• Issue front cover thumbnail

      Volume 43, Issue 3

      July 2018,   pages  a-567

    • Editorial


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    • GFP fluorescence: A few lesser-known nuggets that make it work


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      Use of Green Fluorescent Protein (GFP) as a marker has revolutionized biological research in the last few decades. In this briefcommentary, we reflect upon the success story of GFP and highlight a few lesser-known facets about GFP that add up to itsusefulness.

    • A novel fluorescence microscopic approach to quantitatively analyse protein-induced membrane remodelling


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      Membrane remodelling or the bending and rupture of the lipid bilayer occurs during diverse cellular processes such as celldivision, synaptic transmission, vesicular transport, organelle biogenesis and sporulation. These activities are brought aboutby the localized change in membrane curvature, which in turn causes lipid-packing stress, of a planar lipid bilayer byproteins. For instance, vesicular transport processes are typically characterized by the cooperative recruitment of proteinsthat induce budding of a planar membrane and catalyse fission of the necks of membrane buds to release vesicles. Theanalysis of such membrane remodelling reactions has traditionally been restricted to electron microscopy–based approachesor force spectroscopic analysis of membrane tethers pulled from liposome-based model membrane systems. Our recentwork has demonstrated the facile creation of tubular model membrane systems of supported membrane tubes (SMrTs),which mimic late-stage intermediates of typical vesicular transport reactions. This review addresses the nature of such anassay system and a fluorescence-intensity-based analysis of changes in tube dimensions that is indicative of the membraneremodelling capacity of proteins.

    • Fluorescence microscopy applied to intracellular transport by microtubule motors


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      Long-distance transport of many organelles inside eukaryotic cells is driven by the dynein and kinesin motors onmicrotubule filaments. More than 30 years since the discovery of these motors, unanswered questions include motor–organelle selectivity, structural determinants of processivity, collective behaviour of motors and track selection within thecomplex cytoskeletal architecture, to name a few. Fluorescence microscopy has been invaluable in addressing some of thesequestions. Here we present a review of some efforts to understand these sub-microscopic machines using fluorescence.

    • Single-molecule photobleaching: Instrumentation and applications


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      Single-molecule photobleaching (smPB) technique is a powerful tool for characterizing molecular assemblies. It canprovide a direct measure of the number of monomers constituting a given oligomeric particle and generate the oligomer sizedistribution in a specimen. A major current application of this technique is in understanding protein aggregation, which islinked to many incurable diseases. Quantitative measurement of the size distribution of an aggregating protein in aphysiological solution remains a difficult task, since techniques such as dynamic light scattering or fluorescence correlationspectroscopy (FCS) can provide an average size, but cannot accurately resolve the underlying size distribution. Here wedescribe the smPB method as implemented on a home-built total internal reflection fluorescence microscope (TIRF). Wefirst describe the construction of a TIRF microscope, and then demonstrate the power of smPB by characterizing a solutionof Amylin (hIAPP) oligomers, a 37-residue peptide whose aggregation is associated with Type II diabetes. We compare ourresults with FCS data obtained from the same specimen, and discuss the advantages and disadvantages of the twotechniques.

    • Studying backbone torsional dynamics of intrinsically disordered proteins using fluorescence depolarization kinetics


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      Intrinsically disordered proteins (IDPs) do not autonomously adopt a stable unique 3D structure and exist as an ensemble ofrapidly interconverting structures. They are characterized by significant conformational plasticity and are associated withseveral biological functions and dysfunctions. The rapid conformational fluctuation is governed by the backbone segmentaldynamics arising due to the dihedral angle fluctuation on the Ramachandran φ–ψ conformational space. We discovered thatthe intrinsic backbone torsional mobility can be monitored by a sensitive fluorescence readout, namely fluorescencedepolarization kinetics, of tryptophan in an archetypal IDP such as α-synuclein. This methodology allows us to map thesite-specific torsional mobility in the dihedral space within picosecond-nanosecond time range at a low protein concentrationunder the native condition. The characteristic timescale of * 1.4 ns, independent of residue position, representscollective torsional dynamics of dihedral angles (φ and ψ) of several residues from tryptophan and is independent of overallglobal tumbling of the protein. We believe that fluorescence depolarization kinetics methodology will find broad applicationto study both short-range and long-range correlated motions, internal friction, binding-induced folding, disorder-to-ordertransition, misfolding and aggregation of IDPs.

