scholarly journals Aggregatibacter actinomycetemcomitans LtxA Hijacks Endocytic Trafficking Pathways in Human Lymphocytes

Pathogens ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 74 ◽  
Author(s):  
Edward T Lally ◽  
Kathleen Boesze-Battaglia ◽  
Anuradha Dhingra ◽  
Nestor M Gomez ◽  
Jinery Lora ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Human Lymphocytes ◽  
Membrane Damage ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Jurkat Cells ◽  
Endocytic Trafficking ◽  
Plasma Membrane Damage ◽  
Human T Lymphocyte

Leukotoxin (LtxA), from oral pathogen Aggregatibacter actinomycetemcomitans, is a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.

scholarly journals Aggregatibacter actinomycetemcomitansLtxA hijacks endocytic trafficking pathways in human lymphocytes

2019 ◽  
Author(s):  
Edward T Lally ◽  
Kathleen Boesze-Battaglia ◽  
Anuradha Dhingra ◽  
Nestor M Gomez ◽  
Claire H Mitchell ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Human Lymphocytes ◽  
Membrane Damage ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Jurkat Cells ◽  
Endocytic Trafficking ◽  
Recycling Endosomes ◽  
Plasma Membrane Damage ◽  
Human T Lymphocyte

ABSTRACTLeukotoxin (LtxA) from oral pathogenAggregatibacter actinomycetemcomitansis a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18) expressing leukocytes and ultimately causes cell death; however toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5 knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pH. Our results demonstrate that LtxA/LFA-1 complex gains an access to the cytosol of Jurkat cells without evidence of plasma membrane damage utilizing dynamin-dependent and clathrin-independent mechanism. Upon internalization LtxA follows the LFA-1 endocytic trafficking pathways as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp2) and CD11a. Knockdown of Rab5a resulted in loss of susceptibility of Jurkat cells to LtxA cytotoxicity suggesting that late events of LtxA endocytic trafficking are required for toxicity. The toxin trafficking via the degradation endocytic pathway may culminate in delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.

The Efects of Spaceflight on Mitochondria in Human Lymphocytes (Jurkat).

1999 ◽  
Vol 5 (S2) ◽  
pp. 1118-1119
Author(s):  
Heide Schatten ◽  
Marian Lewis
Keyword(s):  
Mitochondrial Membrane ◽  
Space Shuttle ◽  
Human Lymphocytes ◽  
Jurkat Cells ◽  
Apoptotic Bodies ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Alterations ◽  
Mitochondrial Volume ◽  
Number Of Cells ◽  
Related Increase

Spaceflight induced mitochondrial alterations have been reported for muscle and may be associated with altered physiological functions in space. Mitochondrial alterations are also indicative of preapoptotic events which are seen in greater amounts in cells exposed to spaceflight when compared with cells cultured at 1 g. Preapoptotic mitochondrial changes include alterations of processes at the inner mitochondrial membrane and can result in changes in mitochondrial volume. Higher amounts of oxidative stress during space flight may be one of the causes for changes which lead to apoptosis. Jurkat cells flown on the STS-76 space shuttle mission showed an increase in the number of cells with apoptotic bodies early in the mission and a time-dependent, microgravity-related increase in the Fas/APO-1 cell death factor. Here we investigated the morphology of mitochondria in Jurkat cells exposed to spaceflight during the STS-76 mission.

scholarly journals A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

2021 ◽  
Vol 7 (13) ◽  
pp. eabc6345
Author(s):  
Shrawan Kumar Mageswaran ◽  
Wei Yuan Yang ◽  
Yogaditya Chakrabarty ◽  
Catherine M. Oikonomou ◽  
Grant J. Jensen
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Membrane Damage ◽  
Light And Electron Microscopy ◽  
Actin Dynamics ◽  
Endosomal Sorting ◽  
Plasma Membrane Damage ◽  
Cryo Electron Tomography ◽  
Vacuolar Protein

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

scholarly journals Plasma membrane integrity: implications for health and disease

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Dustin A. Ammendolia ◽  
William M. Bement ◽  
John H. Brumell
Keyword(s):  
Plasma Membrane ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Whole Body ◽  
Plasma Membrane Integrity ◽  
Plasma Membrane Damage ◽  
Cellular Environment ◽  
Health And Disease ◽  
Repair Pathways ◽  
The Impact

AbstractPlasma membrane integrity is essential for cellular homeostasis. In vivo, cells experience plasma membrane damage from a multitude of stressors in the extra- and intra-cellular environment. To avoid lethal consequences, cells are equipped with repair pathways to restore membrane integrity. Here, we assess plasma membrane damage and repair from a whole-body perspective. We highlight the role of tissue-specific stressors in health and disease and examine membrane repair pathways across diverse cell types. Furthermore, we outline the impact of genetic and environmental factors on plasma membrane integrity and how these contribute to disease pathogenesis in different tissues.

