Endosomal trafficking defects can induce calcium dependent azole tolerance inCandida albicans.

The azole antifungals arrest fungal growth through inhibition of ergosterol biosynthesis. We recently reported that avps21Δ/Δmutant, deficient in membrane trafficking through the late endosome/pre-vacuolar compartment (PVC), continues to grow in the presence of the azoles, despite depletion of cellular ergosterol. Herein, we report that thevps21Δ/Δmutant exhibits less plasma membrane damage upon azole treatment than wild-type, as measured by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that thevps21Δ/Δmutant has abnormal levels of intracellular Ca2+, and in the presence of fluconazole, enhanced expression of a calcineurin responsiveRTA2-GFPreporter. Furthermore, the azole tolerance phenotype of thevps21Δ/Δmutant is dependent upon both extracellular calcium levels and calcineurin activity. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment, and indicate that this may occur through modulation of calcium and calcineurin dependent responses.

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Trafficking through the Late Endosome Significantly Impacts Candida albicans Tolerance of the Azole Antifungals

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04239-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2410-2420 ◽

Cited By ~ 17

Author(s):

Arturo Luna-Tapia ◽

Morgan E. Kerns ◽

Karen E. Eberle ◽

Branko S. Jursic ◽

Glen E. Palmer

Keyword(s):

Candida Albicans ◽

Membrane Trafficking ◽

Ergosterol Biosynthesis ◽

Endosomal Trafficking ◽

Membrane Function ◽

Content Type ◽

Azole Antifungals ◽

Trafficking Defects ◽

Lanosterol Demethylase ◽

Cellular Compartments

ABSTRACTThe azole antifungals block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of “toxic” sterol intermediates are both thought to compromise plasma membrane function. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of theCandida albicansvacuole and to determine whether, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of theC. albicansvacuole can be observed as an early consequence of azole treatment, and it precedes significant growth inhibition. In addition, aC. albicansvps21Δ/Δ mutant, blocked in membrane trafficking through the late endosomal prevacuolar compartment (PVC), is able to grow significantly more than the wild type in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, thevps21Δ/Δ mutant is able to grow despite the depletion of cellular ergosterol. This phenotype resembles an exaggerated form of “trailing growth” that has been described for some clinical isolates. In contrast, thevps21Δ/Δ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. On the basis of these results, we propose that endosomal trafficking defects may lead to the cellular “redistribution” of the sterol intermediates that accumulate following inhibition of ergosterol biosynthesis. Furthermore, the destination of these intermediates, or the precise cellular compartments in which they accumulate, may be an important determinant of their toxicity and thus ultimately antifungal efficacy.

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Inactivation of lmpA, Encoding a LIMPII-related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-null Mutants

Molecular Biology of the Cell ◽

10.1091/mbc.11.6.2019 ◽

2000 ◽

Vol 11 (6) ◽

pp. 2019-2031 ◽

Cited By ~ 29

Author(s):

Lesly Temesvari ◽

Linyi Zhang ◽

Brent Fodera ◽

Klaus-Peter Janssen ◽

Michael Schleicher ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Membrane Trafficking ◽

Fluid Phase ◽

Actin Binding ◽

Endosomal Trafficking ◽

Wild Type ◽

Gene Encoding ◽

Biochemical Assays ◽

Endosomal Pathway ◽

Trafficking Defects

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.

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

Science Advances ◽

10.1126/sciadv.abc6345 ◽

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.

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Plasma membrane integrity: implications for health and disease

BMC Biology ◽

10.1186/s12915-021-00972-y ◽

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.

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

Biology Open ◽

10.1242/bio.035287 ◽

2018 ◽

Vol 7 (10) ◽

pp. bio035287 ◽

Cited By ~ 2

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

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

Journal of Investigative Dermatology ◽

10.1046/j.1523-1747.1998.00091.x ◽

1998 ◽

Vol 110 (1) ◽

pp. 79-83 ◽

Cited By ~ 10

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

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

10.1101/747014 ◽

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

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The early endosomal protein Rab21 is critical for enterocyte functions and intestinal homeostasis

10.1101/2020.11.18.378984 ◽

2020 ◽

Author(s):

Sonya Nassari ◽

Dominique Lévesque ◽

François-Michel Boisvert ◽

Steve Jean

Keyword(s):

Membrane Trafficking ◽

Genetic Diseases ◽

Growth Factor Receptor ◽

Small Gtpase ◽

Intestinal Morphology ◽

Endosomal Trafficking ◽

Intestinal Homeostasis ◽

Compensatory Proliferation ◽

Epidermal Growth

ABSTRACTMembrane trafficking is defined as the vesicular transport of molecules into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to genetic diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo model system, we investigated enterocyte-specific functions for the early endosomal trafficking machinery in gut homeostasis. We focused on the small GTPase Rab21, which regulates specific steps in early endosomal trafficking. Rab21-depleted guts showed severe abnormalities in intestinal morphology, with deregulated homeostasis associated with a gain in mitotic cells and increased cell death. Increases in both apoptosis and yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using a RNA interference screen, we identified specific regulators of autophagy and membrane trafficking that phenocopied Rab21 loss. We further showed that Rab21-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling, and identified improperly trafficked cargoes in Rab21-depleted enterocytes. Our data shed light on an important role for early endosomal trafficking, and particularly Rab21, in enterocyte-mediated intestinal homeostasis.

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