell Movement , Collagen/metabolism , Basement Membrane/metabolism , Actins/metabolism , Neoplasm Invasiveness
3.
bioRxiv ; 2023 Sep 15.
Article in English | MEDLINE | ID: mdl-37745366

ABSTRACT

Adverse social determinants of health (aSDoH) are associated with obesity and related comorbidities like diabetes, cardiovascular disease, and cancer. Obesity is also associated with natural killer cell (NK) dysregulation, suggesting a potential mechanistic link. Therefore, we measured NK phenotypes and function in a cohort of African-American (AA) women from resource-limited neighborhoods. Obesity was associated with reduced NK cytotoxicity and a shift towards a regulatory phenotype. In vitro, LDL promoted NK dysfunction, implicating hyperlipidemia as a mediator of obesity-related immune dysregulation. Dual specific phosphatase 1 (DUSP1) was induced by LDL and was upregulated in NK cells from subjects with obesity, implicating DUSP1 in obesity-mediated NK dysfunction. In vitro, DUSP1 repressed LAMP1/CD107a, depleting NK cells of functional lysosomes to prevent degranulation and cytokine secretion. Together, these data provide novel mechanistic links between aSDoH, obesity, and immune dysregulation that could be leveraged to improve outcomes in marginalized populations.

4.
Adv Biol (Weinh) ; 7(10): e2200202, 2023 10.
Article in English | MEDLINE | ID: mdl-37140138

ABSTRACT

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.


Subject(s)
Mitochondria , Mitochondrial Membranes , Mice , Animals , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Microscopy, Electron, Scanning , Cells, Cultured
5.
Nat Commun ; 13(1): 6622, 2022 11 04.
Article in English | MEDLINE | ID: mdl-36333356

ABSTRACT

Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they might affect contractile fiber-type. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm, identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors cut, salm, and H15.


Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Proteomics , Transcription Factors/genetics , Transcription Factors/metabolism , Muscle, Skeletal/metabolism , T-Box Domain Proteins/metabolism
6.
Nat Commun ; 13(1): 6058, 2022 10 13.
Article in English | MEDLINE | ID: mdl-36229433

ABSTRACT

Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.


Subject(s)
Muscle, Striated , Sarcomeres , Actins/metabolism , Adenosine Triphosphate/metabolism , Animals , Humans , Mice , Mitochondria , Muscle Contraction , Muscle, Striated/metabolism , Myosins/metabolism , Sarcomeres/metabolism
7.
Proc Natl Acad Sci U S A ; 119(43): e2209750119, 2022 10 25.
Article in English | MEDLINE | ID: mdl-36252017

ABSTRACT

Mucins are large, highly glycosylated transmembrane and secreted proteins that line and protect epithelial surfaces. However, the details of mucin biosynthesis and packaging in vivo are largely unknown. Here, we demonstrate that multiple distinct mucins undergo intragranular restructuring during secretory granule maturation in vivo, forming unique structures that are spatially segregated within the same granule. We further identify temporally-regulated genes that influence mucin restructuring, including those controlling pH (Vha16-1), Ca2+ ions (fwe) and Cl- ions (Clic and ClC-c). Finally, we show that altered mucin glycosylation influences the dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular segregation of mucins through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct mucins are efficiently packaged into and secreted from secretory granules may provide insight into diseases resulting from defects in mucin secretion.


Subject(s)
Mucins , Secretory Vesicles , Cytoplasmic Granules/metabolism , Glycosylation , Mucins/metabolism , Secretory Vesicles/metabolism
8.
Philos Trans R Soc Lond B Biol Sci ; 377(1864): 20210322, 2022 11 21.
Article in English | MEDLINE | ID: mdl-36189814

