scholarly journals Vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the A36R protein

2005 ◽  
Vol 86 (11) ◽  
pp. 2961-2968 ◽  
Author(s):  
Esteban Herrero-Martínez ◽  
Kim L. Roberts ◽  
Michael Hollinshead ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Core Protein ◽  
Time Lapse ◽  
Cell Periphery ◽  
Wild Type ◽  
Enveloped Virus ◽  
Microtubule Motors ◽  
Specific Proteins ◽  
Green Fluorescent

Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either F12L (vΔF12L) or A36R (vΔA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at ∼60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vΔA36R-infected cells. Moreover, vΔA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vΔA36R IEV moved in a stop–start manner at speeds consistent with microtubular movement, although the mean length of vΔA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.

scholarly journals Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection

1999 ◽  
Vol 73 (5) ◽  
pp. 4110-4119 ◽  
Author(s):  
Gillian Elliott ◽  
Peter O’Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Live Cell ◽  
Cell Periphery ◽  
Green Fluorescent ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.

scholarly journals Visualization of TGN to Endosome Trafficking through Fluorescently Labeled MPR and AP-1 in Living Cells

2003 ◽  
Vol 14 (1) ◽  
pp. 142-155 ◽  
Author(s):  
Satoshi Waguri ◽  
Frédérique Dewitte ◽  
Roland Le Borgne ◽  
Yves Rouillé ◽  
Yasuo Uchiyama ◽  
...  
Keyword(s):  
Hela Cells ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Endosome Trafficking ◽  
Early Endosomes ◽  
Green Fluorescent

We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose γ-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes.

Detection of cytokeratin dynamics by time-lapse fluorescence microscopy in living cells

1999 ◽  
Vol 112 (24) ◽  
pp. 4521-4534 ◽  
Author(s):  
R. Windoffer ◽  
R.E. Leube
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Cell Periphery ◽  
Filament Bundles ◽  
Cytokeratin 13 ◽  
Filament Networks ◽  
Green Fluorescent

To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells. Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin 13 and is therefore a reliable in vivo tag for this polypeptide and the structures formed by it. Time-lapse fluorescence microscopy reveals that the cytokeratin 13-containing network is in constant motion, resulting in continuous restructuring occurring in single and migratory cells, as well as in desmosome-anchored cells. Two major types of movement are distinguished: (i) oscillations of mostly long filaments, and (ii) an inward-directed flow of fluorescence originating as diffuse material at the cell periphery and moving in the form of dots and thin filaments toward the deeper cytoplasm where it coalesces with other filaments and filament bundles. Both movements are energy dependent and can be inhibited by nocodazole, but not by cytochalasin D. Finally, disassembly and reformation of cytokeratin filament networks are documented in dividing cells revealing distinct and rapidly occurring stages of cytokeratin organisation and distribution.

scholarly journals Vaccinia virus utilizes microtubules for movement to the cell surface

2001 ◽  
Vol 154 (2) ◽  
pp. 389-402 ◽  
Author(s):  
Michael Hollinshead ◽  
Gaener Rodger ◽  
Henriette Van Eijl ◽  
Mansun Law ◽  
Ruth Hollinshead ◽  
...  
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cytochalasin D ◽  
Enveloped Virus ◽  
Proposal A ◽  
Virus C ◽  
Green Fluorescent ◽  
Rate Of Movement

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 μm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.

scholarly journals Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails

2001 ◽  
Vol 75 (23) ◽  
pp. 11651-11663 ◽  
Author(s):  
Brian M. Ward ◽  
Bernard Moss
Keyword(s):  
Membrane Protein ◽  
Vaccinia Virus ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Reading Frame ◽  
Extracellular Virus ◽  
Intracellular Movement ◽  
Vaccinia Viruses ◽  
Green Fluorescent

ABSTRACT Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria andShigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr112 and Tyr132 of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 μm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.

scholarly journals Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface

2002 ◽  
Vol 83 (2) ◽  
pp. 323-332 ◽  
Author(s):  
Gaener Rodger ◽  
Geoffrey L. Smith
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Mutant Virus ◽  
Virus Protein ◽  
Plaque Size ◽  
Enveloped Virus ◽  
Egfp Fusion Protein ◽  
Green Fluorescent

A vaccinia virus (VV) recombinant is described in which the outer envelope of extracellular enveloped virus (EEV), cell-associated enveloped virus (CEV) and intracellular enveloped virus (IEV) is labelled with the enhanced green fluorescent protein (EGFP) derived from Aequorea victoria. To construct this virus, EGFP was fused to the VV B5R protein from which the four short consensus repeats (SCRs) of the extracellular domain had been deleted. Cells infected with the recombinant virus expressed a B5R–EGFP fusion protein of 40 kDa that was present on IEV, CEV and EEV, but was absent from IMV. The recombinant virus produced 2- and 3-fold reduced levels of IMV and EEV, respectively. Analysis of infected cells by confocal microscopy showed that actin tail formation by the mutant virus was reduced by 86% compared to wild-type (WT). The virus formed a small plaque compared to WT, consistent with a role for actin tails in promoting cell-to-cell spread of virus. However, the enveloped virions were still transported to the cell surface, confirming that this process is independent of actin tail formation. Lastly, we compared the mutant virus with a recombinant VV in which the B5R SCR domains were deleted and show that, contrary to a previous report, the plaque size of the latter virus was reduced compared to WT. This observation reconciles an inconsistency in the field and confirms that viruses deficient in formation of actin tails form small plaques.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

scholarly journals A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

2004 ◽  
Vol 15 (10) ◽  
pp. 4622-4632 ◽  
Author(s):  
Yasmina Bauer ◽  
Philipp Knechtle ◽  
Jürgen Wendland ◽  
Hanspeter Helfer ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Time Lapse ◽  
Growth Direction ◽  
Ashbya Gossypii ◽  
Growth Guidance ◽  
Characteristic Features ◽  
Green Fluorescent ◽  
Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

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