Project description:Recent developments in Raman spectroscopy instrumentation and data processing algorithms have led to the emergence of Ramanomics - an independent discipline with unprecedented capabilities to map the distribution of distinct molecular groups in live cells. Here, we introduce a method for probing the absolute concentrations of proteins, RNA and lipids in single organelles of live cultured cells by biomolecular component analysis using microRaman data. We found significant cell-to-cell variations in the molecular profiles of organelles, thus providing a physiologically relevant set of markers of cellular heterogeneity. At the same cell the molecular profiles of different organelles can strongly correlate, reflecting tight coordination of their functions. This correlation was significant in WI-38 diploid fibroblasts and weak in HeLa cells, indicating profound differences in the regulation of biochemical processes in these cell lines.
Project description:We have developed an optical trapping method to precisely measure the force generated by motor proteins on single organelles of unknown size in cell extract. This approach, termed VMatch, permits the functional interrogation of native motor complexes. We apply VMatch to measure the force, number and activity of kinesin-1 on motile lipid droplets isolated from the liver of normally fed and food-deprived rats.
Project description:The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.
Project description:We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min(-1) mg(-1)) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min(-1) mg(-1) and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min(-1) mg(-1) resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min(-1) mg(-1) (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues.
Project description:Living cells reside within anisotropic microenvironments that orchestrate a broad range of polarized responses through physical and chemical cues. To unravel how localized chemical signals influence complex behaviors, tools must be developed for establishing patterns of chemical gradients that vary over subcellular dimensions. Here, we present a strategy for addressing this critical need in which an arbitrary number of chemically distinct, subcellular dosing streams are created in real time within a microfluidic environment. In this approach, cells are cultured on a thin polymer membrane that serves as a barrier between the cell-culture environment and a reagent chamber containing multiple reagent species flowing in parallel under low Reynolds number conditions. Focal ablation of the membrane creates pores that allow solution to flow from desired regions within this reagent pattern into the cell-culture chamber, resulting in narrow, chemically distinct dosing streams. Unlike previous dosing strategies, this system provides the capacity to tailor arbitrary patterns of reagents on the fly to suit the geometry and orientation of specific cells.
Project description:Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices.
Project description:Raman microspectroscopy is a rapidly developing technique, which has an unparalleled potential for in situ proteomics, lipidomics, and metabolomics, due to its remarkable capability to analyze the molecular composition of live cells and single cellular organelles. However, the scope of Raman spectroscopy for bio-applications is limited by a lack of software tools for express-analysis of biomolecular composition based on Raman spectra. In this study, we have developed the first software toolbox for immediate analysis of intracellular Raman spectra using a powerful biomolecular component analysis (BCA) algorithm. Our software could be easily integrated with commercial Raman spectroscopy instrumentation, and serve for precise analysis of molecular content in major cellular organelles, including nucleoli, endoplasmic reticulum, Golgi apparatus, and mitochondria of either live or fixed cells. The proposed software may be applied in broad directions of cell science, and serve for further advancement and standardization of Raman spectroscopy.
Project description:Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.
Project description:Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.
Project description:We present here a novel chemical method to continuously analyze intracellular AKT signaling activities at single-cell resolution, without genetic manipulations. A pair of cyclic peptide-based fluorescent probes were developed to recognize the phosphorylated Ser474 site and a distal epitope on AKT. A Förster resonance energy transfer signal is generated upon concurrent binding of the two probes onto the same AKT protein, which is contingent upon the Ser474 phosphorylation. Intracellular delivery of the probes enabled dynamic measurements of the AKT signaling activities. We further implemented this detection strategy on a microwell single-cell platform, and interrogated the AKT signaling dynamics in a human glioblastoma cell line. We resolved unique features of the single-cell signaling dynamics following different perturbations. Our study provided the first example of monitoring the temporal evolution of cellular signaling heterogeneities and unveiled biological information that was inaccessible to other methods.