ABSTRACT

The heart meets the high energy demands of constant muscle contraction and calcium cycling primarily through the conversion of fatty acids into adenosine triphosphate (ATP) by a large volume of mitochondria. As such, the spatial relationships among lipid droplets (LDs), mitochondria, the sarcotubular system and the contractile apparatus are critical to the efficient distribution of energy within the cardiomyocyte. However, the connectivity among components of the cardiac cellular energy distribution system during postnatal development remains unclear. Here, we use volume electron microscopy to demonstrate that the sarcomere branches uniting the myofibrillar network occur more than twice as frequently during early postnatal development as in mature cardiomyocytes. Moreover, we show that the mitochondrial networks arranged in parallel to the contractile apparatus are composed of larger, more compact mitochondria with greater connectivity to adjacent mitochondria in mature as compared with early postnatal cardiomyocytes. Finally, we find that connectivity among mitochondria, LDs and the sarcotubular network is greater in developing than in mature muscles. These data suggest that physical connectivity among cellular structures may facilitate the communication needed to coordinate developmental processes within the cardiac muscle cell. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.


Subject(s)
Calcium , Myocytes, Cardiac , Adenosine Triphosphate/metabolism , Calcium/metabolism , Fatty Acids/metabolism , Mitochondria , Myocytes, Cardiac/metabolism
9.
Int J Mol Sci ; 23(12)2022 Jun 17.
Article in English | MEDLINE | ID: mdl-35743192

ABSTRACT

Lysyl oxidase (LOX) is a copper-binding enzyme that cross-links elastin and collagen. The dominant LOX variation contributes to familial thoracic aortic aneurysm. Previously reported murine Lox mutants had a mild phenotype and did not dilate without drug-induced provocation. Here, we present a new, more severe mutant, Loxb2b370.2Clo (c.G854T; p.Cys285Phe), whose mutation falls just N-terminal to the copper-binding domain. Unlike the other mutants, the C285F Lox protein was stably produced/secreted, and male C57Bl/6J Lox+/C285F mice exhibit increased systolic blood pressure (BP; p < 0.05) and reduced caliber aortas (p < 0.01 at 100mmHg) at 3 months that independently dilate by 6 months (p < 0.0001). Multimodal imaging reveals markedly irregular elastic sheets in the mutant (p = 2.8 × 10−8 for breaks by histology) that become increasingly disrupted with age (p < 0.05) and breeding into a high BP background (p = 6.8 × 10−4). Aortic dilation was amplified in males vs. females (p < 0.0001 at 100mmHg) and ameliorated by castration. The transcriptome of young Lox mutants showed alteration in dexamethasone (p = 9.83 × 10−30) and TGFß-responsive genes (p = 7.42 × 10−29), and aortas from older C57Bl/6J Lox+/C285F mice showed both enhanced susceptibility to elastase (p < 0.01 by ANOVA) and increased deposition of aggrecan (p < 0.05). These findings suggest that the secreted Lox+/C285F mutants produce dysfunctional elastic fibers that show increased susceptibility to proteolytic damage. Over time, the progressive weakening of the connective tissue, modified by sex and blood pressure, leads to worsening aortic disease.


Subject(s)
Elastic Tissue , Protein-Lysine 6-Oxidase , Animals , Aorta/metabolism , Blood Pressure , Copper , Dilatation, Pathologic/pathology , Elastic Tissue/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Protein-Lysine 6-Oxidase/genetics , Protein-Lysine 6-Oxidase/metabolism
10.
Sci Adv ; 8(24): eabm6049, 2022 Jun 17.
Article in English | MEDLINE | ID: mdl-35714180

ABSTRACT

Vesicle fusion at preestablished plasma membrane release sites releases transmitters and hormones to mediate fundamental functions like neuronal network activities and fight-or-flight responses. This half-a-century-old concept-fusion at well-established release sites in excitable cells-needs to be modified to include the sequential compound fusion reported here-vesicle fusion at previously fused Ω-shaped vesicular membrane. With superresolution STED microscopy in excitable neuroendocrine chromaffin cells, we real-time visualized sequential compound fusion pore openings and content releases in generating multivesicular and asynchronous release from single release sites, which enhances exocytosis strength and dynamic ranges in excitable cells. We also visualized subsequent compound fusion pore closure, a new mode of endocytosis termed compound kiss-and-run that enhances vesicle recycling capacity. These results suggest modifying current exo-endocytosis concepts by including rapid release-site assembly at fused vesicle membrane, where sequential compound fusion and kiss-and-run take place to enhance exo-endocytosis capacity and dynamic ranges.

