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

2000 ◽  
Vol 11 (6) ◽  
pp. 2019-2031 ◽  
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.

scholarly journals Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

2012 ◽  
Vol 23 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Andrea L. Marat ◽  
Maria S. Ioannou ◽  
Peter S. McPherson
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Small Gtpase ◽  
Actin Binding ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

scholarly journals G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

1998 ◽  
Vol 141 (7) ◽  
pp. 1529-1537 ◽  
Author(s):  
Barbara Peracino ◽  
Jane Borleis ◽  
Tian Jin ◽  
Monika Westphal ◽  
Jean-Marc Schwartz ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
G Protein ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Fluid Phase ◽  
Β Subunit ◽  
Wild Type ◽  
Fluid Phase Endocytosis ◽  
Green Fluorescent

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

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

2016 ◽  
pp. AAC.01034-16 ◽  
Author(s):  
Arturo Luna-Tapia ◽  
Hélène Tournu ◽  
Tracy L. Peters ◽  
Glen E. Palmer
Keyword(s):  
Membrane Trafficking ◽  
Membrane Damage ◽  
Fungal Growth ◽  
Luciferase Reporter ◽  
Ergosterol Biosynthesis ◽  
Endosomal Trafficking ◽  
Calcium Dependent ◽  
Plasma Membrane Damage ◽  
Trafficking Defects ◽  
Azole Tolerance

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.

scholarly journals Low-Dose Fluvoxamine Modulates Endocytic Trafficking of SARS-CoV-2 Spike Protein: A Potential Mechanism for Anti-COVID-19 Protection by Antidepressants

2021 ◽  
Vol 12 ◽  
Author(s):  
Oleg O. Glebov
Keyword(s):  
Membrane Trafficking ◽  
Low Dose ◽  
Protein A ◽  
Antidepressant Drug ◽  
Fluid Phase ◽  
Spike Protein ◽  
Endosomal Trafficking ◽  
Therapeutic Concentration ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

Commonly prescribed antidepressants may be associated with protection against severe COVID-19. The mechanism of their action in this context, however, remains unknown. Here, I investigated the effect of an antidepressant drug fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking

2002 ◽  
Vol 115 (5) ◽  
pp. 899-911 ◽  
Author(s):  
Maria Kauppi ◽  
Anne Simonsen ◽  
Bjørn Bremnes ◽  
Amandio Vieira ◽  
Judy Callaghan ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Hela Cells ◽  
Membrane Trafficking ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Wild Type ◽  
Dynamic Interactions ◽  
Recycling Endosomes ◽  
Endosomal Membrane ◽  
Gtpase Cycle

Rab22a is a small GTPase that is expressed ubiquitously in mammalian tissues and displays the highest sequence homology to Rab5. In BHK-21 cells,overexpression of the wild-type Rab22a caused formation of abnormally large vacuole-like structures containing the early-endosomal antigen EEA1 but not Rab11, a marker of recycling endosomes or the late-endosomal/lysosomal markers LAMP-1 and lyso-bis-phosphatidic acid. In HeLa cells, overexpressed Rab22a was found on smaller EEA1-positive endosomes, but a portion of the protein was also found in the Golgi complex. Using the yeast two-hybrid system and a biochemical pull-down assay, the GTP-bound form of Rab22a was found to interact with the N-terminus of EEA1. In HeLa cells overexpressing Rab22a or its mutants affected in the GTPase cycle, no significant changes were observed in the uptake of Alexa-transferrin. However, the GTPase-deficient Rab22a Q64L mutant caused a redistribution of transferrin-positive endosomes to the leading edges of cells and a fragmentation of the Golgi complex. In BHK cells,the Q64L mutant caused the accumulation of a fluid phase marker,TRITC-dextran, and a lysosomal hydrolase, aspartylglucosaminidase, in abnormal vacuole-like structures that contained both early and late endosome markers. Both the wild-type Rab22a and the Q64L mutant were found to interfere with the degradation of EGF. These results suggest that Rab22a may regulate the dynamic interactions of endosomal compartments and it may be involved in the communication between the biosynthetic and early endocytic pathways.

scholarly journals A role for the actin cytoskeleton in cell death and aging in yeast

