Measurement of rapid protein diffusion in the cytoplasm by photoconverted intensity profile expansion

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2016.Cataloged from PDF version of thesis.Includes bibliographical references (pages 82-85).Whether at the level of a single protein, or in the cytoplasm as a whole, the diffusive mobility of proteins plays a key role in biological function. To measure protein diffusion in cells, researchers have developed multiple fluorescence microscopy methods, and have tested them rigorously. However, using these methods for precise measurement of diffusion coefficients requires expertise that can be a barrier to broad utilization of these methods. Here, we report on a new method we have developed, which we name Photo-converted Intensity Profile Expansion (PIPE). It is a simple and intuitive technique that works on commercial imaging systems and requires little expertise. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal as diffusion spreads it out. The width of the expanding signal is directly related to the protein ensemble mean-square displacement, from which the diffusion coefficient of the ensemble is calculated. In the main part of the thesis, we demonstrate the success of PIPE in measuring accurate diffusion coefficients in silico, in vitro and in vivo. We then broaden the discussion, and challenge the assumption that the Fickian diffusion equation is the most appropriate model for describing protein motion in the cytoplasm. Since the cytoplasm is crowded with obstacles that trap proteins for a wide range of times, the motion of those proteins may be more accurately described by models of anomalous diffusion. To contribute to the ongoing debate about anomalous diffusion, we show how PIPE can be used to measure the degree of diffusion anomality by examining the temporal scaling of the mean-square displacement. Whether for measuring normal or anomalous diffusion, we suggest that the simplicity and user-friendliness of PIPE could make it a useful tool in molecular and cell biology.by Rotem Gura Sadovsky.Ph. D

Imaging stress

Recent innovations in cell biology and imaging approaches are changing the way we study cellular stress, protein misfolding, and aggregation. Studies have begun to show that stress responses are even more variegated and dynamic than previously thought, encompassing nano-scale reorganization of cytosolic machinery that occurs almost instantaneously, much faster than transcriptional responses. Moreover, protein and mRNA quality control is often organized into highly dynamic macromolecular assemblies, or dynamic droplets, which could easily be mistaken for dysfunctional “aggregates,” but which are, in fact, regulated functional compartments. The nano-scale architecture of stress-response ranges from diffraction-limited structures like stress granules, P-bodies, and stress foci to slightly larger quality control inclusions like juxta nuclear quality control compartment (JUNQ) and insoluble protein deposit compartment (IPOD), as well as others. Examining the biochemical and physical properties of these dynamic structures necessitates live cell imaging at high spatial and temporal resolution, and techniques to make quantitative measurements with respect to movement, localization, and mobility. Hence, it is important to note some of the most recent observations, while casting an eye towards new imaging approaches that offer the possibility of collecting entirely new kinds of data from living cells.European Research Council (European Union's Seventh Framework Programme (FP/2007-2013)/ERC-StG2013 337713 DarkSide starting grant)Israel Science Foundation (Grant ISF 843/11)Israel. Ministry of Health (grant under the framework of E-Rare-2)Niedersachsen-Israel Research ProgramAbisch-Frenkel Foundatio

Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion) to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE’s success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE’s direct measurement and ease of use make it appealing for cell biologists

Modern drug discovery using ethnobotany: A large-scale cross-cultural analysis of traditional medicine reveals common therapeutic uses

Summary: For millennia, numerous cultures and civilizations have relied on traditional remedies derived from plants to treat a wide range of conditions and ailments. Here, we systematically analyzed ethnobotanical patterns across taxonomically related plants, demonstrating that congeneric medicinal plants are more likely to be used for treating similar indications. Next, we reconstructed the phytochemical space covered by medicinal plants to reveal that (i) taxonomically related medicinal plants cover a similar phytochemical space, and (ii) chemical similarity correlates with similar therapeutic usage. Lastly, we present several case scenarios illustrating how mining this information can be used for drug discovery applications, including: (i) investigating taxonomic hotspots around particular indications, (ii) exploring shared patterns of congeneric plants located in different geographic areas, but which have been used to treat the same indications, and (iii) showing the concordance between ethnobotanical patterns among non-taxonomically related plants and the presence of shared bioactive phytochemicals