scholarly journals The Rab6 Effector Bicaudal D1 Associates with Chlamydia trachomatis Inclusions in a Biovar-Specific Manner

2006 ◽  
Vol 75 (2) ◽  
pp. 781-791 ◽  
Author(s):  
A. R. Moorhead ◽  
K. A. Rzomp ◽  
M. A. Scidmore
Keyword(s):  
Gene Expression ◽  
Chlamydia Trachomatis ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Rab Gtpases ◽  
Specific Manner ◽  
Guanine Nucleotide Binding ◽  
Obligate Intracellular Bacteria ◽  
Green Fluorescent

ABSTRACT Chlamydia species are obligate intracellular bacteria that replicate within a membrane-bound vacuole, the inclusion, which is trafficked to the peri-Golgi region by processes that are dependent on early chlamydial gene expression. Although neither the host nor the chlamydial proteins that regulate the intracellular trafficking have been clearly defined, several enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, including Rab6, are recruited to Chlamydia trachomatis inclusions. To further characterize the association of Rab6 with C. trachomatis inclusions, we examined the intracellular localization of guanine nucleotide-binding mutants of Rab6 and demonstrated that only active GTP-bound and not inactive GDP-bound EGFP-Rab6 mutants were recruited to the inclusion, suggesting that EGFP-Rab6 interacts with the inclusion via a host Rab6 effector or a chlamydial protein that mimics a Rab6 effector. Using EGFP-tagged fusion proteins, we also demonstrated that the Rab6 effector Bicaudal D1 (BICD1) localized to C. trachomatis inclusions in a biovar-specific manner. In addition, we demonstrated that EGFP-Rab6 and its effector EGFP-BICD1 are recruited to the inclusion in a microtubule- and Golgi apparatus-independent but chlamydial gene expression-dependent mechanism. Finally, in contrast to the Rab6-dependent Golgi apparatus localization of endogenous BICD1, EGFP-BICD1 was recruited to the inclusion by a Rab6-independent mechanism. Collectively, these data demonstrate that neither Rab6 nor BICD1 is trafficked to the inclusion via a Golgi apparatus-localized intermediate, suggesting that each protein is trafficked to the C. trachomatis serovar L2 inclusion by a unique, but as-yet-undefined, mechanism.

scholarly journals Multiple Host Proteins That Function in Phosphatidylinositol-4-Phosphate Metabolism Are Recruited to the Chlamydial Inclusion

2010 ◽  
Vol 78 (5) ◽  
pp. 1990-2007 ◽  
Author(s):  
Andrew M. Moorhead ◽  
Joo-Yong Jung ◽  
Asya Smirnov ◽  
Susanne Kaufer ◽  
Marci A. Scidmore
Keyword(s):  
Golgi Complex ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Host Proteins ◽  
Oculocerebrorenal Syndrome ◽  
Green Fluorescent ◽  
Phosphatidylinositol 4 ◽  
Chlamydial Inclusion

ABSTRACT Chlamydiae replicate within a nonacidified vacuole, termed an inclusion. As obligate intracellular bacteria, chlamydiae actively modify their vacuole to exploit host signaling and trafficking pathways. Recently, we demonstrated that several Rab GTPases are actively targeted to the inclusion. To define the biological roles of inclusion localized Rab GTPases, we have begun to identify inclusion-localized Rab effectors. Here we demonstrate that oculocerebrorenal syndrome of Lowe protein 1 (OCRL1), a Golgi complex-localized phosphatidylinositol (PI)-5-phosphatase that binds to multiple Rab GTPases, localizes to chlamydial inclusions. By examining the intracellular localization of green fluorescent protein (GFP) fusion proteins that bind to unique phosphoinositide species, we also demonstrate that phosphatidylinositol-4-phosphate (PI4P), the product of OCRL1, is present at the inclusion membrane. Furthermore, two additional host proteins, Arf1, which together with PI4P mediates the recruitment of PI4P-binding proteins to the Golgi complex, and PI4KIIα, a major producer of Golgi complex-localized PI4P, also localize to chlamydial inclusions. Depletion of OCRL1, Arf1, or PI4KIIα by small interfering RNA (siRNA) decreases inclusion formation and the production of infectious progeny. Infectivity is further decreased in cells simultaneously depleted for all three host proteins, suggesting partially overlapping functions in infected cells. Collectively, these data demonstrate that Chlamydia species create a unique replication-competent vacuolar environment by modulating both the Rab GTPase and the PI composition of the chlamydial inclusion.