2004 ◽  
Vol 164 (6) ◽  
pp. 803-809 ◽  
Author(s):  
Campbell W. Gourlay ◽  
Lindsay N. Carpp ◽  
Paul Timpson ◽  
Steven J. Winder ◽  
Kathryn R. Ayscough
Keyword(s):  
Cell Death ◽  
Actin Cytoskeleton ◽  
Mitochondrial Membrane ◽  
Actin Dynamics ◽  
Ros Production ◽  
Oxygen Species ◽  
Wild Type ◽  
Metabolic Capacity ◽  
Gene Encoding ◽  
Yeast Strains

Several determinants of aging, including metabolic capacity and genetic stability, are recognized in both yeast and humans. However, many aspects of the pathways leading to cell death remain to be elucidated. Here we report a role for the actin cytoskeleton both in cell death and in promoting longevity. We have analyzed yeast strains expressing mutants with either increased or decreased actin dynamics. We show that decreased actin dynamics causes depolarization of the mitochondrial membrane and an increase in reactive oxygen species (ROS) production, resulting in cell death. Important, however, is the demonstration that increasing actin dynamics, either by a specific actin allele or by deletion of a gene encoding the actin-bundling protein Scp1p, can increase lifespan by over 65%. Increased longevity appears to be due to these cells producing lower than wild-type levels of ROS. Homology between Scp1p and mammalian SM22/transgelin, which itself has been isolated in senescence screens, suggests a conserved mechanism linking aging to actin stability.

scholarly journals Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

2021 ◽  
Author(s):  
Lin Zeng ◽  
Alejandro R Walker ◽  
Kyulim Lee ◽  
Zachary A Taylor ◽  
Robert A Burne
Keyword(s):  
Central Carbon Metabolism ◽  
Arginine Deiminase ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Gene Encoding ◽  
Streptococcus Sanguinis ◽  
Biochemical Assays ◽  
Production Of Hydrogen ◽  
Spontaneous Mutants ◽  
Production Of Ammonia

Genetic truncations in a gene encoding a putative glucose-PTS protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. Using an engineered mutant of manL and its complemented derivative, we showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage (~10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. Significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2.

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

2015 ◽  
Vol 59 (4) ◽  
pp. 2410-2420 ◽  
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.

scholarly journals DictyosteliumLvsB Mutants Model the Lysosomal Defects Associated with Chediak-Higashi Syndrome

2002 ◽  
Vol 13 (2) ◽  
pp. 656-669 ◽  
Author(s):  
Edward Harris ◽  
Ning Wang ◽  
Wei-l Wu ◽  
Alisha Weatherford ◽  
Arturo De Lozanne ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Genetic Disorder ◽  
Fluid Phase ◽  
Negative Regulator ◽  
Cysteine Proteinases ◽  
Gene Encoding ◽  
Lysosomal Trafficking ◽  
Lysosome Biogenesis ◽  
Genes Encoding ◽  
Chediak Higashi Syndrome

Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans (“lysosomal trafficking regulator”) or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideumcontains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes,lvsA (large volumesphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, onlylvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, theDictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal inlvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal α-mannosidase were normal, although lvsB mutants inefficiently retained α-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes inlvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis.

scholarly journals Inactivation of Two Dictyostelium discoideum Genes, DdPIK1 and DdPIK2, Encoding Proteins Related to Mammalian Phosphatidylinositide 3-kinases, Results in Defects in Endocytosis, Lysosome to Postlysosome Transport, and Actin Cytoskeleton Organization

1997 ◽  
Vol 136 (6) ◽  
pp. 1271-1286 ◽  
Author(s):  
Greg Buczynski ◽  
Bryon Grove ◽  
Anson Nomura ◽  
Maurice Kleve ◽  
John Bush ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Dictyostelium Discoideum ◽  
Mutant Strain ◽  
Fluorescent Microscopy ◽  
Membrane Traffic ◽  
Fluid Phase ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization ◽  
Endosomal Pathway ◽  
Mutant Cells

Phosphatidylinositide 3-kinases (PI 3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum, DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (Δddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, Δddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme α-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that Δddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1–3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, Δddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

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