11.
J Clin Invest ; 132(14)2022 07 15.
Article in English | MEDLINE | ID: mdl-35700042

ABSTRACT

Mitochondrial stress triggers a response in the cell's mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a knockin (KI) mouse model and found that mutant CHCHD10 aggregated in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, the survival of CHCHD10-KI mice depended on a protective stress response mediated by the mitochondrial metalloendopeptidase OMA1. The OMA1 stress response acted both locally within mitochondria, causing mitochondrial fragmentation, and signaled outside the mitochondria, activating the integrated stress response through cleavage of DAP3-binding cell death enhancer 1 (DELE1). We additionally identified an isoform switch in the terminal complex of the electron transport chain as a component of this response. Our results demonstrate that OMA1 was critical for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.


Subject(s)
Metalloproteases , Mitochondrial Myopathies , Mitochondrial Proteins , Animals , Metalloproteases/genetics , Metalloproteases/metabolism , Mice , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Myopathies/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mutation , Protein Folding
12.
Nat Commun ; 13(1): 2661, 2022 05 13.
Article in English | MEDLINE | ID: mdl-35562354

ABSTRACT

Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. We recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. Here, we demonstrate the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster. Moreover, we find that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and we show that sarcomere branching can be turned on in IF muscles by salm-mediated conversion to tubular muscles. Finally, we demonstrate that neurochondrin misexpression results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms.


Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Regulation , Mammals/metabolism , Muscle, Skeletal/metabolism , Sarcomeres/metabolism , T-Box Domain Proteins/metabolism , Transcription Factors/metabolism
13.
J Physiol ; 600(3): 547-567, 2022 02.
Article in English | MEDLINE | ID: mdl-34837710

ABSTRACT

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused ion beam electron microscopy imaging and tandem mass tag mass spectrometry proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart, permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogeneous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism. KEY POINTS: Least shrews were studied to explore the relationship between metabolic function, mitochondrial morphology and protein content in different tissues. Liver and kidney mitochondrial content and enzymatic activity approaches that of the heart, indicating similar metabolic demand among tissues that contribute to basal and maximum metabolism. This allows an examination of mitochondrial structure and composition in tissues with similar maximum metabolic demands. Mitochondrial networks only occur in striated muscle. In contrast, the liver and kidney maintain individual mitochondria with limited reticulation. Muscle mitochondrial reticulation is the result of dense ATPase activity and cell-spanning myofibrils which require networking for adequate metabolic support. In contrast, liver and kidney ATPase activity is localized to the endoplasmic reticulum and basolateral membrane, respectively, generating a locally balanced energy conversion and utilization. Mitochondrial morphology is not driven by maximum metabolic demand, but by the cytosolic distribution of energy-utilizing systems set by the functions of the tissue.


Subject(s)
Muscle, Striated , Shrews , Animals , Energy Metabolism/physiology , Mitochondria/metabolism , Muscle, Skeletal/physiology , North America , Shrews/anatomy & histology
14.
Neuron ; 109(19): 3119-3134.e5, 2021 10 06.
Article in English | MEDLINE | ID: mdl-34411513

ABSTRACT

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis = exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation.


Subject(s)
Chromaffin Cells/physiology , Chromaffin Cells/ultrastructure , Endocytosis/physiology , Neuroendocrine Cells/physiology , Neuroendocrine Cells/ultrastructure , Animals , Calcium Signaling , Cattle , Cell Fusion , Cell Membrane/physiology , Cell Membrane/ultrastructure , Computer Systems , Dynamins/physiology , Exocytosis/physiology , Membrane Fusion , Primary Cell Culture , Synaptic Vesicles/metabolism
15.
J Virol ; 95(13): e0246620, 2021 06 10.
Article in English | MEDLINE | ID: mdl-33853959

