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 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 Actin dynamics in living mammalian cells

1998 ◽  
Vol 111 (12) ◽  
pp. 1649-1658 ◽  
Author(s):  
C. Ballestrem ◽  
B. Wehrle-Haller ◽  
B.A. Imhof
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Dynamics ◽  
Mammalian Cell Lines ◽  
Cytoskeletal Dynamics ◽  
Green Fluorescent

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

scholarly journals Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

1998 ◽  
Vol 143 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Miri Yoon ◽  
Robert D. Moir ◽  
Veena Prahlad ◽  
Robert D. Goldman
Keyword(s):  
Intermediate Filament ◽  
Cell Shape ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
A Cell ◽  
Filament Networks ◽  
Green Fluorescent ◽  
Vimentin Intermediate Filament

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

scholarly journals Two Putative α-Helical Domains of Human Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by at Least Two Mechanisms

2000 ◽  
Vol 74 (15) ◽  
pp. 7179-7186 ◽  
Author(s):  
Masakazu Kamata ◽  
Yoko Aida
Keyword(s):  
Nuclear Localization ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Expression Vectors ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Entire Cell ◽  
Green Fluorescent ◽  
Triton X

ABSTRACT To identify the domains of Vpr that are involved nuclear localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within αH1 or αH2 resulted, by contrast, in diffuse expression over the entire cell. In addition, double mutations within both of these α-helical domains led to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the αH1 and αH2 domains fused individually with green fluorescent protein (GFP) and a Flag tag and extracted them with digitonin and Triton X-100 prior to fixation. Flag-αH1-GFP was detected in the nucleus but not in the cytoplasm, while Flag-αH2-GFP was retained predominantly in the nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two α-helical domains might be involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative α-helical domains mediate the nuclear localization of Vpr by at least two mechanisms.

scholarly journals Dynamics of DNA Replication Factories in Living Cells

2000 ◽  
Vol 149 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Heinrich Leonhardt ◽  
Hans-Peter Rahn ◽  
Peter Weinzierl ◽  
Anje Sporbert ◽  
Thomas Cremer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Time Lapse ◽  
Living Cells ◽  
Nuclear Antigen ◽  
Central Component ◽  
Nuclear Speckles ◽  
Replication Foci ◽  
Green Fluorescent

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.

scholarly journals Spindle Formation inAspergillusIs Coupled to Tubulin Movement into the Nucleus

2003 ◽  
Vol 14 (5) ◽  
pp. 2192-2200 ◽  
Author(s):  
Yulia Ovechkina ◽  
Paul Maddox ◽  
C. Elizabeth Oakley ◽  
Xin Xiang ◽  
Stephen A. Osmani ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Mitotic Spindle ◽  
Essential Role ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Interphase Nuclei ◽  
Spindle Formation ◽  
Spinning Disk ◽  
Green Fluorescent

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/α-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.

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 A Selective Transport Route from Golgi to Late Endosomes That Requires the Yeast Gga Proteins

2000 ◽  
Vol 151 (3) ◽  
pp. 587-600 ◽  
Author(s):  
Michael W. Black ◽  
Hugh R.B. Pelham
Keyword(s):  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Mutant Strains ◽  
Direct Delivery ◽  
Green Fluorescent ◽  
Exocytic Pathway

Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes. By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.

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