scholarly journals Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles

2000 ◽  
Vol 113 (24) ◽  
pp. 4421-4426 ◽  
Author(s):  
M. Kaksonen ◽  
H.B. Peng ◽  
H. Rauvala
Keyword(s):  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Actin Binding ◽  
Common Mechanism ◽  
Membrane Protrusions ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Endosomal Vesicles

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network

2000 ◽  
Vol 113 (18) ◽  
pp. 3151-3159 ◽  
Author(s):  
R. Blum ◽  
D.J. Stephens ◽  
I. Schulz
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Temperature Sensitive ◽  
Long Distance ◽  
Anterograde Transport ◽  
Network Transport ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Tubular Membranes

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www.biologists.com/JCS/movies/jcs1334.html

scholarly journals The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

2000 ◽  
Vol 150 (6) ◽  
pp. 1321-1334 ◽  
Author(s):  
Zhengshan Dai ◽  
Xiaoyan Luo ◽  
Hongbo Xie ◽  
H. Benjamin Peng
Keyword(s):  
Actin Cytoskeleton ◽  
Acetylcholine Receptor ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Actin Binding ◽  
Heparin Binding ◽  
Actin Assembly ◽  
Achr Cluster ◽  
Green Fluorescent ◽  
Receptor Clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin–binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule–coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead–muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein–coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

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 Migration of primordial germ cells during late embryogenesis of pikeperch Sander lucioperca relative to blastomere transplantation

2017 ◽  
Vol 62 (No. 3) ◽  
pp. 121-129 ◽  
Author(s):  
H. Güralp ◽  
K. Pocherniaieva ◽  
M. Blecha ◽  
T. Policar ◽  
M. Pšenička ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Somatic Cells ◽  
Time Lapse ◽  
Sander Lucioperca ◽  
Initial Appearance ◽  
Green Fluorescent ◽  
Time Lapse Imaging

Pikeperch Sander lucioperca is a valuable fish in Europe, and basic information about its embryonic development, especially primordial germ cell (PGC) migration, is important for use in biotechnology. We categorized pikeperch embryonic development into six stages as in other fish species: zygote, cleavage, blastula, gastrula, segmentation, and hatching and described PGC migration. PGCs were visualized by injection of synthesized green fluorescent protein (GFP) within the 3’untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to location at the gonadal ridge. We conducted blastomere transplantation (BT) at the blastula stage. Donor embryos were labelled with GFP-nos3 3’UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve BT chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that BT can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.

scholarly journals Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis

2004 ◽  
Vol 164 (5) ◽  
pp. 759-768 ◽  
Author(s):  
Concepción Gómez-Moutón ◽  
Rosa Ana Lacalle ◽  
Emilia Mira ◽  
Sonia Jiménez-Baranda ◽  
Domingo F. Barber ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Cholesterol Depletion ◽  
Dependent Manner ◽  
Gradient Sensing ◽  
Raft Domains ◽  
Green Fluorescent

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol–anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide–differentiated HL60 cells in a pertussis toxin–sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase γ in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.

scholarly journals Two-Color Green Fluorescent Protein Time-Lapse Imaging

BioTechniques ◽  
1998 ◽  
Vol 25 (5) ◽  
pp. 838-846 ◽  
Author(s):  
Jan Ellenberg ◽  
Jennifer Lippincott-Schwartz ◽  
John F. Presley
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Green Fluorescent ◽  
Time Lapse Imaging

scholarly journals Differential Dynamics of α5 Integrin, Paxillin, and α-Actinin during Formation and Disassembly of Adhesions in Migrating Cells

2001 ◽  
Vol 153 (7) ◽  
pp. 1427-1440 ◽  
Author(s):  
Christina M. Laukaitis ◽  
Donna J. Webb ◽  
Karen Donais ◽  
Alan F. Horwitz
Keyword(s):  
Green Fluorescent Protein ◽  
Transferrin Receptor ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Perinuclear Region ◽  
Membrane Protrusions ◽  
Green Fluorescent ◽  
Endocytic Vesicles ◽  
Migrating Cells

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed α5 integrin, α-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. α5-GFP also rescued the adhesive defects in CHO B2 cells, which are α5 integrin deficient. In ruffling cells, α5-GFP and α-actinin–GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. α-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of α-actinin–GFP and paxillin-GFP, which grew in size. Subsequently, α5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. α5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, α5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The α5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, α5-GFP was found in fibrous structures behind the cell, whereas α-actinin–GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.

scholarly journals Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

Endocrinology ◽  
2015 ◽  
Vol 157 (2) ◽  
pp. 831-843 ◽  
Author(s):  
Brian S. Edwards ◽  
An K. Dang ◽  
Dilyara A. Murtazina ◽  
Melissa G. Dozier ◽  
Jennifer D. Whitesell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Binding ◽  
Erk Phosphorylation ◽  
Green Fluorescent ◽  
Ca2 Channels

Abstract We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3–1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3–1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca2+ influx via the L-type Ca2+ channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca2+ influx through L-type Ca2+ channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca2+ influx via L-type channels to facilitate ERK phosphorylation.

scholarly journals Toxofilin, a Novel Actin-binding Protein from Toxoplasma gondii, Sequesters Actin Monomers and Caps Actin Filaments

2000 ◽  
Vol 11 (1) ◽  
pp. 355-368 ◽  
Author(s):  
Olivier Poupel ◽  
Haralabia Boleti ◽  
Sophie Axisa ◽  
Evelyne Couture-Tosi ◽  
Isabelle Tardieux
Keyword(s):  
Green Fluorescent Protein ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Green Fluorescent

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.

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.

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