Imbalances in protein homeostasis caused by mutant desmin.

AIMS: We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. METHODS: We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy. RESULTS: We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB-crystallin and Hsp27. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies. CONCLUSIONS: Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

N-cadherin-functionalized polymer-tethered multi-bilayer: a cell surface-mimicking substrate to probe cellular mechanosensitivity.

Fate and function of anchorage-dependent cells depend on a variety of environmental cues, including those of mechanical nature. Previous progress in the understanding of cellular mechanosensitivity has been closely linked to the availability of artificial cell substrates of adjustable viscoelasticity, allowing for a direct correlation between substrate stiffness and cell response. Exemplary, polymeric gel substrates with polymer-conjugated cell-substrate linkers provided valuable insight into the role of mechanical signals during cell migration in an extracellular matrix environment. In contrast, less is known about the role of external mechanical signals across cell-cell interfaces, in part, due to the limitations of traditional polymeric substrates to mimic the remarkable dynamics of cell-cell linkages. To overcome this shortcoming, we introduce a cell surface-mimicking cell substrate of adjustable stiffness, which is comprised of a polymer-tethered lipid multi-bilayer stack with N-cadherin linkers. Unlike traditional polymeric cell substrates with polymer-conjugated linkers, this novel artificial cell substrate is able to replicate the dynamic assembly/disassembly of cadherin linkers into linker clusters and the long-range movements of cadherin-based cell-substrate linkages observed at cell-cell interfaces. Moreover, substrate stiffness can be changed by adjusting the number of bilayers in the multi-bilayer stack, thus enabling the analysis of cellular mechanosensitivity in the presence of artificial cell-cell linkages. The presented biomembrane-mimicking cell substrate provides a valuable tool to explore the functional role of mechanical cues from neighboring cells.

Anti-PR3 immune responses induce segmental and necrotizing glomerulonephritis.

Wegener's granulomatosis (WG) is a life-threatening autoimmune vasculitis that affects lungs, kidneys and other organs. A hallmark of WG is the presence of classic anti-neutrophil cytoplasmic antibodies (c-ANCA) against self-proteinase 3 (PR3). Little is known about the role of these antibodies and PR3-specific immune responses in disease development. In this study, we demonstrate that PR3-specific autoimmune responses are pathogenic in non-obese diabetic (NOD) mice with an impaired regulatory arm of the immune response. Immunization of autoimmunity prone NOD mice with rmPR3 (recombinant mouse PR3) in complete Freund's adjuvant (CFA) resulted in high levels of c-ANCA, without detectable disease development. However, when splenocytes from these immunized mice were transferred into immunodeficient NOD-severe combined immunodeficiency (SCID) mice, the recipient mice developed vasculitis and severe segmental and necrotizing glomerulonephritis. No disease developed in NOD-SCID mice that received splenocytes from the CFA-alone-immunized donors (controls), indicating that disease development depends upon PR3-specific immune responses. In contrast to the pathology observed in NOD-SCID mice, no disease was observed when splenocytes from rmPR3-immunized C57BL/6 mice were transferred into immunodeficient C57BL/6-RAG-1(-/-) mice, suggesting that complex and probably multi-genetic factors play a role in the regulation of disease development.

The IsoStretcher: An isotropic cell stretch device to study mechanical biosensor pathways in living cells.

Mechanosensation in many organs (e.g. lungs, heart, gut) is mediated by biosensors (like mechanosensitive ion channels), which convert mechanical stimuli into electrical and/or biochemical signals. To study those pathways, technical devices are needed that apply strain profiles to cells, and ideally allow simultaneous live-cell microscopy analysis. Strain profiles in organs can be complex and multiaxial, e.g. in hollow organs. Most devices in mechanobiology apply longitudinal uniaxial stretch to adhered cells using elastomeric membranes to study mechanical biosensors. Recent approaches in biomedical engineering have employed intelligent systems to apply biaxial or multiaxial stretch to cells. Here, we present an isotropic cell stretch system (IsoStretcher) that overcomes some previous limitations. Our system uses a rotational swivel mechanism that translates into a radial displacement of hooks attached to small circular silicone membranes. Isotropicity and focus stability are demonstrated with fluorescent beads, and transmission efficiency of elastomer membrane stretch to cellular area change in HeLa/HEK cells. Applying our system to lamin-A overexpressing fibrosarcoma cells, we found a markedly reduced stretch of cell area, indicative of a stiffer cytoskeleton. We also investigated stretch-activated Ca(2+) entry into atrial HL-1 myocytes. 10% isotropic stretch induced robust oscillating increases in intracellular Fluo-4 Ca(2+) fluorescence. Store-operated Ca(2+) entry was not detected in these cells. The Isostretcher provides a useful versatile tool for mechanobiology.

