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A spatially resolved total internal reflection fluorescence correlation microscopy (TIR-FCM) system is constructed with an electron multiplying charge-coupled device (EMCCD) camera. The system was used to determine diffusion coefficients of lipid molecules in a planar lipid bilayer, and lipids and epidermal growth factor receptor (EGFR) proteins on cell membranes of Chinese Hamster Ovary (CHO) cells. The evaluation of the 'cross talk' between neighboring pixels suggests that a higher degree of multiplexing can be achieved than was previously proposed [Kannan, B. et al. Anal Chem. 2006, 78, 344451] using the same camera with a focused laser excitation. The best time resolution possible with this system is 4 ms for a region of interest comprising 20 lines in the CCD and is good enough to determine membrane diffusion in lipid bilayers and of membrane proteins in living cells. In this work, using a TIR-FCM setup, 1600 autocorrelation functions were measured simultaneously with a time resolution of 4.8 ms. This area corresponds to a 40 x 40 pixel region of interest with a dimension of 11.3 x 11.3 [micro][m.sup.2] and is sufficiently large to allow the measurement of the lower membrane of a whole cell simultaneously.

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]

Rif induces dorsal filopodia but the signaling pathway responsible for this has not been identified. We show here that Rif interacts with the I-BAR family protein IRTKS (also known as BAIAP2L1) through its I-BAR domain. Rif also interacts with Pinkbar (also known as BAIAP2L2) in N1E-115 mouse neuroblastoma cells. IRTKS and Rif induce dorsal membrane ruffles and filopodia. Dominant-negative Rif inhibits the formation of IRTKS-induced morphological structures, and Rif activity is blocked in IRTKS-knockout (KO) cells. To further define the Rif-IRTKS signaling pathway, we identify Eps8 and WAVE2 (also known as WASF2) as IRTKS interactors. We find that Eps8 regulates the size and number of dorsal filopodia and membrane ruffles downstream of Rif-IRTKS signaling, whereas WAVE2 modulates dorsal membrane ruffling. Furthermore, our data suggests that Tir, a protein essential for enterohemorrhagic Escherichia coli infection, might compete for Rif for interaction with the I-BAR domain of IRTKS. Based on this evidence, we propose a model in which Rho family GTPases use the I-BAR proteins, IRSp53 (also known as BAIAP2), IRTKS and Pinkbar, as a central mechanism to modulate cell morphology. [ABSTRACT FROM AUTHOR]

A facile method for the synthesis of highly fluorescent manganese-doped zinc sulfide (ZnS:Mn) nanocrystals covalently functionalized with polyethylene glycol conjugated graphene oxide (GO-PEG) for drug delivery and cell labeling is reported. First, covalently functionalized GO with PEG-bis(amine) to enhance the solubility and biocompatibility in water and physiological buffers. Second, glutathione (GSH)-coated ZnS:Mn-doped nanocrystals were covalently grafted onto GO-PEG. An acid-amidation process was employed to obtain GO-PEG/ZnS:Mn nanocomposites, which were characterized by UV/Vis, photoluminescence, and Fourier transform infrared spectroscopies, and transmission electron microscopy. Finally, the anticancer drug doxorubicin (DOX) was noncovalently loaded onto these GO-PEG/ZnS:Mn composite particles. High drug entrapment efficiency (100 % due to more GO surface available for binding), slow in vitro release of drug (ca. 40 % at acidic pH), better HeLa cancer cell killing efficiency (ca. 85 %), and cell labeling capability are the important traits of these DOX-loaded nanocomposites. It is believed these novel fluorescent [GO-PEG/ZnS:Mn]-DOX composite particles have great potential as theranostic agents in cancer diagnosis and therapy. [ABSTRACT FROM AUTHOR]

The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.