ABSTRACT

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus responsible for several diseases, including cancers of lymphoid and epithelial cells. EBV cancers typically exhibit viral latency; however, the production and release of EBV through its lytic phase are essential for cancer development. Antiviral agents that specifically target EBV production do not currently exist. Previously, we reported that the proton pump inhibitor tenatoprazole, which blocks the interaction of ubiquitin with the ESCRT-1 factor Tsg101, inhibits production of several enveloped viruses, including EBV. Here, we show that three structurally distinct prazoles impair mature particle formation postreactivation and identify the impact on stages of replication. The prazoles did not impair expression of lytic genes representative of the different kinetic classes but interfered with capsid maturation in the nucleus as well as virion transport from the nucleus. Replacement of endogenous Tsg101 with a mutant Tsg101 refractory to prazole-mediated inhibition rescued EBV release. These findings directly implicate Tsg101 in EBV nuclear egress and identify prazoles as potential therapeutic candidates for conditions that rely on EBV replication, such as chronic active EBV infection and posttransplant lymphoproliferative disorders. IMPORTANCE Production of virions is necessary for the ubiquitous Epstein-Barr virus (EBV) to persist in humans and can set the stage for development of EBV cancers in at-risk individuals. In our attempts to identify inhibitors of the EBV lytic phase, we previously found that a prazole proton pump inhibitor, known to block the interaction of ubiquitin with the ESCRT-1 factor Tsg101, blocks production of EBV. We now find that three structurally distinct prazoles impair maturation of EBV capsids and virion transport from the nucleus and, by interfering with Tsg101, prevent EBV release from lytically active cells. Our findings not only implicate Tsg101 in EBV production but also identify widely used prazoles as candidates to prevent development of posttransplant EBV lymphomas.


Subject(s)
2-Pyridinylmethylsulfinylbenzimidazoles/pharmacology , Antiviral Agents/pharmacology , DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Rabeprazole/pharmacology , Transcription Factors/metabolism , Virus Release/drug effects , A549 Cells , Cell Line, Tumor , Epstein-Barr Virus Infections/prevention & control , HEK293 Cells , Herpesvirus 4, Human/drug effects , Herpesvirus 4, Human/growth & development , Humans , Proton Pump Inhibitors/pharmacology , Viral Load/drug effects , Virus Activation/drug effects , Virus Latency/drug effects , Virus Replication/drug effects
16.
Environ Sci Technol ; 55(9): 6197-6205, 2021 05 04.
Article in English | MEDLINE | ID: mdl-33856208

ABSTRACT

An individual virion was long believed to act as an independent infectious unit in virology, until the recent discovery of vesicle-cloaked virus clusters which has greatly challenged this central paradigm. Vesicle-cloaked virus clusters (also known as viral vesicles) are phospholipid-bilayer encapsulated fluid sacs that contain multiple virions or multiple copies of viral genomes. Norovirus is a global leading causative agent of gastroenteritis, and the reported prevalence of vesicle-cloaked norovirus clusters in stool has raised concerns whether the current disinfection, sanitation, and hygiene practices can effectively control environmental pollution by these pathogenic units. In this study, we have demonstrated that vesicle-cloaked murine norovirus (MNV-1) clusters were highly persistent under temperature variation (i.e., freeze-thaw) and they were partially resistant to detergent decomposition. MNV-1 vesicles were 1.89-3.17-fold more infectious in vitro than their free virus counterparts. Most importantly, MNV-1 vesicles were up to 2.16-times more resistant to UV254 disinfection than free MNV-1 at a low viral load in vitro. Interestingly, with the increase of the viral load, free MNV-1 and MNV-1 vesicles showed equivalent resistance to UV254 disinfection. We show that the increased multiplicity of infection provided by vesicles is in part responsible for these attributes. Our study, for the first time, sheds light on the environmental behavior of vesicle-cloaked virus clusters as unique emerging pathogenic units. Our study highlights the need to revisit current paradigms of disinfection, sanitation, and hygiene practices for protecting public health.


Subject(s)
Caliciviridae Infections , Norovirus , Animals , Disinfection , Feces , Mice
17.
Nat Commun ; 12(1): 1463, 2021 03 05.
Article in English | MEDLINE | ID: mdl-33674590

ABSTRACT

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.