Peptide-specific antibodies localize the major lipid binding sites of talin dimers to oppositely arranged N-terminal 47 kDa subdomains.

Using ultrastructural analysis and labeling with polyclonal antibodies that recognize peptide sequences specific for phospholipid binding, we mapped the functional domain structure of intact platelet talin and its proteolytic fragments. The talin dimer, which is crucial for actin and lipid binding, is built of a backbone containing the 200 kDa rod portions, at both ends of which a 47 kDa globular domain is attached. Peptide-specific polyclonal antibodies were raised against three potential lipid binding sequences residing within the N-terminal 47 kDa domain (i.e. S19, amino acids 21-39; H18, amino acids 287-304; and H17, amino acids 385-406). Antibodies H17 and H18 localize these lipid binding sequences within the N-terminal 47 kDa globular talin subdomains opposed at the outer 200 kDa rod domains within talin dimers. Hence, we conclude that in its dimeric form, which is used in actin and lipid binding, talin is a dumbbell-shaped molecule built of two antiparallel subunits.

Examination of temperature-induced 'gel-sol' transformation of alpha-actinin/cross-linked actin networks by static light scattering.

We studied the gel-sol transformation of F-actin/alpha-actinin solutions. Cross-linking of actin filaments by alpha-actinin shows a temperature-dependent increase in light scatter signal, (I)T. Higher F-actin/alpha-actinin molar ratios, r(A alpha) as well as increases in F-actin concentration, [A], and reduction of actin filament lengths, rAG, augment the maximal light intensity, I and shift the gel-sol transition point, Tg to higher temperatures. This behavior is interpreted in terms of the model developed by Tempel, M., Isenberg, G. and Sackmann, E. (1996) (Physical Review E 54, 1802-1810) based on the percolation theory. Using the temperature-dependent binding model of this theory allows instant prediction of the equilibrium constant, K for F-actin/alpha-actinin solutions at temperatures T < Tg.

Analysis of filamin and alpha-actinin binding to actin by the stopped flow method.

We ascertained by the stopped flow method the overall association rate constant, k+1, of filamin and alpha-actinin to fluorescently labelled filamentous actin of approximately 1.3 x 10(6) M-1.s-1 and approximately 1.0 x 10(6) M-1.s-1 as well as the overall dissociation rate constant, k-1, of approximately 0.6 s-1 and approximately 0.4 s-1, respectively. The overall equilibrium constant, K, for filamin and alpha-actinin to actin deduced from the relation K = k+1/k-1 agree well with published data.

Computer analyses suggest interactions of non-muscle filamin with lipid membranes.

It is concluded from structure predictions of the primary amino acid sequence by computer analyses that two segments of non-muscle filamin could facilitate lipid membrane attachment or anchoring. Residues 49-71 of the amino-terminal may attach to phospholipid membranes, and residues 131-155 may anchor in the hydrophobic region of lipid membranes.

Internal actin filament dynamics in the presence of vinculin: a dynamic light scattering study.

Analyses of dynamic light scattering data by stretched exponential fit show that vinculin has a negligible influence on internal actin filament dynamics and actin bending stiffness which contrasts with our previous observations with talin, another actin and vinculin-binding protein from focal adhesions. The results here agree with kinetic and rheologic measurements.

Talin binds to actin and promotes filament nucleation.

Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non-interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase-I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G-buffer conditions. The overall binding constant was approximately 0.25 microM.

Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine-phalloidin labelling of actin filaments. Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein-lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of actin filament growth at lipid interfaces have been visualized.

Determination of the affinity of talin and vinculin to charged lipid vesicles: a light scatter study.

Recent experimental findings have demonstrated that both talin and vinculin bind to phospholipids and insert into the hydrophobic region of lipid membranes. Here, we show that the light scatter method can be used for measuring the affinity of proteins to phospholipid membranes. Large unilamellar DMPC/DMPG vesicles were produced by the extrusion technique (LUVETs). We have used repeated heating/cooling scans between 15 degrees C and 35 degrees C to ensure protein-lipid interaction/insertion. A molar affinity of talin, K = 2.9 x 10(6) M-1 and of vinculin, K = 3.3 x 10(5) M-1 to lipid vesicles, respectively, was determined from the plot; light scatter signal at 380 nm against protein concentrations by fitting the term, ln (Io/I-1) = A-K x c to the data.