    • Fluorescence-based approaches for monitoring membrane receptor oligomerization


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      Membrane protein structures are highly under-represented relative to water-soluble protein structures in the protein databank.This is especially the case because membrane proteins represent more than 30% of proteins encoded in the humangenome yet contribute to less than 10% of currently known structures (Torres et al. in Trends Biol Sci 28:137–144, 2003).Obtaining high-resolution structures of membrane proteins by traditional methods such as NMR and x-ray crystallographyis challenging, because membrane proteins are difficult to solubilise, purify and crystallize. Consequently, development ofmethods to examine protein structure in situ is highly desirable. Fluorescence is highly sensitive to protein structure anddynamics (Lakowicz in Principles of fluorescence spectroscopy, Springer, New York, 2007). This is mainly because of thetime a fluorescence probe molecule spends in the excited state. Judicious choice and placement of fluorescentmolecule(s) within a protein(s) enables the experimentalist to obtain information at a specific site(s) in the protein (complex)of interest. Moreover, the inherent multi-dimensional nature of fluorescence signals across wavelength, orientation, spaceand time enables the design of experiments that give direct information on protein structure and dynamics in a biologicalsetting. The purpose of this review is to introduce the reader to approaches to determine oligomeric state or quaternarystructure at the cell membrane surface with the ultimate goal of linking the oligomeric state to the biological function. In thefirst section, we present a brief overview of available methods for determining oligomeric state and compare theiradvantages and disadvantages. In the second section, we highlight some of the methods developed in our laboratory toaddress contemporary questions in membrane protein oligomerization. In the third section, we outline our approach todetermine the link between protein oligomerization and biological activity.

    • Applications of STED fluorescence nanoscopy in unravelling nanoscale structure and dynamics of biological systems


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      Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking theoptical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has usheredin the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biologicalsystems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique whichprovides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-establishedfluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure ofcellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED anddifferent modalities of operation in order to achieve the best resolution. Various applications of this technique in cellimaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging inunravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recentstudies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed bycombination of STED and FCS techniques.

    • Ultrafast dynamics-driven biomolecular recognition where fast activities dictate slow events


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      In general, biological macromolecules require significant dynamical freedom to carry out their different functions, includingsignal transduction, metabolism, catalysis and gene regulation. Effectors (ligands, DNA and external milieu, etc) areconsidered to function in a purely dynamical manner by selectively stabilizing a specific dynamical state, thereby regulatingbiological function. In particular, proteins in presence of these effectors can exist in several dynamical states with distinctbinding or enzymatic activity. Here, we have reviewed the efficacy of ultrafast fluorescence spectroscopy to monitor thedynamical flexibility of various proteins in presence of different effectors leading to their biological activity. Recent studiesdemonstrate the potency of a combined approach involving picosecond-resolved Fo¨ rster resonance energy transfer,polarisation-gated fluorescence and time-dependent stokes shift for the exploration of ultrafast dynamics in biomolecularrecognition of various protein molecules. The allosteric protein–protein recognition following differential protein–DNAinteraction is shown to be a consequence of some ultrafast segmental motions at the C-terminal of Gal repressor proteindimer with DNA operator sequences OE and OI. Differential ultrafast dynamics at the C-terminal of k-repressor protein withtwo different operator DNA sequences for the protein–protein interaction with different strengths is also reviewed. We havealso systemically briefed the study on the role of ultrafast dynamics of water molecules on the functionality of enzymeproteins a-chymotrypsin and deoxyribonuclease I. The studies on the essential ultrafast dynamics at the active site of theenzyme a-chymotrypsin by using an anthraniloyl fluorescent extrinsic probe covalently attached to the serine-195 residuefor the enzymatic activity at homeothermic condition has also been reviewed. Finally, we have highlighted the evidence thata photoinduced dynamical event dictates the molecular recognition of a photochromic ligand, dihydroindolizine with theserine protease a-chymotrypsin and with a liposome (L-a-phosphatidylcholine).

