Cytoplasm-to-Nucleus Translocation of a Herpesvirus Tegument Protein during Cell Division

ABSTRACT We have previously shown that the herpes simplex virus tegument protein VP22 localizes predominantly to the cytoplasm of expressing cells. We have also shown that VP22 has the unusual property of intercellular spread, which involves the movement of VP22 from the cytoplasm of these expressing cells into the nuclei of nonexpressing cells. Thus, VP22 can localize in two distinct subcellular patterns. By utilizing time-lapse confocal microscopy of live cells expressing a green fluorescent protein-tagged protein, we now report in detail the intracellular trafficking properties of VP22 in expressing cells, as opposed to the intercellular trafficking of VP22 between expressing and nonexpressing cells. Our results show that during interphase VP22 appears to be targeted exclusively to the cytoplasm of the expressing cell. However, at the early stages of mitosis VP22 translocates from the cytoplasm to the nucleus, where it immediately binds to the condensing cellular chromatin and remains bound there through all stages of mitosis and chromatin decondensation into the G1stage of the next cycle. Hence, in VP22-expressing cells the subcellular localization of the protein is regulated by the cell cycle such that initially cytoplasmic protein becomes nuclear during cell division, resulting in a gradual increase over time in the number of nuclear VP22-expressing cells. Importantly, we demonstrate that this process is a feature not only of VP22 expressed in isolation but also of VP22 expressed during virus infection. Thus, VP22 utilizes an unusual pathway for nuclear targeting in cells expressing the protein which differs from the nuclear targeting pathway used during intercellular trafficking.

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Changes in the Min Oscillation Pattern before and after Cell Birth

Journal of Bacteriology ◽

10.1128/jb.00364-10 ◽

2010 ◽

Vol 192 (16) ◽

pp. 4134-4142 ◽

Cited By ~ 24

Author(s):

Jennifer R. Juarez ◽

William Margolin

Keyword(s):

Cell Division ◽

Fluorescent Protein ◽

Time Lapse ◽

The Other ◽

Cell Pole ◽

Daughter Cells ◽

Division Site ◽

Before And After ◽

Dividing Cells ◽

Green Fluorescent

ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.

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

Journal of Virology ◽

10.1128/jvi.73.5.4110-4119.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4110-4119 ◽

Cited By ~ 135

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.

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Microtubule-Independent Motility and Nuclear Targeting of Adenoviruses with Fluorescently Labeled Genomes

Journal of Virology ◽

10.1128/jvi.75.5.2421-2434.2001 ◽

2001 ◽

Vol 75 (5) ◽

pp. 2421-2434 ◽

Cited By ~ 39

Author(s):

Jolanta B. Glotzer ◽

Anne-Isabelle Michou ◽

Adam Baker ◽

Mediyha Saltik ◽

Matt Cotten

Keyword(s):

Fluorescent Protein ◽

Host Cells ◽

Live Cells ◽

Wild Type ◽

Nuclear Targeting ◽

Microtubule Network ◽

Entry Pathway ◽

Green Fluorescent ◽

Affect Gene Expression ◽

Intracellular Motility

ABSTRACT A novel adenovirus system for analyzing the adenovirus entry pathway has been developed that contains green fluorescent protein bound to the encapsidated viral DNA (AdLite viruses). AdLite viruses enter host cells and accumulate around the nuclei and near the microtubule organizing centers (MTOC). In live cells, individual AdLite particles were observed trafficking both toward and away from the nucleus. Depolymerization of microtubules during infection prevented AdLite accumulation around the MTOC; however, it did not abolish perinuclear localization of AdLite particles. Furthermore, depolymerization of microtubules did not affect AdLite motility and did not affect gene expression from wild-type adenovirus and adenovirus-derived vectors. These data revealed that adenovirus intracellular motility and nuclear targeting can be supported by a mechanism that does not rely on the microtubule network.

