Cortactin deficiency causes increased RhoA/ROCK1-dependent actomyosin contractility, intestinal epithelial barrier dysfunction, and disproportionately severe DSS-induced colitis.

The intestinal epithelium constitutes a first line of defense of the innate immune system. Epithelial dysfunction is a hallmark of intestinal disorders such as inflammatory bowel diseases (IBDs). The actin cytoskeleton controls epithelial barrier integrity but the function of actin regulators such as cortactin is poorly understood. Given that cortactin controls endothelial permeability, we hypothesized that cortactin is also important for epithelial barrier regulation. We found increased permeability in the colon of cortactin-KO mice that was accompanied by reduced levels of ZO-1, claudin-1, and E-cadherin. By contrast, claudin-2 was upregulated. Cortactin deficiency increased RhoA/ROCK1-dependent actomyosin contractility, and inhibition of ROCK1 rescued the barrier defect. Interestingly, cortactin deficiency caused increased epithelial proliferation without affecting apoptosis. KO mice did not develop spontaneous colitis, but were more susceptible to dextran sulfate sodium colitis and showed severe colon tissue damage and edema formation. KO mice with colitis displayed strong mucus deposition and goblet cell depletion. In healthy human colon tissues, cortactin co-localized with ZO-1 at epithelial cell contacts. In IBDs patients, we observed decreased cortactin levels and loss of co-localization with ZO-1. Thus, cortactin is a master regulator of intestinal epithelial barrier integrity in vivo and could serve as a suitable target for pharmacological intervention in IBDs.

Dynamic MRI of the TMJ under physical load.

OBJECTIVES: The objective of this study was to examine the kinematics of structures of the temporomandibular joint (TMJ) under physiological load while masticating. METHODS: Radial MRI was chosen as a fast imaging method to dynamically capture the motions of the joint's anatomy. The technique included a golden ratio-based increment angle and a sliding window reconstruction. The measurements were performed on 22 subjects with and without deformation/displacement of the intra-articular disc while they were biting on a cooled caramel toffee. RESULTS: The reconstructed dynamic images provided sufficient information about the size and localization of the disc as well as the change of the intra-articular distance with and without loading. CONCLUSIONS: The feasibility of the golden ratio-based radial MRI technique to dynamically capture the anatomy of the TMJ under physical load was demonstrated in this initial study.

Influence of dental materials on dental MRI.

OBJECTIVES: To investigate the potential influence of standard dental materials on dental MRI (dMRI) by estimating the magnetic susceptibility with the help of the MRI-based geometric distortion method and to classify the materials from the standpoint of dMRI. METHODS: A series of standard dental materials was studied on a 1.5 T MRI system using spin echo and gradient echo pulse sequences and their magnetic susceptibility was estimated using the geometric method. Measurements on samples of dental materials were supported by in vivo examples obtained in dedicated dMRI procedures. RESULTS: The tested materials showed a range of distortion degrees. The following materials were classified as fully compatible materials that can be present even in the tooth of interest: the resin-based sealer AH Plus(®) (Dentsply, Maillefer, Germany), glass ionomer cement, gutta-percha, zirconium dioxide and composites from one of the tested manufacturers. Interestingly, composites provided by the other manufacturer caused relatively strong distortions and were therefore classified as compatible I, along with amalgam, gold alloy, gold-ceramic crowns, titanium alloy and NiTi orthodontic wires. Materials, the magnetic susceptibility of which differed from that of water by more than 200 ppm, were classified as non-compatible materials that should not be present in the patient's mouth for any dMRI applications. They included stainless steel orthodontic appliances and CoCr. CONCLUSIONS: A classification of the materials that complies with the standard grouping of materials according to their magnetic susceptibility was proposed and adopted for the purposes of dMRI. The proposed classification can serve as a guideline in future dMRI research.

Three-dimensional localization of impacted teeth using magnetic resonance imaging.

Impacted teeth remain embedded in the jawbone beyond the normal eruption time with completed root growth. They can often get infected or damage neighboring teeth. Information about the three-dimensional position of impacted teeth is invaluable in orthodontic diagnosis and treatment planning. The purpose of this prospective study was to assess the feasibility of using magnetic resonance imaging (MRI) for the three-dimensional localization of impacted teeth in children and adults. The study included 39 patients from the pediatric age group with different tooth impactions and seven adults with impacted wisdom teeth. MRI yielded a clear separation between impacted teeth and the surrounding tissue, and the position and angulation of impacted teeth in all three spatial dimensions could be assessed. Compared to conventional radiography, dental MRI provides the advantage of full volumetric morphology accompanied by complete elimination of ionizing radiation, which is particularly relevant for repeated examinations of the pediatric group.

A novel post-and-core restoration system--results of three years of clinical application of the "Wuerzburg Post".

