A panel of monoclonal antibodies to rat plectin: Distinction by epitope mapping and immunoreactivity with different tissues and cell lines

Foisner, Roland; Фельдман, Б В; Sander, Laura; Seifert, Georg; Artlieb, U; Wiche, Gerhard

doi:10.1016/s0065-1281(11)80029-2

Cited by 47 publications

(37 citation statements)

References 23 publications

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“…Murine monoclonal antibodies were used for detection of plectin (clone 10F6; kindly provided by Gerhard Wiche, Biocenter, Vienna, Austria) (Foisner et al, 1994) and α-tubulin (Amersham Pharmacia Biotech, Freiburg, Germany). Monoclonal CK epitope antibodies were generously provided by Bishr Omary and Nam-On Ku (Stanford University, Palo Alto, CA), reacting with CKs 8 and 18 (L2A1) , phospho-S73 of CK8 (LJ4) (Liao et al, 1997), phospho-S431 of CK8 (5B3) and phospho-S33 of CK18 (IB4) .…”

Section: Methodsmentioning

confidence: 99%

Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

Strnad

Windoffer

Leube

2002

Journal of Cell Science

105

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The cytokeratin filament network is intrinsically dynamic, continuously exchanging subunits over its entire surface, while conferring structural stability on epithelial cells. However, it is not known how cytokeratin filaments are remodeled in situations where the network is temporarily and spatially restricted. Using the tyrosine phosphatase inhibitor orthovanadate we observed rapid and reversible restructuring in living cells, which may provide the basis for such dynamics. By examining cells stably expressing fluorescent cytokeratin chimeras, we found that cytokeratin filaments were broken down and then formed into granular aggregates within a few minutes of orthovanadate addition. After drug removal, gradual reincorporation of granules into the filament network was observed for aggregates that were either part of residual filaments or stayed in close apposition to remaining filaments. Even when cytokeratin filaments were no longer detectable, granules with low mobility were still able to reestablish a cytokeratin filament network. This process took less than 30 minutes and occurred at multiple foci throughout the cytoplasm without apparent correlation to alterations in the actin- and tubulin-based systems. Interestingly, the short-lived and rather small orthovanadate-induced cytokeratin granules contained the cytoskeletal crosslinker plectin but lacked the cytokeratin-solubilising 14-3-3 proteins. By contrast, the long-lived and larger cytokeratin aggregates generated after treatment with the serine/threonine phosphatase inhibitor okadaic acid were negative for plectin but positive for 14-3-3 proteins. Taken together, our observations in living orthovanadate-treated interphase cells revealed modes of cytokeratin remodeling that qualify as basic mechanisms capable of rapidly adapting the cytokeratin filament cytoskeleton to specific requirements.

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Section: Methodsmentioning

confidence: 99%

Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

Strnad

Windoffer

Leube

2002

Journal of Cell Science

105

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“…Cell lysates, supernatant fractions, and pellet fractions were then either mixed with SDS sample buffer (Laemmli, 1970) and subjected to SDS-PAGE and immunoblot analyses, or supplemented with 1% SDS and 5 mM EDTA, diluted 1:10 in immunoprecipitation buffer (50 mM Tris/ HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA), and processed for immunoprecipitation. One milliliter samples were incubated with 100 p.l protein A-Sepharose (Pharmacia Biotech, Brussels, Belgium; 10% (w/v) in immunoprecipitation buffer) for 1 h, briefly centrifuged in an Eppendorf centrifuge, and supernatants were incubated with 10 p.l of plectin antiserum (Wiche and Baker, 1982), or with 10 ,ug/ml purified monoclonal antibodies to c-myc (Evan et al, 1985;ATCC CRL 1229), or with 100 p.l of Sepharose beads coupled to monoclonal antibody to plectin 7A8 (0.25 mg/ml beads; Foisner et al, 1991aFoisner et al, , 1994 or to the myc antibody (1-2 mg/ml) overnight at 4°C. After addition of 10% protein A Sepharose to the former two samples and 4 h of incubation, all samples were centrifuged for 5 min in an Eppendorf centrifuge and pellets were washed three times in immunoprecipitation buffer and solubilized in SDS-PAGE sample buffer (Laemmli, 1970).…”

Section: Methodsmentioning

confidence: 99%

M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase.

