Photoconvertible Protein Research Articles

New Tools for Imaging of Immune Systems: Visualization of Cell Cycle, Cell Death, and Cell Movement by Using the Mice Lines Expressing Fucci, SCAT3.1, and Kaede and KikGR.

Visualization of biological events in real time in vivo has become a crucial to understand immune responses. We have been established novel visualization tools for life of immune cells: proliferation, cell death, and migration. Fucci-transgenic mice allow us to visualize cell cycle phases by reciprocal expression of mKusabira-Orange2 in G1 phase and mAzami-Green in S/G2/M phase. Caspase-3 indicator SCAT3.1 knock-in mice visualize cell death by changing color. Photoconvertible proteins, Kaede and KikGR expressing mice track cell movement between organs by labeling immune cells as red color. Here, I will introduce how to use and visualize these mice. These techniques will help to understand immune system in the living whole body.

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells.

307 Identification of resolvin E1, an omega-3 poly-unsaturated fatty acids-derived lipid mediator, as an inhibitor for psoriatic dermatitis by downregulating migration of IL-17A-producing cutaneous γδ T cells and IL-23 production from dendritic cells

The potential of omega-3 poly-unsaturated fatty acids (PUFAs) as a therapeutic target for psoriasis is a long-disputed question, since various epidemiological studies have suggested that intake of omega-3 PUFAs have ameliorating effect on the development of psoriasis. However, its actual significance and the molecular mechanisms have not been explored. In this study, we examined the effect of resolvin E1 (RvE1), a metabolite derived from omega-3 PUFAs, on psoriatic dermatitis using an imiquimod-induced mouse psoriasis model. RvE1 potently suppressed the inflammatory cell infiltration and epidermal hyperplasia in the psoriatic skin. In addition, RvE1 decreased the mRNA expression of key cytokines including IL-23 and IL-17A, which are produced by cutaneous dendritic cells (DCs) and γδ T cells, respectively. Consistently, RvE1 inhibited IL-23p19 and IL-23p40 mRNA expression in bone marrow-derived DCs in vitro. Furthermore, an in vivo cell migration assay using Kaede (a photo-convertible protein)-transgenic mice revealed that RvE1 significantly inhibited migration of both cutaneous DCs and γδ T cells from skin to draining lymph nodes (dLNs), an essential step for γδ T cell expansion in dLNs and their subsequent recirculation into skin to accelerate the psoriatic dermatitis. We have also revealed that this suppressive effect of RvE1 on γδ T cell migration was mediated by RvE1’s antagonistic function on BLT1, a receptor of leukotriene B4. Taken together, these results indicate that RvE1 exerts anti-inflammatory effects on psoriatic dermatitis in two manners; the attenuation of γδ T cell migration from skin to dLNs and the inhibition of IL-23 production from DCs. Our results have clarified a novel mechanism of omega-3 PUFA-mediated amelioration of psoriasis, and suggest a potential of RvE1 as a therapeutic target for psoriasis.

Abstract 1590: High intranuclear mobility of AR-v7 reveals distinct mode of transcriptional activity in prostate cancer with important therapeutic implications