ABSTRACT Luciferase from Indian firefly Luciola praeusta (Coleoptera: Lampyridae: Luciolinae) was isolated and the properties compared with that of the North American firefly, Photinus pyralis. Luciola praeusta luciferase was purified using acetone extraction, gel-filtration column chromatography, ammonium sulfate precipitation and anion exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a homogeneous preparation and the molecular mass was slightly higher than that of Photinus pyralis. The effect of pH, buffer composition and metal ions on the spectral characteristics was studied. The maximum bioluminescence activity of luciferase was observed in ACES buffer at pH 6.5. The emission maximum of 562 nm (in crude extract) was red shifted to 570 nm in Tricine buffer at pH 7.8. In addition, the effect of bovine serum albumin on the storage stability of the protein was investigated. Based on the unique spectral characteristics observed, we propose that Luciola praeusta luciferase in the native form is suitable for the assay of biochemical metabolites in acidic pH. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Oct4 and Sox2 are two essential transcription factors that co-regulate target genes for the maintenance of pluripotency. However, it is unclear whether they interact prior to DNA binding or how the target sites are accessed in the nucleus. By generating fluorescent protein fusions of Oct4 and Sox2 that are functionally capable of producing iPSCs (induced pluripotent stem cells), we show that their interaction is dependent on the presence of cognate DNA-binding elements, based on diffusion time, complex formation and lifetime measurements. Through fluorescence correlation spectroscopy, the levels of Oct4 and Sox2 in the iPSCs were quantified in live cells and two diffusion coefficients, corresponding to free and loosely bound forms of the protein, were distinguished. Notably, the fraction of slow-diffusing molecules in the iPSCs was found to be elevated, similar to the profile in embryonic stem cells, probably due to a change in the nuclear milieu during reprogramming. Taken together, these findings have defined quantitatively the amount of proteins pertinent to the pluripotent state and revealed increased accessibility to the underlying DNA as a mechanism for Oct4 and Sox2 to find their target binding sites and interact, without prior formation of heterodimer complexes. [ABSTRACT FROM AUTHOR]

Filopodia are cellular protrusions important for axon guidance, embryonic development, and wound healing. The Rho GTPase Cdc42 is the best studied inducer of filopodium formation, and several of its effectors and their interacting partners have been linked to the process. These include IRSp53, N-WASP, Mena, and Eps8. The Rho GTPase, Rif, also drives filopodium formation. The signaling pathway by which Rif induces filopodia is poorly understood, with mDia2 being the only protein implicated to date. It is thus not clear how distinct the Rif-driven pathway for filopodium formation is from the one mediated by Cdc42. In this study, we characterize the dynamics of Rif-induced filopodia by time lapse imaging of live neuronal cells and show that Rif drives filopodium formation via an independent pathway that does not involve the Cdc42 effectors N-WASP and IRSp53, the IRSp53 binding partner Mena, or the Rac effectors WAVE1 and WAVE2. Rif formed filopodia in the absence of N-WASP or Mena and when IRSp53, WAVE1, or WAVE2 was knocked down by RNAi. Rif-mediated filopodial protrusion was instead reduced by silencing mDial expression or overexpressing a dominant negative mutant ofmDial. mDial on its own was able to form filopodia. Data from acceptor photobleaching FRET studies of protein-protein interaction demonstrate that Rif interacts directly with mDial in filopodia but not with mDia2. Taken together, these results suggest a novel pathway for filopodia formation via Rif and mDial. [ABSTRACT FROM AUTHOR]

Transducer of Cdc42-dependent actin assembly (Toca-1) consists of an F-BAR domain, a Cdc42 binding site and an SH3 domain. Toca-1 interacts with N-WASP, an activator of actin nucleation that binds Cdc42. Cdc42 may play an important role in regulating Toca-1 and N-WASP functions. We report here that the cellular expression of Toca-1 and N-WASP induces membrane tubulation and the formation of motile vesicles. Marker and uptake analysis suggests that the tubules and vesicles are associated with clathrin-mediated endocytosis. Forster resonance energy transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) analysis shows that Cdc42, N-WASP and Toca-1 form a trimer complex on the membrane tubules and vesicles and that Cdc42 interaction with N-WASP is critical for complex formation. Modulation of Cdc42 interaction with Toca-1 and/or N-WASP affects membrane tubulation, vesicle formation and vesicle motility. Thus Cdc42 may influence endocytic membrane trafficking by regulating the formation and activity of the Toca-1/N-WASP complex. [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]

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-offunction actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics. [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]

Multiphoton microscopy has emerged as a powerful modality for noninvasive, spatial, and temporal imaging of biological tissues without the use of labels and/or dyes. It provides complimentary imaging modalities, which include two-photon excited fluorescence (2PEF) and second harmonic generation (SHG). 2PEF from endogenous chromophores such as nicotinamide adenine dinucleotides (NADH), flavins and keratin enable visualization of cellular and subcellular structures. SHG provides visualization of asymmetric macromolecular structures such as collagen. These modalities enable the visualization of biochemical and biological alterations within live tissues in their native state.Organotypic cultures of the skin and oral mucosa equivalents have been increasingly used across basic and translational research. However, assessment of the skin and oral mucosa equivalents is predominantly based on histological techniques which are not suited for real-time imaging and longitudinal studies of the tissues in their native state. 2PEF from endogenous chromophores and SHG from collagen can be effectively used as an imaging tool for noninvasive and label-free acquisition of cellular and matrix structures of live skin and oral mucosa cultures.In this chapter, the methods for noninvasive and label-free imaging of monolayer and organotypic cultures of the skin and oral mucosa using multiphoton microscopy are described.