Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells

In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5’ leader and 3’ trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNA^Ile was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNA^Ile within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNA^Ile also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNA^Ile with long 5’ leader and long 3’ trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNA^Ile could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus.     

膜锚定Gaussia萤光素酶的细胞标记及生物荧光成像

目的:制备表达膜锚定Gaussia萤光素酶(extGluc)报告基因的慢病毒,用于标记细胞。方法:将报告基因extGluc克隆至慢病毒载体pCCsin.PPT.SFFV.IRES.eGFP.Wpre(VeGFP)中,以聚乙烯亚胺(PEI)介导,将慢病毒包装所需4种质粒(pVeGFP-extGLuc、pMDL、pRev、pVSVG),转染293FT细胞,72 h后收集病毒上清进行浓缩,感染293FT细胞,并用流式细胞仪检测病毒滴度,生物荧光成像和化学发光分析extGluc的表达;之后,用收集的慢病毒感染人单核细胞白血病细胞株U937。结果:对经PCR筛选出的阳性克隆所含质粒进行酶切鉴定,表明extGlu报告基因插入载体中;重组慢病毒包装成功且病毒滴度为5×106 TU/mL;用包装的病毒颗粒感染293FT细胞,生物荧光成像和化学发光证实extGluc的膜定位,且酶活性与细胞数目呈线性相关;病毒颗粒能够感染悬浮细胞U937。结论:包装了extGluc标记的重组慢病毒,可用于标记细胞,为体内监测细胞迁移、聚集和变化提供了一种方法。     

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.     

Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50-100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2-3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen-antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.     

Investigation of the Internalization of Fluorescently Labeled Lipophilic siRNA into Cultured Tumor Cells

Russian Journal of Bioorganic Chemistry - The attachment of lipophilic molecules of natural origin, which have natural means for cell internalization, to small interfering RNA (siRNA) is an...     

Imaging Cellular Inorganic Phosphate in Caenorhabditis elegans Using a Genetically Encoded FRET-Based Biosensor

Inorganic phosphate (Pi) has central roles in metabolism, cell signaling and energy conversion. The distribution of Pi to each cell and cellular compartment of an animal must be tightly coordinated with its dietary supply and with the varied metabolic demands of individual cells. An analytical method for monitoring Pi dynamics with spatial and temporal resolution is therefore needed to gain a comprehensive understanding of mechanisms governing the transport and recycling of this essential nutrient. Here we demonstrate the utility of a genetically encoded FRET-based Pi sensor to assess cellular Pi levels in the nematode Caenorhabditis elegans. The sensor was expressed in different cells and tissues of the animal, including head neurons, tail neurons, pharyngeal muscle, and the intestine. Cytosolic Pi concentrations were monitored using ratiometric imaging. Injection of phosphate buffer into intestinal cells confirmed that the sensor was responsive to changes in Pi concentration in vivo. Live Pi imaging revealed cell-specific and developmental stage-specific differences in cytosolic Pi concentrations. In addition, cellular Pi levels were perturbed by food deprivation and by exposure to the respiratory inhibitor cyanide. These results suggest that Pi concentration is a sensitive indicator of metabolic status. Moreover, we propose that live Pi imaging in C. elegans is a powerful approach to discern mechanisms that govern Pi distribution in individual cells and throughout an animal.     

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (Aβ) is the major constituent of senile plaques found in the brains of Alzheimer’s disease patients. Aβ is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by β and γ-secretases. Despite the importance of Aβ to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the γ-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.     

A Transformation-induced Alteration of Cellular Membranes in Crown Gall Tumor Cells

Phospholipids were utilized as a membrane marker to test for transformation-induced alteration of cellular membranes of cultured crown gall cells of Vinca rosea L. Fully transformed cells contained less than half the amount of phospholipids (7.8 micrograms lipid P per gram fresh weight) of normal V. rosea cells (21.4 micrograms lipid P per gram fresh weight). The normal V. rosea callus cells were not significantly different (P > 0.05) in phospholipid content from partially transformed crown gall cells (20.7 micrograms lipid P per gram fresh weight). Stimulation to rapid growth of the partially transformed cells by adding higher concentrations of inorganic salts and auxin did not significantly alter their phospholipid content (23.1 micrograms lipid P per gram fresh weight). These findings suggest that the transformation process is directly responsible for an alteration of the cellular membranes and that the membrane alteration cannot be attributed to secondary effects associated with the rapid growth of these neoplastic cells.     

The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus

To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs. The cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured. The cdc2Ms kinase is activated at the G1/S transition when phosphate-starved cells from the G0 phase re-enter the cell cycle and remain active as cells transit the S, G2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa. In contrast, when cdc2Ms kinase was purified by binding to p13suc1, it was active only in the G2 and M phases. In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus. By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible. Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added. Furthermore, purified CDK inhibitors, such as the mouse p27kip1 and the yeast p40sic1, blocked the purified plant CDK activity.     

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization.     

Exploring Dynamics of Molybdate in Living Animal Cells by a Genetically Encoded FRET Nanosensor

Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.     

Plasmid Stability in Dried Cells of the Desert Cyanobacterium Chroococcidiopsis and its Potential for GFP Imaging of Survivors on Earth and in Space

Two GFP-based plasmids, namely pTTQ18-GFP-pDU1(mini) and pDUCA7-GFP, of about 7?kbp and 15?kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18?months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1(mini)-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2?months of dry storage, the presence of dried cells with a GFP-RecA(Syn) distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecA(Syn) structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.     

Imaging and tracking of single GFP molecules in solution

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.     

Breast Tumor Targeting in Mice Bearing 4T1 Tumor with Labeled CXCR4 Antagonist Analogue

International Journal of Peptide Research and Therapeutics - Chemokine receptor belongs to G-protein-coupled cell-surface receptors. CXCR4 as a chemokine receptor is over-expressed in many type of...     

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (<10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t_1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.     

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.     

Coherent Neutron Scattering and Collective Dynamics in the Protein,GFP

Collective dynamics are considered to be one of the major properties of soft materials, including biological macromolecules. We present coherent neutron scattering studies of the low-frequency vibrations, the so-called boson peak, in fully deuterated green fluorescent protein (GFP). Our analysis revealed unexpectedly low coherence of the atomic motions in GFP. This result implies a low amount of in-phase collective motion of the secondary structural units contributing to the boson peak vibrations and fast conformational fluctuations on the picosecond timescale. These observations are in contrast to earlier studies of polymers and glass-forming systems, and suggest that random or out-of-phase motions of the β-strands contribute greater than two-thirds of the intensity to the low-frequency vibrational spectra of GFP.