Abstract It is well established that androgen receptor (AR) signaling, is a key driver of prostate cancer (PC) growth and metastatic progression. Therefore, androgen deprivation therapy (ADT) is the first line of treatment for PC. However, most patients develop castration resistant prostate cancer (CRPC). Interestingly, AR signaling remains active in CRPC, due to the expression of transcriptionally active AR splice variants (AR-Vs), which lack the ligand binding domain (LBD) and constitutively translocate to the nucleus even in castrate conditions. AR-v7 is the most prevalent AR-V expressed in about 60% of CRPC tumors. AR-v7 expression was clinically correlated with poor prognosis of CRPC patients and with resistance to next-generation AR signaling inhibitors, which are part of standard clinical care. Therefore, inhibition of AR-v7 function is urgently needed for the treatment of CRPC. Currently, there is no therapeutic modality that can inhibit AR-v7 expression or activity. Mechanistically, AR-v7 transcriptional targets largely overlap with those of AR-fl, with the exception of a few AR-v7 unique targets. However, the exact mechanism by which transcription is activated by AR-v7 is not known. In this study we sought to investigate the mechanisms underlying the transcriptional activity of the ligand-independent AR-v7 in comparison to liganded AR-fl. We used live cell imaging to monitor the dynamics and intranuclear mobility of fluorescently-tagged AR-v7 or AR-fl. Fluorescent recovery after photobleaching (FRAP) revealed that AR-v7 intranuclear mobility was significantly faster than that of liganded AR-fl, with t1/2 3s versus several minutes, respectively. To precisely map the spatial distribution and chromatin-binding kinetics of AR-fl and AR-v7, we generated expression plasmids with AR tagged with green-to-red mEos4 photo-convertible proteins. Photoconversion of a discrete, subnuclear pool of AR-fl in the presence of its ligand (R1881) followed by 1 hr of time-lapse imaging in 5 min intervals, revealed absence of mobility indicating tight chromatin binding and active transcription. In contrast, similar photoconversion experiment for AR-v7, revealed immediate redistribution throughout the nucleus in less than 9 s, suggesting a “hit-and-run” mode of interaction with DNA with uncertain transcriptional output. QRT-PCR of endogenous target genes and ARE-mcherry reporter assays showed similar transcriptional activity of the two proteins. We are currently investigating the relationship between rates of intranuclear mobility and transcriptional activity and the mechanisms underlying the distinct mobility patterns. These data suggest that AR-v7 has a distinct mode of interaction with DNA and gene promoters, which may identify novel targetable pathways for its inhibition in CRPC. Citation Format: Seaho Kim, Mohd Azrin Jamalruddin, Paraskevi Giannakakou. High intranuclear mobility of AR-v7 reveals distinct mode of transcriptional activity in prostate cancer with important therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1590. doi:10.1158/1538-7445.AM2017-1590

A rare subset of skin-tropic regulatory T cells expressing Il10/Gzmb inhibits the cutaneous immune response

Abstract We reported that Foxp3+ regulatory T cells (Tregs) migrating from the skin to the draining lymph node have a strong immunosuppressive effect on contact hypersensitivity (CHS) response. However, the Treg subsets that regulate the CHS response remain poorly defined. In this study, we used a combination of single-cell real-time PCR high-dimensional gene expression profiling data with spatiotemporal information about cellular movement from the mice expressing the photoconvertible protein KikGR to elucidate the role and characteristics of Treg subsets in CHS response. We found that although immunosuppressive genes Ctla4 and Tgfb1 were expressed in the majority of Tregs, Il10-expressing Tregs were rare and unexpectedly, the majority of Il10-expressing Tregs co-expressed Gzmb and displayed Th1-skewing. Furthermore, Gzmb-/Il10-expressing Treg subsets expressed the novel markers CD43 and CCR5. CD43+ CCR5+ CXCR3− Tregs highly expressed skin-tropic chemokine receptors CCR4 and CCR8. We also measured the retention of Treg subsets within inflamed skin using KikGR mice, and found that the subset of Tregs that was most highly retained in the skin, CD43+ CCR5+ CXCR3− Tregs, had superior in vivo inhibitory function to other Treg subsets. These results suggested that even if only present in small numbers, highly activated Tregs that co-express Gzmb and Il10 and that have the capacity to remain in inflamed tissue are likely to be clinically relevant due to the role of these molecules in the control of excessive immune responses. Taken together, the identification of a rare Treg subset co-expressing multiple immunosuppressive molecules and having tissue-remaining capacity offers a novel strategy for the control of skin inflammatory responses.

3D Protein Dynamics in the Cell Nucleus

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.