Subject(s)
Kinesins/chemistry , Kinesins/metabolism , Microtubules/metabolism , Adenosine Triphosphate , Animals , Binding Sites , Golgi Apparatus/metabolism , HEK293 Cells , HeLa Cells , Humans , Kinesins/genetics , Mice , Paclitaxel , Protein Transport , RAW 264.7 Cells
18.
Blood ; 137(22): 3116-3126, 2021 06 03.
Article in English | MEDLINE | ID: mdl-33661274

ABSTRACT

The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.


Subject(s)
Anemia, Sickle Cell/blood , Cell-Free Nucleic Acids/blood , DNA Methylation , DNA, Mitochondrial/blood , Adult , Aged , Biomarkers/blood , Extracellular Traps/metabolism , Female , Humans , Inflammation/blood , Male , Membrane Proteins/metabolism , Middle Aged , Nucleotidyltransferases/metabolism , Signal Transduction
19.
Autophagy ; 17(11): 3707-3724, 2021 11.
Article in English | MEDLINE | ID: mdl-33629936

ABSTRACT

The mechanisms orchestrating recycling of lysosomes through autophagic lysosome reformation (ALR) is incompletely understood. Previous data show that genetic depletion of BLOC1S1/GCN5L1/BORCS1 increases autolysosome (AL) accumulation. We postulated that this phenotype may manifest due to perturbed ALR. We explored this in control and bloc1s1 liver-specific knockout (LKO) mouse hepatocytes, showing that in response to nutrient-deprivation LKO's fail to initiate ALR due to blunted lysosomal tubulation. As kinesin motor proteins and the intracellular cytoskeleton are requirements for tubular formation from ALs, we explored the interaction of BLOC1S1 with motor proteins and cytoskeletal factors. BLOC1S1 interacts with the ARL8B-KIF5B (GTPase and kinesin motor protein) complex to recruit KIF5B to ALs. Furthermore, BLOC1S1 interacts with the actin nucleation promoting factor WHAMM, which is an essential structural protein in the initiation of lysosomal tubulation (LT). Interestingly, the genetic reintroduction of BLOC1S1 rescues LT in LKO hepatocytes, but not when KIF5B is concurrently depleted. Finally, given the central role of MTORC1 signaling in ALR initiation, it was interesting that MTORC1 activity was increased despite the absence of LT in LKO hepatocytes. Concurrently, inhibition of MTORC1 abolished BLOC1S1 reconstitution-mediated rescue of LT in LKO hepatocytes. Taken together these data demonstrate that the functional interaction of BLOC1S1 with the kinesin binding complex and the actin cytoskeleton are a requirement for LT which, in parallel with MTORC1 signaling, initiate lysosome recycling via ALR.Abbreviations: 3-MA: 3-methyladenine; AL: autolysosome; ALR: autophagic lysosome reformation; ARL8B: ADP-ribosylation factor-like protein 8B; ARPC2: actin related protein 2/3 complex, subunit 2; ATAT1/αTAT1: alpha tubulin acetyltransferase 1; AVd: autophagic vacuoles, degradative; BLOC1S1/GCN5L1: biogenesis of lysosomal organelles complex-1, subunit 1; CQ: chloroquine; KIF5B: kinesin family member 5B; KLC1: kinesin light chain 1; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LC3B-I: cytosolic form of LC3B; LC3B-II: lipidated form of LC3B; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; LKO: liver-specific knockout; LIs: lysosome inhibitors; LT: lysosomal tubulation; Ly: lysosome; MTORC1: mechanistic target of rapamycin kinase complex 1; PLEKHM2/SKIP: pleckstrin homology domain containing, family M (with RUN domain) member 2; Snapin: SNAP-associated protein; SQSTM1/p62: sequestosome 1; SVPs: synaptic vesicle precursors; TFEB: transcription Factor EB; TFE3: transcription factor E3; WHAMM: WAS protein homolog associated with actin, golgi membranes and microtubules.


Subject(s)
Lysosomes/metabolism , Microtubules/metabolism , Mitochondrial Proteins/metabolism , Nerve Tissue Proteins/metabolism , Actin Cytoskeleton/metabolism , Animals , Autophagy , Hepatocytes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout
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