Visualization of Prosomes (MCP-Proteasomes), Intermediate Filament and Actin Networks by “Instantaneous Fixation” Preserving the Cytoskeleton

Arcangeletti, Cristina; Sütterlin, Rosmarie; Aebi, Ueli; Conto, Flora De; Missorini, S.; Chezzi, Carlo; Scherrer, Klaus

doi:10.1006/jsbi.1997.3871

Cited by 50 publications

(38 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Third, recently we and other groups found that Nrf2 is degraded rapidly and efficiently with the 26S proteasome under unstressed conditions (24)(25)(26)(27). Because the proteasome is also known to colocalize with the actin filaments and intermediate filaments (28), we envisage that Keap1 may transfer the newly synthesized Nrf2 to the proteasome localized nearby, resulting in the rapid turnover of Nrf2.…”

Section: Discussionmentioning

confidence: 76%

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes

Kang

Kobayashi

Wakabayashi

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

473

323

View full text Add to dashboard Cite

Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection-transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.

show abstract

Section: Discussionmentioning

confidence: 76%

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes

Kang

Kobayashi

Wakabayashi

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

473

323

View full text Add to dashboard Cite

show abstract

“…In experiments designed for staining of cytoskeletal structures, cells were fixed and permeabilized simultaneously in cytoskeleton-stabilizing buffer CSB (10 mM PIPES pH 6.9, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 , 1 mM EGTA) as described in Ref. 21.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Distinct Plasma Membrane Macrodomain in Differentiated Adipocytes

Parton

Molero

Floetenmeyer

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol. Chem. 277, 25867-25869) described novel caveolin-and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed "caves," using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

show abstract

“…Because proteasomes have been observed in association with actin filaments (AFs) and actin-myosin complexes in animal cells (Arcangeletti et al, 1997(Arcangeletti et al, , 2000, we hypothesized that inhibition of proteasome activity would disrupt the organization of AFs, which play a central role in the polarized tip growth of pollen tubes. To examine this possibility, the actin cytoskeleton in control and MG132-treated pollen tubes was compared using fluorescein isothiocyanate (FITC)-phalloidin staining.…”

Section: Mg132 Treatment Disrupts Cytoskeleton Organization and Cytopmentioning

confidence: 99%

Roles of the Ubiquitin/Proteasome Pathway in Pollen Tube Growth with Emphasis on MG132-Induced Alterations in Ultrastructure, Cytoskeleton, and Cell Wall Components

Sheng

Lü

et al. 2006

Plant Physiology

View full text Add to dashboard Cite

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose-and time-dependent manner, while hardly similar effects were detected when cysteineprotease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanatephalloidin and b-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca 21 gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.Most cellular proteins are continuously synthesized and degraded within the lifespan of a cell. Efficient protein turnover is essential for many aspects of cell physiology and development (Hellmann and Estelle, 2002). As one of the most important proteolytic pathways in eukaryotic cells, the ubiquitin/proteasome pathway (UPP) is believed to be involved in the degradation of the bulk of intracellular proteins, including misfolded proteins and short-and long-lived regulatory proteins (Glickman and Ciechanover, 2002;Ciechanover, 2005;Hershko, 2005;Varshavsky, 2005). Therefore, the biological functions of the UPP in both animal and plant systems have received considerable attention.Pollen tubes are responsible for delivering sperm cells to the egg for fertilization and are therefore essential for higher plant sexual reproduction. The emergence and elongation of pollen tubes is a complex and intriguing example of cell morphogenesis. The control of this type of growth requires a number of factors and activities to be integrated in space and time

show abstract

Visualization of Prosomes (MCP-Proteasomes), Intermediate Filament and Actin Networks by “Instantaneous Fixation” Preserving the Cytoskeleton

Cited by 50 publications

References 51 publications

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes

Characterization of a Distinct Plasma Membrane Macrodomain in Differentiated Adipocytes

Roles of the Ubiquitin/Proteasome Pathway in Pollen Tube Growth with Emphasis on MG132-Induced Alterations in Ultrastructure, Cytoskeleton, and Cell Wall Components

Contact Info

Product

Resources

About