Actin-binding domain of mouse plectin. Crystal structure and binding to vimentin.

Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2alpha) from mouse, to a resolution of 1.95 and 2.0 A. Comparison of pleABD/2alpha with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2alpha has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks.     

Crystal structure of a tandem pair of fibronectin type III domains from the cytoplasmic tail of integrin alpha6beta4.

The integrin alpha6beta4 is an essential component of hemidesmosomes but it also plays a dynamic role in invasive carcinoma cells. The cytoplasmic tail of the beta4 subunit is uniquely large among integrins and includes two pairs of fibronectin type III domains separated by a connecting segment. Here we describe the crystal structure of the first tandem domain pair, a module that is critical for alpha6beta4 function. The structure reveals a novel interdomain interface and candidate protein-binding sites, including a large acidic cleft formed from the surfaces of both domains and a prominent loop that is reminiscent of the RGD integrin-binding loop of fibronectin. This is the first crystal structure of either a hemidesmosome component or an integrin cytoplasmic domain, and it will enable the intracellular functions of alpha6beta4 to be dissected at the atomic level.     

Plectin-RACK1 (receptor for activated C kinase 1) scaffolding: a novel mechanism to regulate protein kinase C activity

Agonist-induced translocation of protein kinase C (PKC) isozymes is mediated by receptors for the activated form of the kinase, shuttling it from one intracellular site to another and enhancing its catalytic activity. It is however unknown whether the receptors themselves are anchored to certain intracellular structures prior to their engagement with PKC. We show here sequestering of receptor for activated C kinase 1 (RACK1) to the cytoskeleton through the cytoskeletal linker protein plectin during the initial stages of cell adhesion. We found that upon PKC activation, RACK1 was released from the cytoskeleton and transferred to the detergent-soluble cell compartment, where it formed an inducible triple complex with one of the PKC isozymes, PKCdelta, and with plectin. In plectin-deficient cells the cytoskeleton-associated RACK1 fraction was reduced, and the protein was found predominantly at sites to which it normally translocated upon PKC activation. Concomitantly, dislocation of PKCdelta and elevated enzymatic activity were observed in these cells. PKCdelta was also more rapidly degraded, likely due to its overactivation. We propose a previously unrecognized function of plectin as cytoskeletal regulator of PKC signaling, and possibly other signaling events, through sequestration of the scaffolding protein RACK1.     

Cytoplasmic network arrays demonstrated by immunolocalization using antibodies to a high molecular weight protein present in cytoskeletal preparations from cultured cells

Antisera were raised in rabbits against the two major components of intermediate filament preparations from glia-derived C6 cells, polypeptides of M_r around 300 000 and 58 000 (vimentin). These, and a third antiserum raised against microtubule proteins from hog brain, were shown to be specific for their respective immunogens. The assay employed involved the separation of components of crude cell extracts or filament preparations by SDS-polyacrylamide gel electrophoresis, and their subsequent transfer to and immobilization on nitrocellulose sheets. Cross-reacting counterparts of the immunogens were found in various cell lines, including C6, BALB/c 3T3, SV101, CHO, HeLa and PtK2 cells. In indirect immunofluorescence studies, antibodies to the high-M_r polypeptide component stained dense cytoplasmic network arrays of seemingly short, irregularly oriented fibres and lines of dots, in fibroblasts and in HeLa cells, but not in PtK2 cells. In well spread cells these networks were clearly distinguishable in morphology from the fibres decorated by antibodies to either microtubule protein or vimentin. The network arrays were resistant towards treatments with Triton X-100 and colcemid. By double immunofluorescence microscopy of single cells, using an additional antibody preparation to vimentin raised in guinea pigs, it was shown that after prolonged colcemid treatment of BALB/c 3T3 cells both; vimentin filaments and the structures stained by antibodies to the high-M_r component, accumulated in corresponding areas of the cytoplasm. The possibilities are discussed that this novel network-like structure is of the intermediate filament type and that it might function as a cross-linker of cytoplasmic—in particular cytoskeletal—elements. To signify its fluorescent localization and its possible linking role it is proposed to call the high-M_r component of intermediate filament preparations from cultured cells ‘plectin’.     

Intermediate filament-associated proteins

The term intermediate filament-associated proteins refers to a growing number of proteins whose ability to interact with intermediate filaments has been either directly demonstrated or inferred from indirect evidence. Here we discuss recently published data on the identification and characterization of such proteins, with emphasis on their tissue/cell type-specific expression, subcellular distribution and possible function(s).     