Foisner

Malecz

Dressel

et al. 1996

MBoC

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Plectin, a widespread and abundant cytoskeletal cross-linking protein, serves as a target for protein kinases throughout the cell cycle, without any significant variation in overall phosphorylation level. One of the various phosphorylation sites of the molecule was found to be phosphorylated preferentially during mitosis. By in vivo phosphorylation of ectopically expressed plectin domains in stably transfected Chinese hamster ovary cells, this site was mapped to the C-terminal repeat 6 domain of the polypeptide. The same site has been identified as an in vitro target for p34cdc2 kinase. Mitosis-specific phosphorylation of plectin was accompanied by a rearrangement of plectin structures, changing from a filamentous, largely vimentin-associated state in interphase to a diffuse vimentin-independent distribution in mitosis as visualized by immunofluorescence microscopy. Subcellular fractionation studies showed that in interphase cells up to 80% of cellular plectin was found associated with an insoluble cell fraction mostly consisting of intermediate filaments, while during mitosis the majority of plectin (> 75%) became soluble. Furthermore, phosphorylation of purified plectin by p34cdc2 kinase decreased plectin's ability to interact with preassembled vimentin filaments in vitro. Together, our data suggest that a mitosis-specific phosphorylation involving p34cdc2 kinase regulates plectin's cross-linking activities and association with intermediate filaments during the cell cycle.

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“…Samples from mouse were incubated (1 hour, at RT) with normal goat serum (1:30 in PBS) before incubation with primary antibodies. Immunoreagents and processing of specimens for microscopy were similar to those of cells, except that in the case of rat tissue mouse mAb 10F6 to plectin (Foisner et al, 1994) and anti-mouse-TR (Jackson Laboratories) were used as primary and secondary antibodies, respectively. All immunolabelled specimens were visualised using an Axiovert 100M laser-scanning microscope (Zeiss).…”

Section: Immunofluorescence Microscopy Of Cells and Tissuesmentioning

confidence: 99%

Plectin scaffolds recruit energy-controlling AMP-activated protein kinase (AMPK) in differentiated myofibres

Gregor

Zeöld

Oehler

et al. 2006

Journal of Cell Science

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Plectin, a cytolinker protein greater than 500 kDa in size, has an important role as a mechanical stabiliser of cells. It interlinks the various cytoskeletal filament systems and anchors intermediate filaments to peripheral junctional complexes. In addition, there is increasing evidence that plectin acts as a scaffolding platform that controls the spatial and temporal localisation and interaction of signaling proteins. In this study we show that, in differentiated mouse myotubes, plectin binds to the regulatory γ1 subunit of AMP-activated protein kinase (AMPK), the key regulatory enzyme of energy homeostasis. No interaction was observed in undifferentiated myoblasts, and plectin-deficient myotubes showed altered positioning of γ1-AMPK. In addition we found that plectin affects the subunit composition of AMPK, because isoform α1 of the catalytic subunit decreased in proportion to isoform α2 during in vitro differentiation of plectin-/- myotubes. In plectin-deficient myocytes we could also detect a higher level of activated (Thr172-phosphorylated) AMPK, compared with wild-type cells. Our data suggest a differentiation-dependent association of plectin with AMPK, where plectin selectively stabilises α1-γ1 AMPK complexes by binding to the γ1 regulatory subunit. The distinct plectin expression patterns in different fibre types combined with its involvement in the regulation of isoform compositions of AMPK complexes could provide a mechanism whereby cytoarchitecture influences energy homeostasis.

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A panel of monoclonal antibodies to rat plectin: Distinction by epitope mapping and immunoreactivity with different tissues and cell lines

Cited by 47 publications

References 23 publications

Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase.

Plectin scaffolds recruit energy-controlling AMP-activated protein kinase (AMPK) in differentiated myofibres

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