The Wuerzburg Post is a new post-and-core restoration system designed to eliminate the weak parts of post-and-core restorations and the associated problems, respectively. In contrast to conventional posts, the Wuerzburg Post is a short and thick post, which no longer relies on cementation or luting for retention in the root, but on stress-free positive locking, which it achieves by means of a post which can be spread into a predefined and form-congruent undercut cavity. The second key feature is an annular groove which runs in the dentin, girded by a corresponding structure, ensuring regular force transmission and stress dissipation, as opposed to the classic ferrule design. There are two versions: one with a machined core which can be prepared like a classic build-up to support crowns and bridges, and another one with a 2.25 mm ball end to connect to common dies which can be integrated into removable prostheses. As the system utilizes prefabricated parts made from Titanium, a precise fit is ensured, enabling the user to restore teeth quickly and easily. Over the course of the past three years, 129 posts were inserted, most commonly on upper and lower incisors and canines. The main application was restoration of fractured telescopes. During the observation period, five failures were observed. Two of the failiures did not cause significant damage to the tooth, and were subsequently immediately repairable. The survival rate amounts to over 95% after three years under risk.

Filopodia formation induced by active mDia2/Drf3.

Filopodia are rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3DeltaDAD) induced the formation of filopodia and accumulated at their tips. Low expression of Drf3DeltaDAD induced rod-shaped or tapered filopodia, whereas over-expression resulted in multiple, club-shaped filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3DeltaDAD at their tips. These data suggest Drf3-induced filopodia form and extend by de novo nucleation of actin filaments instead of convergent elongation. Finally, Drf3DeltaDAD also induced the formation of unusual, lamellipodia-like structures, which contained both lamellipodial markers and the prominent filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines, reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.

Effect of bone conditioning on primary stability of FRIALIT-2 implants.

BACKGROUND: Primary stability is crucial to implants used for orthodontic anchorage. Bone condensing to enhance primary stability is controversial. MATERIAL AND METHODS: Fourteen Frialit-2-stepped screw and cylinder implants were placed in the median palatine sutures of 22 cadaveric human heads. In half of both types, the implant bed was prepared using a Frialit Bone Condenser. Primary implant stability was evaluated using non-invasive resonance frequency analysis. Moreover, the bone-implant contact area was examined histomorphometrically and radiographically. RESULTS: Bone condensing yielded a slightly, yet not significantly increased implant stability quotient compared with a conventional technique. In spongy bone, a significant histomorphometric increase of bone-implant contact (P<0.0001) and a significant increase of radiographic density was revealed for both implant types, while no significant changes were observed within the compact area. CONCLUSION: The study shows that bone condensing yields an improved histologic implant-bone contact only in spongy bone, which was paralleled by radiographic-densitometric findings.

Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement.

Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.

Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions.

Actin polymerization is accompanied by the formation of protein complexes that link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these processes is the LIM domain protein zyxin, which can bind both Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the actin filament cross-linking protein alpha-actinin. To characterize the localization and dynamics of zyxin in detail, we generated both monoclonal antibodies and a green fluorescent protein (GFP)-fusion construct. The antibodies colocalized with ectopically expressed GFP-VASP at focal adhesions and along stress fibers, but failed to label lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin antibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal adhesion protein vinculin revealed that both proteins incorporated simultaneously into newly formed adhesions. However, during spontaneous or induced focal adhesion disassembly, zyxin delocalization preceded that of either vinculin or paxillin. Together, these data identify zyxin as an early target for signals leading to adhesion disassembly, but exclude its role in recruiting Ena/VASP proteins to the tips of lamellipodia and filopodia.

Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells.

In mammalian cells, actin dynamics is tightly controlled through small GTPases of the Rho family, WASP/Scar proteins and the Arp2/3 complex. We employed Cre/loxP-mediated gene targeting to disrupt the ubiquitously expressed N-WASP in the mouse germline, which led to embryonic lethality. To elucidate the role of N-WASP at the cellular level, we immortalized embryonic fibroblasts and selected various N-WASP-defective cell lines. These fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia, but failed to support the motility of Shigella flexneri. In addition, enteropathogenic Escherichia coli were incapable of inducing the formation of actin pedestals in N-WASP-defective cells. Our results prove the essential role of this protein for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N-WASP is dispensable for filopodia formation.

The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia.

Cell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia [1], these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins, targets of Abl tyrosine kinases [2-4], have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation [5]. Here, we describe the unique localization of Abi proteins in living, motile cells. We show that Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. We identify the targeting domain as the homologous N terminus of these two proteins. Our findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility.

Visualising the actin cytoskeleton.

The actin cytoskeleton is a dynamic filamentous network whose formation and remodeling underlies the fundamental processes of cell motility and shape determination. To serve these roles, different compartments of the actin cytoskeleton engage in forming specific coupling sites between neighbouring cells and with the underlying matrix, which themselves serve signal transducing functions. In this review, we focus on methods used to visualise the actin cytoskeleton and its dynamics, embracing the use of proteins tagged with conventional fluorophores and green fluorescent protein. Included also is a comparison of cooled CCD technology, confocal and 2-photon fluorescence microscopy of living and fixed cells, as well as a critique of current procedures for electron microscopy.