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Distinct Sequence Elements of Cyclin B1 Promote Localization to Chromatin, Centrosomes, and Kinetochores during Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.e06-06-0539 ◽

2007 ◽

Vol 18 (12) ◽

pp. 4847-4858 ◽

Cited By ~ 50

Author(s):

Anna M. Bentley ◽

Guillaume Normand ◽

Jonathan Hoyt ◽

Randall W. King

Keyword(s):

Cell Division ◽

Fluorescent Protein ◽

Cyclin B1 ◽

Vital Role ◽

Live Cells ◽

Nuclear Envelope Breakdown ◽

M Phase ◽

Sequence Elements ◽

Distinct Sequence ◽

Green Fluorescent

The mitotic cyclins promote cell division by binding and activating cyclin-dependent kinases (CDKs). Each cyclin has a unique pattern of subcellular localization that plays a vital role in regulating cell division. During mitosis, cyclin B1 is known to localize to centrosomes, microtubules, and chromatin. To determine the mechanisms of cyclin B1 localization in M phase, we imaged full-length and mutant versions of human cyclin B1-enhanced green fluorescent protein in live cells by using spinning disk confocal microscopy. In addition to centrosome, microtubule, and chromatin localization, we found that cyclin B1 also localizes to unattached kinetochores after nuclear envelope breakdown. Kinetochore recruitment of cyclin B1 required the kinetochore proteins Hec1 and Mad2, and it was stimulated by microtubule destabilization. Mutagenesis studies revealed that cyclin B1 is recruited to kinetochores through both CDK1-dependent and -independent mechanisms. In contrast, localization of cyclin B1 to chromatin and centrosomes is independent of CDK1 binding. The N-terminal domain of cyclin B1 is necessary and sufficient for chromatin association, whereas centrosome recruitment relies on sequences within the cyclin box. Our data support a role for cyclin B1 function at unattached kinetochores, and they demonstrate that separable and distinct sequence elements target cyclin B1 to kinetochores, chromatin, and centrosomes during mitosis.

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The Capsid and Tegument of the Alphaherpesviruses Are Linked by an Interaction between the UL25 and VP1/2 Proteins

Journal of Virology ◽

10.1128/jvi.01113-07 ◽

2007 ◽

Vol 81 (21) ◽

pp. 11790-11797 ◽

Cited By ~ 105

Author(s):

Kelly Elizabeth Coller ◽

Joy I-Hsuan Lee ◽

Aki Ueda ◽

Gregory Allan Smith

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Protein Product ◽

Tegument Protein ◽

Viral Propagation ◽

Tegument Proteins ◽

Green Fluorescent ◽

Simplex Virus ◽

Carboxy Terminal ◽

Hsv 1

ABSTRACT How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.

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Two-Step Assembly Dynamics of the Bacillussubtilis Divisome

Journal of Bacteriology ◽

10.1128/jb.01758-08 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4186-4194 ◽

Cited By ~ 116

Author(s):

Pamela Gamba ◽

Jan-Willem Veening ◽

Nigel J. Saunders ◽

Leendert W. Hamoen ◽

Richard A. Daniel

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Fluorescent Protein ◽

Time Lapse ◽

Localization Pattern ◽

Ftsz Ring ◽

Time Lapse Microscopy ◽

Green Fluorescent ◽

Posttranscriptional Level

ABSTRACT Cell division in bacteria is carried out by about a dozen proteins which assemble at midcell and form a complex known as the divisome. To study the dynamics and temporal hierarchy of divisome assembly in Bacillus subtilis, we have examined the in vivo localization pattern of a set of division proteins fused to green fluorescent protein in germinating spores and vegetative cells. Using time series and time-lapse microscopy, we show that the FtsZ ring assembles early and concomitantly with FtsA, ZapA, and EzrA. After a time delay of at least 20% of the cell cycle, a second set of division proteins, including GpsB, FtsL, DivIB, FtsW, Pbp2B, and DivIVA, are recruited to midcell. Together, our data provide in vivo evidence for two-step assembly of the divisome. Interestingly, overproduction of FtsZ advances the temporal assembly of EzrA but not of DivIVA, suggesting that a signal different from that of FtsZ polymerization drives the assembly of late divisome proteins. Microarray analysis shows that FtsZ depletion or overexpression does not significantly alter the transcription of division genes, supporting the hypothesis that cell division in B. subtilis is mainly regulated at the posttranscriptional level.

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Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

Applied and Environmental Microbiology ◽

10.1128/aem.06644-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8310-8317 ◽

Cited By ~ 27

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.

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

Blood ◽

10.1182/blood-2010-01-264382 ◽

2010 ◽

Vol 116 (6) ◽

pp. 909-914 ◽

Cited By ~ 114

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0104 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4622-4632 ◽

Cited By ~ 54

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.

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