Creating new fluorescent probes for cell biology Tsien, Roger Y; Zhang, Jin; Campbell, Robert E ...
Nature reviews. Molecular cell biology,
200212, 2002-Dec, 2002-12-01, 20021201, Letnik:
3, Številka:
12
Journal Article
Recenzirano
Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally ...perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein-protein interactions.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we ...introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.
Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful ...tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn²⁺ ions binds tightly but reversibly to hexahistidine (His₆) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca²⁺ from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His₆ tags are accessible to the dye or antibodies, demonstrating externalization.
A glowing new era in cancer surgery may be dawning. Using fluorescently labelled markers, surgical molecular navigation means that tumours and nerves can be displayed in real time intra-operatively ...in contrasting pseudocolours, which allows more complete tumour resection while preserving important structures. These advances can potentially cause a paradigm shift in cancer surgery, improving patient outcome and decreasing overall health-care costs.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The endoplasmic reticulum (ER) serves as a cellular storehouse for Ca2+, and Ca2+released from the ER plays a role in a host of critical signaling reactions, including exocytosis, contraction, ...metabolism, regulation of transcription, fertilization, and apoptosis. Given the central role played by the ER, our understanding of these signaling processes could be greatly enhanced by the ability to image Ca2+ERdirectly in individual cells. We created a genetically encoded Ca2+indicator by redesigning the binding interface of calmodulin and a calmodulin-binding peptide. The sensor has improved reaction kinetics and a Kdideal for imaging Ca2+in the ER and is no longer perturbed by large excesses of native calmodulin. Importantly, it provides a significant improvement over all previous methods for monitoring Ca2+ERand has been used to directly show that, in MCF-7 breast cancer cells, the antiapoptotic protein B cell lymphoma 2 (Bcl-2) (i) lowers Ca2+ERby increasing Ca2+leakage under resting conditions and (ii) alters Ca2+oscillations induced by ATP, and that acute inhibition of Bcl-2 by the green tea compound epigallocatechin gallate results in an increase in Ca2+ERdue to inhibition of Bcl-2-mediated Ca2+leakage.
Matrix metalloproteinases-2 and -9 (MMP-2/-9) are key tissue remodeling enzymes that have multiple overlapping activities critical for wound healing and tumor progression in vivo. To overcome issues ...of redundancy in studying their functions in vivo, we created MMP-2/-9 double knockout (DKO) mice in the C57BL/6 background to examine wound healing. We then bred the DKO mice into the polyomavirus middle T (PyVmT) model of breast cancer to analyze the role of these enzymes in tumorigenesis. Breeding analyses indicated that significantly fewer DKO mice were born than predicted by Mendelian genetics and weaned DKO mice were growth compromised compared with wild type (WT) cohorts. Epithelial wound healing was dramatically delayed in adult DKO mice and when the DKO was combined with the PyVmT oncogene, we found that the biologically related process of mammary tumorigenesis was inhibited in a site-specific manner. To further examine the role of MMP-2/-9 in tumor progression, tumor cells derived from WT or DKO PyVmT transgenic tumors were grown in WT or DKO mice. Ratiometric activatable cell penetrating peptides (RACPPs) previously used to image cancer based on MMP-2/-9 activity were used to understand differences in MMP activity in WT or knockout syngeneic tumors in WT and KO animals. Analysis of an MMP-2 selective RACPP in WT or DKO mice bearing WT and DKO PyVmT tumor cells indicated that the genotype of the tumor cells was more important than the host stromal genotype in promoting MMP-2/-9 activity in the tumors in this model system. Additional complexities were revealed as the recruitment of host macrophages by the tumor cells was found to be the source of the tumor MMP-2/-9 activity and it is evident that MMP-2/-9 from both host and tumor is required for maximum signal using RACPP imaging for detection. We conclude that in the PyVmT model, the majority of MMP-2/-9 activity in mammary tumors is associated with host macrophages recruited into the tumor rather than that produced by the tumor cells themselves. Thus therapies that target tumor-associated macrophage functions have the potential to slow tumor progression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. ...Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein “toolkit”.
The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed ...evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.
Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background ...staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to β-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MG and SRB aptamers, which are short RNA sequences originally selected only for binding to malachite green or sulforhodamine B, can greatly enhance the fluorescence of normally nonfluorescent ...triphenylmethane dyes. MG aptamer enhances the quantum yields of malachite green (MG) and a novel rigidized derivative, indolinyl malachite green (IMG) by >2000-fold. SRB aptamer brightens patent blue V and VF by >90-fold. These enhancements are specific because MG aptamer has no effect on patent blue dyes and SRB aptamer has little or no effect on MG and IMG. Such sequence-specific fluorescence labeling of short RNA motifs is a first step toward genetically encodable fusion tags for imaging selected RNAs in vitro and in cells.
PDF