scholarly journals Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore

Biology Open ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. bio035287 ◽  
Author(s):  
Lars Nygård Skalman ◽  
Mikkel R. Holst ◽  
Elin Larsson ◽  
Richard Lundmark
Keyword(s):  
Plasma Membrane ◽  
Membrane Damage ◽  
Listeriolysin O ◽  
Membrane Pore ◽  
Plasma Membrane Damage

scholarly journals Benzoyl Peroxide Increases UVA-Induced Plasma Membrane Damage and Lipid Oxidation in Murine Leukemia L1210 Cells

1998 ◽  
Vol 110 (1) ◽  
pp. 79-83 ◽  
Author(s):  
Sally H. Ibbotson ◽  
Christopher R. Lambert ◽  
Michael N. Moran ◽  
Mary C. Lynch ◽  
Irene E. Kochevar
Keyword(s):  
Plasma Membrane ◽  
Lipid Oxidation ◽  
Benzoyl Peroxide ◽  
Membrane Damage ◽  
Plasma Membrane Damage ◽  
L1210 Cells ◽  
Murine Leukemia

scholarly journals Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

2019 ◽  
Author(s):  
Kai S. Beckwith ◽  
Marianne S. Beckwith ◽  
Sindre Ullmann ◽  
Ragnhild Sætra ◽  
Haelin Kim ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Plasma Membrane ◽  
Membrane Damage ◽  
Common Mechanism ◽  
Mycobacterium Tuberculosis Infection ◽  
Plasma Membrane Damage ◽  
Cytosolic Side ◽  
Infected Cells ◽  
Mediated Contact ◽  
Spread Of Infection

AbstractMycobacterium tuberculosis (Mtb) is a major global health problem and causes extensive cytotoxicity in patient cells and tissues. Here we define an NLRP3, caspase-1 and gasdermin D-mediated pathway to pyroptosis in human monocytes following exposure to Mtb. We demonstrate an ESX-1 mediated, contact-induced plasma membrane (PM) damage response that occurs during phagocytosis or from the cytosolic side of the PM after phagosomal rupture in Mtb infected cells. This PM injury in turn causes K+ efflux and activation of NLRP3 dependent IL-1β release and pyroptosis, facilitating the spread of Mtb to neighbouring cells. Further we reveal a dynamic interplay of pyroptosis with ESCRT-mediated PM repair. Collectively, these findings reveal a novel mechanism for pyroptosis and spread of infection acting through dual PM disturbances both during and after phagocytosis. We also highlight dual PM damage as a common mechanism utilized by other NLRP3 activators that have previously been shown to act through lysosomal damage.Graphical abstract

scholarly journals HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

2018 ◽  
Author(s):  
Angela Steinauer ◽  
Jonathan R. LaRochelle ◽  
Rebecca Wissner ◽  
Samuel Berry ◽  
Alanna Schepartz
Keyword(s):  
Cell Penetrating Peptides ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Endosomal Escape ◽  
Stapled Peptides ◽  
Critical Determinant ◽  
Small Proteins ◽  
Primary Means ◽  
The Difference

AbstractProtein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered that cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine motif traffic readily to the cytosol and nucleus with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using a broad spectrum of techniques, including an RNA interference (RNAi) screen, fluorescence correlation spectroscopy (FCS), and confocal imaging, we identify VPS39—a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex—as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. Subsequent multi-color confocal imaging studies identify CPMPs within the endosomal lumen, particularly within the intraluminal vesicles (ILVs) of Rab7+ and Lamp1+ endosomes that are the products of HOPS-mediated fusion. These results suggest that CPMPs require HOPS to reach ILVs—an environment that serves as a prerequisite for efficient endosomal escape.

Membrane outer leaflet is the primary regulator of membrane damage induced by silica nanoparticles in vesicles and erythrocytes

2019 ◽  
Vol 6 (4) ◽  
pp. 1219-1232 ◽  
Author(s):  
Saeed Nazemidashtarjandi ◽  
Amir M. Farnoud
Keyword(s):  
Plasma Membrane ◽  
Silica Nanoparticles ◽  
Membrane Damage ◽  
Engineered Nanoparticles ◽  
Cell Toxicity ◽  
Outer Leaflet ◽  
Plasma Membrane Damage

Plasma membrane damage is one of the primary mechanisms through which engineered nanoparticles induce cell toxicity.

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