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Background and Objectives: Ageing is associated with an impaired cellular function that can affect tissue architecture and wound healing in gingival and periodontal tissues. However, the impact of oral fibroblast ageing on the structural organization of the extracellular matrix (ECM) proteins is poorly understood. Hence, in this study, we investigated the impact of cellular ageing of oral fibroblasts on the production and structural organization of collagen and other ECM proteins. Methods: Oral fibroblasts were serially subcultured, and replicative cellular senescence was assessed using population doubling time, Ki67 counts and expression of P21WAFI. The production and structural organization of ECM proteins were assessed at early (young‐oFB) and late (aged‐oFB) passages. The thickness and pattern of collagen produced by live cultures of young‐ and aged‐oFB were assessed using a label‐free and non‐invasive second harmonic generation (SHG)‐based multiphoton imaging. Expression of other ECM proteins (fibronectin, fibrillin, collagen‐IV and laminins) was evaluated using immunocytochemistry and confocal microscopy‐based depth profile analysis. Results: Aged‐oFB displayed a higher population doubling time, lower Ki67+ cells and higher expression of P21WAFI indicative of slower proliferation rate and senescence phenotype. SHG imaging demonstrated that young‐oFB produced a thick, interwoven network of collagen fibres, while the aged‐oFB produced thin and linearly organized collagen fibres. Similarly, analysis of immunostained cultures showed that young‐oFB produced a rich, interwoven mesh of fibronectin, fibrillin and collagen‐IV fibres. In contrast, the aged‐oFB produced linearly organized fibronectin, fibrillin and collagen‐IV fibres. Lastly, there was no observable difference in production and organization of laminins among the young‐ and aged‐oFB. Conclusion: Our results suggest that oral fibroblast ageing impairs ECM production and more importantly the organization of ECM fibres, which could potentially impair wound healing in the elderly. [ABSTRACT FROM AUTHOR]

Protein transport is an important phenomenon in biological systems. Proteins are transported via several mechanisms to reach their destined compartment of cell for its complete function. One such mechanism is the microtubule mediated protein transport. Up to now, there are no reports on synthetic systems mimicking the biological protein transport mechanism. Here we report a highly efficient method of mimicking the microtubule mediated protein transport using newly designed biotinylated peptides encompassing a microtubule-associated sequence (MTAS) and a nuclear localization signaling (NLS) sequence, and their final conjugation with streptavidin-coated CdSe/ZnS quantum dots (QDs). Our results demonstrate that these novel bio-conjugated QDs enhance the endosomal escape and promote targeted delivery into the nucleus of human mesenchymal stem cells via microtubules. Mimicking the cellular transport mechanism in stem cells is highly desirable for diagnostics, targeting and therapeutic applications, opening up new avenues in the area of drug delivery. [ABSTRACT FROM AUTHOR]

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane- bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (KD) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine KD of Cdc42- effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured KD for Cdc42Vl2-N-WASP, Cdc42Vl2-CRIB, and Cdc42Vl2-IRSp53 was 27, 250, and 391 nM, respectively. The determination of KD for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells. [ABSTRACT FROM AUTHOR]

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15Δ95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis. [ABSTRACT FROM AUTHOR]

The Cdc42 effector IRSp53 is a strong inducer of filopodia formation and consists of an Src homology domain 3 (SH3), a potential WW-binding motif, a partial-Cdc42/Rac interacting binding region motif, and an lnverse-Bin-Amphiphysins-Rvs (I-BAR) domain. We show that IRSp53 interacts directly with neuronal Wiskott-Aldrich syndrome protein (N-WASP) via its SH3 domain and furthermore that N-WASP is required for filopodia formation as IRSp53 failed to induce filopodia formation in N-WASP knock- out (KO) fibroblasts. IRSp53-induced filopodia formation can be reconstituted in N-WASP KO fibroblasts by full-length N-WASP, by N-WASPΔWA (a mutant unable to activate the Arp2/3 complex), and by N-WASPH208D (a mutant unable to bind Cdc42). IRSp53 failed to induce filopodia in mammalian enabled (Mena)/ VASP KO cells, and N-WASP failed to induce filopodia when IRSp53 was knocked down with RNA interference. The IRSp53 I-BAR domain alone induces dynamic membrane protrusions that lack actin and are smaller than normal filopodia ("partial-filopodia") in both wild-type N-WASP and N-WASP KO cells. We propose that IRSp53 generates filopodia by coupling membrane protrusion through its I-BAR domain with actin dynamics through SH3 domain binding partners, including N-WASP and Mena. [ABSTRACT FROM AUTHOR]