Using Photoconvertible and Extractable Fluorescent Proteins to Study Autophagy in Plants.

Several methodologies have been employed to understand the kinetics of induced autophagic degradation in plants, but most of them are not capable of distinguishing the autophagic cargo proteins before and after induction of autophagy in cells. Here, we designed a mass photoconverter that allowed us to simultaneously monitor protein synthesis and degradation in tobacco BY-2 cells using a photoconvertible fluorescence marker protein, Kikume Green Red (KikGR). An example of a new protocol for the analysis of autophagy progression using a fusion protein of cytochrome b5 and KikGR under phosphate starvation is described. The other example described is the analysis of the proliferation of Golgi apparatus in tobacco BY-2 cells using the fusion protein of a prolyl 4-hydroxylase NtP4H1.1 and monomeric KikGR. A detailed protocol on key analysis, as well as tips and notes for experiments using KikGR proteins, are described.

Dynamic mRNA Transport and Local Translation in Radial Glial Progenitors of the Developing Brain

In the developing brain, neurons are produced fromneural stem cells termed radial glia [1, 2]. Radial glial progenitors span the neuroepithelium, extending long basal processes to form endfeet hundreds of micrometers away from the soma. Basal structures influence neuronal migration, tissue integrity, and proliferation [3-7]. Yet, despite the significance of these distal structures, theircell biology remains poorly characterized, impeding our understanding of how basal processes and endfeet influence neurogenesis. Here we use live imaging of embryonic brain tissue to visualize, for the first time, rapid mRNA transport in radial glia, revealing that the basal process is a highway for directed molecular transport. RNA- and mRNA-binding proteins, including the syndromic autism protein FMRP, move in basal processes at velocities consistent with microtubule-based transport, accumulating in endfeet. We develop an exvivo tissue preparation to mechanically isolate radial glia endfeet from the soma, and we use photoconvertible proteins to demonstrate that mRNA is locally translated. Using RNA immunoprecipitation and microarray analyses of endfeet, we discover FMRP-bound transcripts, which encode signaling and cytoskeletal regulators, including many implicated in autism and neurogenesis. We show FMRP controls transport and localization of one target, Kif26a. These discoveries reveal a rich, regulated local transcriptome in radial glia, far from the soma, and establish a tractable mammalian model for studying mRNA transport and local translation invivo. We conclude that cytoskeletal and signaling events at endfeet may be controlled through translation of specific mRNAs transported from thesoma, exposing new mechanistic layers within stem cells of the developing brain.

Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins.

The application of green-to-red photoconvertible fluorescent proteins (PCFPs) for in vivo studies in complex 3D tissue structures has remained limited because traditional near-UV photoconversion is not confined in the axial dimension, and photomodulation using axially confined, pulsed near-IR (NIR) lasers has proven inefficient. Confined primed conversion is a dual-wavelength continuous-wave (CW) illumination method that is capable of axially confined green-to-red photoconversion. Here we present a protocol to implement this technique with a commercial confocal laser-scanning microscope (CLSM); evaluate its performance on an in vitro setup; and apply primed conversion for in vivo labeling of single cells in developing zebrafish and mouse preimplantation embryos expressing the green-to-red photoconvertible protein Dendra2. The implementation requires a basic understanding of laser-scanning microscopy, and it can be performed within a single day once the required filter cube is manufactured.