Microheterogeneity of microtubule-associated proteins, MAP-1 and MAP-2, and differential phosphorylation of individual subcomponents

High molecular weight microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), prepared by copolymerization with tubulin, were electrophorectically separated into three and two major subcomponents, respectively, using 5% sodium dodecyl sulfate-polyacrylamide gels. By two-dimensional gel electrophoresis, all five MAP components were shown to possess a pI of around 5. Four of these proteins, MAP-1A, MAP-1C, MAP-2A, and MAP-2B, present in comparable amounts, were iodinated after electrophoretic separation and analyzed by two-dimensional peptide mapping. With both trypsin and V8 protease, almost identical patterns were obtained from MAP-2A and MAP-2B. MAP-1A and MAP-1C, too, gave similar digestion patterns, although some differences were noted. Incubation with [gamma-32P]ATP demonstrated that endogeneous protein kinase activities phosphorylated individual subcomponents at different rates. MAP-2A, the highest labeled component, was phosphorylated 2.5-fold compared to MAP-2B both in the presence and the absence of cAMP. Labeling of MAP-1 subcomponents was 4 times less than that of MAP-2A in the absence and 16 times less in the presence of cAMP. 32P-labeled MAP-2A and MAP-2B bands were indistinguishable by one-dimensional peptide mapping, as were the three MAP-1 bands. For both MAP-1 and MAP-2 subcomponents, cAMP induced phosphorylation at new molecular sites. Incubation of radiolabeled microtubule proteins with 1 mM ATP effected, upon electrophoresis, a clear shift of MAP-2A and MAP-2B bands to positions of higher apparent molecular weights, while only slightly affecting MAP-1 bands.     

Rapid microwave fixation of cell monolayers preserves microtubule-associated cell structures.

Microwave (MW) fixation has been suggested as a method to rapidly immobilize cellular dynamics for fine structural studies in the electron microscope. To show its suitability for studies on cell monolayers, one has to apply MW fixation systematically in correlation with samples on the light microscopy level. Examples for MW fixation of cell monolayers, however, are still rare. MW-accelerated fixation for relatively long periods of time (1-2 min) has been reported without showing its suitability at the fine structural level. Here, we provide a rapid MW fixation protocol for cell monolayers on a subminute time scale. The impact of the MW-accelerated glutaraldehyde fixation on temperature-sensitive cytoskeletal components such as microtubules was evaluated. For testing the effectiveness of MW-assisted primary fixation, saponin treatment of the monolayers was included. Simultaneous MW-accelerated fixation and extraction by saponin was necessary to achieve a gradual improvement in visualization of cytoskeletal aspects in association with cell junctions, mitochondria, and centrioles. To establish a valuable routine program for fine structural studies of resin-embedded cell models on substrata, a protocol combining MW fixation with automatic processing in a tissue processor is provided.     

Oxidation and nitrosylation of cysteines proximal to the intermediate filament (IF)-binding site of plectin: effects on structure and vimentin binding and involvement in IF collapse

As an intermediate filament (IF)-based cytolinker protein, plectin plays a key role in the maintenance of cellular cytoarchitecture and serves at the same time as a scaffolding platform for signaling cascades. Consisting of six structural repeats (R1-6) and harboring binding sites for different IF proteins and proteins involved in signaling, the plectin C-terminal domain is of strategic functional importance. Depending on the species, it contains at least 13 cysteines, 4 of which reside in the R5 domain. To investigate the structural and biological functions of R5 cysteines, we used cysteine-to-serine mutagenesis and spectroscopic, biochemical, and functional analyses. Urea-induced unfolding experiments indicated that wild-type R5 in the oxidized, disulfide bond-mediated conformation was more stable than its cysteine-free mutant derivative. The binding affinity of R5 for vimentin was significantly higher, however, when the protein was in the reduced, more relaxed conformation. Of the four R5 cysteines, one (Cys4) was particularly reactive as reflected by its ability to form disulfide bridges with R5 Cys1 and to serve as a target for nitrosylation in vitro. Using immortalized endothelial cell cultures from mice, we show that endogenous plectin is nitrosylated in vivo, and we found that NO donor-induced IF collapse proceeds dramatically faster in plectin-deficient compared with wild-type cells. Our data suggest an antagonistic role of plectin in nitrosylation (oxidative stress)-mediated alterations of IF cytoarchitecture and a possible role of R5 Cys4 as a regulatory switch.