Intima-like smooth muscle cells: developmental link between endothelium and media?

The presence of non-contractile smooth muscle cells within the arterial wall raises questions as to their origin and function. These cells abound within the aortae of murine and porcine neonates, but are also present within the intimal and medial layers of adult arteries. They are largely devoid of smooth muscle-associated proteins and manifest an epithelioid form. Their morphological resemblance to endothelial cells prompted us to explore this potential relationship and to investigate their angiogenic properties in three-dimensional collagen gels. Using well-characterized smooth muscle cell lines, displaying either the intima-like (epithelioid) or media-like (spindle-shaped) morphology, we were able to show that intima-like cells share several features in common with endothelial ones and can transform into a media-like phenotype, whereby they irreversibly lose their characteristic pattern of protein expression. Intima-like, but not media-like, vascular smooth muscle cells are capable of forming capillary tubes, and, in co-cultures, can induce media-like ones to participate in this process. Such capillaries consist of a randomly-organized, mixed population of endothelial cells with intima-like or media-like smooth muscle ones. The functional significance of this diversity in smooth muscle cell type is not well understood, but phenotypic plasticity could conceivably figure as an important adaptive response to changes in the local environment.

Cytoskeleton cross-talk during cell motility.

Cell crawling entails the co-ordinated creation and turnover of substrate contact sites that interface with the actin cytoskeleton. The initiation and maturation of contact sites involves signalling via the Rho family of small G proteins, whereas their turnover is under the additional influence of the microtubule cytoskeleton. By exerting relaxing effects on substrate contact assemblies in a site- and dose-specific manner, microtubules can promote both protrusion at the front and retraction at the rear, and thereby control cell polarity.

Interplay between Rac and Rho in the control of substrate contact dynamics.

BACKGROUND: Substrate anchorage and cell locomotion entail the initiation and development of different classes of contact sites, which are associated with the different compartments of the actin cytoskeleton. The Rho-family GTPases are implicated in the signalling pathways that dictate contact initiation, maturation and turnover, but their individual roles in these processes remain to be defined. RESULTS: We monitored the dynamics of peripheral, Rac-induced focal complexes in living cells in response to perturbations of Rac and Rho activity and myosin contractility. We show that focal complexes formed in response to Rac differentiated into focal contacts upon upregulation of Rho. Focal complexes were dissociated by inhibitors of myosin-II-dependent contractility but not by an inhibitor of Rho-kinase. The downregulation of Rac promoted the enlargement of focal contacts, whereas a block in the Rho pathway not only caused a dissolution of focal contacts but also stimulated membrane ruffling and formation of new focal complexes, which were associated with the advance of the cell front. CONCLUSIONS: Rac functions to signal the creation of new substrate contacts at the cell front, which are associated with the induction of ruffling lamellipodia, whereas Rho serves in the maturation of existing contacts, with both contact types requiring contractility for their formation. The transition from a focal complex to a focal contact is associated with a switch to Rho-kinase dependence. Rac and Rho also influence the development of focal contacts and focal complexes, respectively, through mutually antagonistic pathways.

Functional design in the actin cytoskeleton.

Changes in cell shape, anchorage and motility are all associated with the dynamic reorganisation of the architectural arrays of actin filaments that make up the actin cytoskeleton. The relative expression of these functionally different actin filament arrays is intimately linked to the pattern of contacts that a cell develops with its extracellular substrate. Cell polarity is acquired by the development of an asymmetric pattern of substrate contacts, effected in a specific, site-directed manner by the delivery of adhesion-site modulators along microtubules.

Targeting, capture, and stabilization of microtubules at early focal adhesions.

By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a "cross talk" between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.

Sex steroid-opioid interactions associated with the temporal component of avian calling patterns.

Isolation from conspecifics in young, precocial birds predictably induces distress vocalizations (DV) and androgens change this type of vocalization into male typical "crowing" (CR). In addition, opioid peptides are known to exert potent effects on avian vocal behavior. Here we investigate the organizational and activational correlates of sex-steroid actions on opioid-receptor organization and their relevance to the temporal evolution of DV and CR. From the effects of pre- and postnatal steroid applications and postnatal [3H]etorphin binding studies, we find that early steroidal effects become manifested at the behavioral level by changing the characteristic duration of vocalizations. In the male quail this extension of calling duration is accompanied by a clear decrease in opiate binding, whereas in the female there is a moderate increase in binding sites. The transition from DV to CR (within hours) induced by testosterone is correlated with "upregulation" of opiate receptor sites within unilateral brainstem areas of young male quail. Based on these findings, we suggest that organizational steroid effects change the characteristic duration of isolation-induced vocalizations and these effects appear to be manifested at the level of opioid-receptor distribution.