Abstract A065: Oscillatory genes in lymphocyte retention and egress

Abstract The adaptive immune system attacks cancer cells by initiating immune responses in lymphoid organs and egressing of activated effector cells into circulation to reach the tumor. Several G protein-coupled receptors have been reported to recruit lymphocytes to the lymphoid organ or to promote exit. However, how these signals crosstalk to ensure efficient lymphocyte recruitment and transit is not well understood. To further define how cell transit through the lymphoid tissue is controlled, we designed two parallel RNAseq to identify novel regulators for lymphocyte transit. We compared the transcriptome of naïve B cells from the blood and peripheral lymph node. Also, by utilizing photoconvertible proteins, we isolated naive B cells that newly enter the lymphoid organ and those that have been dwelling for several hours. We identified multiple genes whose transcript levels were correlated with lymphoid dwelling time. One of the genes, KLF11, encoding a transcription factor, is expressed more highly in the blood than in the lymph node, and the expression decreases with the dwelling time in the lymphoid organ. Analysis of the KLF11 knock-out mouse identified strong lymphocyte transit and developmental defects, but we found the strong phenotypes segregated independently of the KLF11 knock-out allele. The phenotypes were the result of a mutated DOCK2 allele in the commercial B6 background. We have now generated a KLF11 knock-out with wildtype DOCK2. The progress of characterizing the function of KLF11 in lymphocyte recruitment and egress will be reported. Citation Format: Hsin Chen, Timothy Schmidt, Ying Xu, Erick Lu, Jason G. Cyster. Oscillatory genes in lymphocyte retention and egress [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A065.

Two-photon excited photoconversion of cyanine-based dyes.

The advent of phototransformable fluorescent proteins has led to significant advances in optical imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine-based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal. These findings are expected to be useful for applications involving rapid labeling of cells deep in tissue.

Abstract P3-01-22: Sentinel lymph node metastases in breast cancer: A contributor to distant metastases?

Abstract Breast cancer metastasis remains a major cause of mortality in patients. Although significant progress has been made in understanding the mechanisms of this complex process, the findings have yet to be translated into improved survival rates in patients with metastatic disease. The presence of lymph node metastasis in most breast cancer patients is associated with tumor aggressiveness, poorer prognosis and often results in the need for systemic therapy. However, whether tumor cells in the lymph node exit and contribute to distant metastases remains controversial. To track the fate of tumor cells that metastasized to the lymph node, we engineered breast tumor cell lines to express Dendra2, a photo-convertible protein that fluoresces green in the native state and on light activation converts to red fluorescence. Using a novel chronic lymph node window that allows time-lapse imaging over a period of 10 days, we were able to track the movement of cancer cells in the dynamic microenvironment using high- resolution multi-photon microscopy. Our studies show that tumor cells entering the lymph node through the afferent lymphatic vessel proliferate in the sub-capsular sinus and later begin to invade the lymph node parenchyma. Importantly, by photo-conversion of tumor cells in the lymph node, we are able to track the tumor cells that escaped the lymph node and entered the blood circulation. The circulating tumor cells that transited through the lymph node were viable and proliferated in vitro. Our data indicate that metastatic breast cancer cells can exit the node and potentially colonize distant organs. Citation Format: Pereira ER, Kedrin D, Jones D, Beech E, Taghian A, Padera TP. Sentinel lymph node metastases in breast cancer: A contributor to distant metastases?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-01-22.

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

February 2016 - This Month in JoVE: Photoconvertible Proteins, Gold Nanoparticles, PET Principles, and Bone Marrow Microenvironments