    • Dynamics of water and ions around DNA: What is so special about them?


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      Water around biomolecules is special for behaving strangely – both in terms of structure and dynamics, while ions are foundto control various interactions in biomolecules such as DNA, proteins and lipids. The questions that how water and ionsaround these biomolecules behave in terms of their structure and dynamics, and how they affect the biomolecular functionshave triggered tremendous research activities worldwide. Such activities not only unfolded important static and dynamicproperties of water and ions around these biomolecules, but also provoked heated debate regarding their explanation androle in biological functions. DNA, being negatively charged, interacts strongly with the surrounding dipolar water andpositively charged counterions, leading to complex electrostatic coupling of water and ions with the DNA. Recent timeresolvedfluorescence Stokes shift experiments and related computer simulation studies from our and other laboratorieshave unfolded some unique dynamic characteristics of water and ions near different structures of DNA. These results arediscussed here to showcase the specialty of water and ion dynamics around DNA.

    • Single-molecule fluorescence imaging: Generating insights into molecular interactions in virology


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      Single-molecule fluorescence methods remain a challenging yet information-rich set of techniques that allow one to probethe dynamics, stoichiometry and conformation of biomolecules one molecule at a time. Viruses are small (nanometers) insize, can achieve cellular infections with a small number of virions and their lifecycle is inherently heterogeneous with alarge number of structural and functional intermediates. Single-molecule measurements that reveal the complete distributionof properties rather than the average can hence reveal new insights into virus infections and biology that are inaccessibleotherwise. This article highlights some of the methods and recent applications of single-molecule fluorescence in the field ofvirology. Here, we have focused on new findings in virus–cell interaction, virus cell entry and transport, viral membranefusion, genome release, replication, translation, assembly, genome packaging, egress and interaction with host immuneproteins that underline the advantage of single-molecule approach to the question at hand. Finally, we discuss the challenges,outlook and potential areas for improvement and future use of single-molecule fluorescence that could further aidour understanding of viruses.

    • Fluorescence techniques in developmental biology


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      Advanced fluorescence techniques, commonly known as the F-techniques, measure the kinetics and the interactions ofbiomolecules with high sensitivity and spatiotemporal resolution. Applications of the F-techniques, which were initiallylimited to cells, were further extended to study in vivo protein organization and dynamics in whole organisms. The integrationof F-techniques with multi-photon microscopy and light-sheet microscopy widened their applications in the field of developmentalbiology. It became possible to penetrate the thick tissues of living organisms and obtain good signal-to-noise ratiowith reduced photo-induced toxicity. In this review, we discuss the principle and the applications of the three most commonlyused F-techniques in developmental biology: Fluorescence Recovery After Photo-bleaching (FRAP), Fo¨ rster ResonanceEnergy Transfer (FRET), and Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS).

    • Fluorescence spectroscopy for revealing mechanisms in biology: Strengths and pitfalls


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      This article describes the basic principles of steady-state and time-resolved fluorescence. The formal equivalence of the twomethodologies is described first, followed by the extra advantages of time-resolved methods in revealing populationheterogeneity in complex systems encountered in biology. Several examples from the author’s work are described insupport of the above contention. Finally, several misinterpretations and pitfalls in the interpretation of fluorescence data andtheir remedy are described.

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