scholarly journals Rab GTPases Are Recruited to Chlamydial Inclusions in Both a Species-Dependent and Species-Independent Manner

2003 ◽  
Vol 71 (10) ◽  
pp. 5855-5870 ◽  
Author(s):  
Kimberly A. Rzomp ◽  
Luella D. Scholtes ◽  
Benjamin J. Briggs ◽  
Gary R. Whittaker ◽  
Marci A. Scidmore
Keyword(s):  
Membrane Trafficking ◽  
Chlamydia Pneumoniae ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Specific Interaction ◽  
Rab Gtpases ◽  
Independent Manner ◽  
Inclusion Membrane ◽  
Recycling Endosomes ◽  
Green Fluorescent

ABSTRACT Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.

scholarly journals Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus

2002 ◽  
Vol 76 (2) ◽  
pp. 865-874 ◽  
Author(s):  
Patrice Dunoyer ◽  
Christophe Ritzenthaler ◽  
Odile Hemmer ◽  
Pierre Michler ◽  
Christiane Fritsch
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
De Novo ◽  
Intracellular Localization ◽  
Viral Rna ◽  
Replication Complex ◽  
Green Fluorescent

ABSTRACT RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5′-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas.

Important role for the V-type H+-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor

2004 ◽  
Vol 286 (5) ◽  
pp. E704-E710 ◽  
Author(s):  
Hesham A. W. Tawfeek ◽  
Abdul B. Abou-Samra
Keyword(s):  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Receptor Recycling ◽  
Bafilomycin A1 ◽  
Coated Pits ◽  
Concanamycin A ◽  
Hydrogen Atpase ◽  
Green Fluorescent ◽  
Pthrp Receptor

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.

scholarly journals Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242640
Author(s):  
Jianying Zhang ◽  
Daibang Nie ◽  
Kelly Williamson ◽  
Arthur McDowell ◽  
MaCalus V. Hogan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Treadmill Running ◽  
Whole Body ◽  
Tendon Cells ◽  
Elevated Expression ◽  
Green Fluorescent

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

Intracellular localization and in vivo trafficking of p24A and p23

1999 ◽  
Vol 112 (4) ◽  
pp. 537-548 ◽  
Author(s):  
R. Blum ◽  
F. Pfeiffer ◽  
P. Feick ◽  
W. Nastainczyk ◽  
B. Kohler ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Maximum Speed ◽  
Heterotrimeric G Proteins ◽  
P24 Protein ◽  
Intermediate Compartment ◽  
Copi Vesicle ◽  
Green Fluorescent ◽  
Golgi Network

Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction. Both p24A and p23 cycle continuously between intermediate compartment (IC) elements and the cis-Golgi network. In vivo trafficking of p24A and p23 tagged to green fluorescent protein (GFP) revealed that both proteins travel by large (up to 1 micrometer in length) microtubule-dependent pre-Golgi carriers with a maximum speed of up to 1.6 micrometer s-1 from the IC to the Golgi cisternae. Aluminum fluoride, a general activator of heterotrimeric G-proteins, blocked peripheral pre-Golgi movements of GFP-p24A/p23 and inhibited fluorescence recovery after photobleaching in the perinuclear Golgi area. p24A and p23 are predominantly colocalized. Overexpression of GFP-p24A, to an extent which did not destroy the Golgi complex, induced delocalization of part of the proteins into ER elements. This study therefore gives new insights into the localization and trafficking behavior of the two COPI-binding proteins p24A and p23.

In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members

1998 ◽  
Vol 64 (2) ◽  
pp. 721-732 ◽  
Author(s):  
Søren Møller ◽  
Claus Sternberg ◽  
Jens Bo Andersen ◽  
Bjarke Bak Christensen ◽  
Juan Luis Ramos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microbial Communities ◽  
Pure Culture ◽  
Fluorescent Protein ◽  
Confocal Laser ◽  
Specific Gene ◽  
Community Members ◽  
Metabolic Interactions ◽  
Green Fluorescent

ABSTRACT Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of anAcinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and themeta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied inP. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from thePu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pmpromoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction ofPm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.

Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene‐expression differences betweenMut+andMutSstrains ofPichia pastoris

Yeast ◽  
2019 ◽  
Vol 36 (5) ◽  
pp. 285-296 ◽  
Author(s):  
Chrispian W. Theron ◽  
Julio Berrios ◽  
Sébastien Steels ◽  
Samuel Telek ◽  
Renaud Lecler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Foreign Gene ◽  
Foreign Gene Expression ◽  
Green Fluorescent ◽  
Insight Into
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