Probing phosphatidylinositolphosphates and adenosinenucleotides on talin nucleated actin polymerization.

We have investigated the binding of PI, PIP and PIP2 to talin and the effect of phosphoinositides and adenosinenucleotides on talin-induced actin polymerization. At physiological salt concentrations, talin coprecipitates with liposomes when containing phosphoinositides but not when containing PI. The nucleating effect of talin as reflected by a twofold increase of fluorescence during the polymerization of actin labelled with NBD is not inhibited by phosphoinositides. The polymerization of ADP-actin versus ATP-actin was investigated in the presence and absence of talin by NBD fluorescence. ADP-actin nucleation induced by talin is comparably efficient as with ATP-actin. These experimental findings in summary have implications when evaluating the role of talin during cell activation.

Interaction of NBD-talin with lipid monolayers. A film balance study.

Fluorescently labelled smooth muscle talin like native talin interacts with negatively or partly negatively charged lipid monolayers. This was measured in time/area diagrams using the film balance technique combined with fluorescence imaging after double photolabelling of talin and phospholipids.

Differences in F9 and 5.51 cell elasticity determined by cell poking and atomic force microscopy.

We studied the elasticity of both a wild type (F9) mouse embryonic carcinoma and a vinculin-deficient (5.51) cell line, which was produced by chemical mutagenesis. Using cell poking, we measured the effects of loss of vinculin on the elastic properties of these cells. F9 cells were about 20% more resistant to indentation by the cell poker (a glass stylus) than were 5.51 cells. Using the atomic force microscope to map the elasticity of wild type and vinculin-deficient cells by 128 X 128 force scans, we observed a correlation of elasticity with cell poking elastometric measurements. These findings, as well as previous atomic force, rheologic, and magnetometric measurements [Goldmann and Ezzell, Exp. Cell Res. 226 (1996) 234-237; Ezzell et al., Exp. Cell Res. 231 (1997) 14-26], indicate that vinculin is an integral part of the cytoskeletal network.

Binding of tropomyosin-troponin to actin increases filament bending stiffness.

Rheologic measurements show that the association of tropomyosin-troponin with actin filaments is responsible for the reduction of the internal chain dynamic and increase in the mechanical rigidity of actin filaments. Basing calculations on the linear relation between the plateau modulus, G(N)('), and bending modulus, kappa, I find that tropomyosin-troponin at r(AT) = 7 increases actin filament stiffness by approximately 50%. This is confirmed by dynamic light scattering. Further increases are observed at rising F-actin and constant tropomyosin-troponin concentrations. Tropomyosin-troponin also delays actin assembly and subsequent network formation and increases filament stiffness over time.

Phosphorylation of filamin (ABP-280) regulates the binding to the lipid membrane, integrin, and actin.

Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm, where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, the hypothesis that it serves as a substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins is considered. The results suggest conformationally-induced regulation of filamin (ABP-280).

Kinetic determination of focal adhesion protein formation.

I examined the binding kinetics between integrin (alpha(IIb)beta(3)) and purified focal adhesion proteins, including alpha-actinin, filamin, vinculin, talin, and F-actin. Using static light-scatter technique, I observed affinities of the order talin > filamin > F-actin > alpha-actinin > (talin when bound to vinculin) which were lower when integrin was complexed with fibronectin. No binding between integrin and vinculin was detected. The calculated dissociation constants (K(d)) ranged between 0.4 microM and 5 microM. These results in part confirm previously published data using different methods. The modest affinity with which the focal adhesion proteins interact in vitro might be indicative of how cells, e.g., thrombocytes, gain a high degree of versatility and velocity.

Viscoelasticity in wild-type and vinculin-deficient (5.51) mouse F9 embryonic carcinoma cells examined by atomic force microscopy and rheology.

We have been studying mouse F9 embryonic carcinoma cells which contain no detectable vinculin protein (5.51 cells), and compared them with F9 wild-type cells. Employing atomic force microscopy, we probed the elastic properties of individual F9 wild-type and 5.51 cells by measuring the dynamic response of controlled loads of the cantilever tip. An elastic modulus (Young) of approximately 3.8 and approximately 2.5 kPa was calculated for wild-type and 5.51 cells, respectively. Using disc rheometry, we detected a marked change in shear of a 1000g pellet of approximately 55 x 10(6) cells between wild-type and 5.51 mutants. These differences are attributed to the loss of vinculin and altered cytoskeletal organization in these cells.