Here's a look at what's coming up in the February 2016 issue of JoVE: The Journal of Visualized Experiments. In JoVE Developmental Biology, the transparent, rapidly developing zebrafish embryo is ideal for visualizing developmental processes. When cells of interest are labeled with fluorescent photoconvertible proteins, they allow precise tracking of defined structures-highlighting specific cells while leaving other transgenic cells in the dark. Beretta et al. have established the photoswitchable monomeric orange (PSmOrange) system for zebrafish. This protein's orange-to-red spectrum allows it to visible in existing transgenic lines expressing green fluorescent protein (GFP). Microinjection of nuclear-targeted PSmOrange mRNA labels all cell nuclei with orange/red fluorescence, and targeted photoconversion switches its emission spectrum to far red. The quantum efficiency and stability of PSmOrange makes it a superb cell-tracking tool for living zebrafish during embryonic development and disease. In JoVE Chemistry, few materials have found as many uses in so many diverse fields as gold nanoparticles. Their applications range from biological sensors to radio frequency-based cancer treatments. Gold nanoparticles are valued for their unique structural, optical and electronic properties. These special attributes caught the interest of Oliver Smithies, who won the Nobel Prize in Physiology or Medicine in 2007. This month, he and his colleagues describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles, and present models that can predict particle size with great accuracy. In JoVE Engineering, we look at the principles of positron emission tomography (PET), a non-invasive technique for imaging the body's inner tissues and organs. Montano-Zetina and Villalobos-Mora present a guide for constructing a simple, homemade PET system for fully characterizing its basic working principles. This prototype demonstrates the primary functions of PET, and serves as an elegant model for teaching its principles to the academic public. In JoVE Medicine, it is well established that the bone marrow microenvironment provides a haven for hematopoietic diseases. This month, Slone et al. use cell types from the bone marrow niche in an in vitro co-culture model. This supports the generation of a subpopulation of chemoresistant tumor cells. These calls can be used to investigate the underlying pathways of tumor development and to test novel therapeutic strategies. You've just had a sneak peek of the February 2016 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

The Emergence of Y-junctions in the Zebrafish Cone Mosaic

Cone photoreceptor cells in the retina of teleost fish are arranged in a remarkably ordered, crystalline pattern. Just like an atomic crystal, this “cellular crystal” exhibits topological defects; they are known as Y-junctions in the fish literature and correspond to edge dislocations in the terminology of materials science. Although the presence of Y-junctions in the cone mosaic has previously been noted, they have never been studied systematically. We have characterized Y-junctions within the zebrafish cone mosaic using two methods. The first is a flat mount retina prepared from transgenic zebrafish in which the UV-opsin promoter drives expression of GFP in UV cones. With image processing software, we characterized the distribution of Y-junctions across the retina. We found that Y-junctions coalesce into grain boundaries and are distributed throughout the retina. Using this information, we developed a second live-imaging method that allows us to study the dynamics of Y-junctions. We generated a transgenic construct that expresses a photoconvertible protein, mEOS, in the nucleus of UV cones. Multiphoton microscopy allows us to visualize Y-junctions in live juvenile fish and photoconvert the cones around an identified Y-junction. We are able to track the Y-junction defect in the cone mosaic pattern over time to determine whether the defect is immobile after it forms or whether the pattern is reorganized as the retina grows.

Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed.

Intermediate filament dynamics: What we can see now and why it matters.

The mechanical properties of vertebrate cells are largely defined by the system of intermediate filaments (IF). As part of a dense network, IF polymers are constantly rearranged and relocalized in the cell to fulfill their duty as cells change shape, migrate, or divide. With the development of new imaging technologies, such as photoconvertible proteins and super-resolution microscopy, a new appreciation for the complexity of IF dynamics has emerged. This review highlights new findings about the transport of IF, the remodeling of filaments by a process of severing and re-annealing, and the subunit exchange that occurs between filament precursors and a soluble pool of IF. We will also discuss the unique dynamic features of the keratin IF network. Finally, we will speculate about how the dynamic properties of IF are related to their functions.

Local translation of cell adhesion molecules in axons.

During development and regeneration, axonal growth depends on a rapid response to extracellular growth and guidance molecules. One mechanism underlying this rapid response is local protein synthesis (Jung et al., 2012). Local protein synthesis is a highly tuned, multi-step process by which mRNA binding proteins bind to specific sequences within the untranslated regions of particular mRNAs in the soma; mRNAs are then transported to axons and growth cones and translated at the appropriate spatial location in response to specific guidance cues. Many studies have now demonstrated that stimulus-induced local translation is critical to appropriate axon growth, guidance and regeneration. Multiple types of proteins are locally translated within axons, including those regulating the cytoskeleton, retrograde signaling, regeneration and cellular survival.

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.