Conformation, thermodynamics and stoichiometry of HSA adsorbed to colloidal CdSe/ZnS quantum dots

Water-soluble luminescent colloidal quantum dots (QDs) have attracted great attention in biological and medical applications. In particular, for any potential in vivo application, the interaction of QDs with human serum albumin (HSA) is crucial. As a step toward the elucidation of the fate of QDs introduced to organism, the interactions between QDs and HSA were systematically investigated by various spectroscopic techniques under the physiological conditions. It was proved that binding of QDs and HSA is a result of the formation of QDs-HSA complex and electrostatic interactions play a major role in stabilizing the complex. The modified Stern-Volmer quenching constant K(a) at different temperatures and corresponding thermodynamic parameters DeltaH, DeltaG and DeltaS were calculated. Furthermore, the site marker competitive experiments revealed that the binding location of QDs with HSA is around site I, centered at Lys199. The conformational changes of HSA induced by QDs have been analyzed by means of CD and FT-IR. The results suggested that HSA underwent substantial conformational changes at both secondary and tertiary structure levels. The stoichiometry of HSA attached to QDs was obtained by dynamic light scattering (DLS) and zeta-potential.     

Real-time visualization of prion transport in single live cells using quantum dots

Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrP^C to the infectious scrapie isoform PrP^Sc. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, we have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addition, using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, we show that lipid rafts play an important role in locating cellular PrP^C to the cell membrane and in initiating PrP^C endocytosis.     

JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein σ1 and membrane penetration protein μ1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a σ1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L × T3D reassortant viruses revealed that the μ1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of σ1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the μ1 protein plays an essential role in stimulating proapoptotic signaling.     

Depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells

NPM (nucleophosmin; also known as B23) is an abundantly and ubiquitously expressed multifunctional nucleolar phosphoprotein, which is involved in numerous cellular processes, including ribosome biogenesis, protein chaperoning and centrosome duplication; however, the role of NPM in the cell cycle still remains unknown. In the present study, we show dynamic localization of NPM throughout the cell cycle of HeLa cells. Using a combination of RNAi (RNA interference) and three-dimensional microscopy we show that NPM is localized at the chromosome periphery during mitosis. We also demonstrate that depletion of NPM causes distortion of nucleolar structure as expected and leads to unexpected dramatic changes in nuclear morphology with multiple micronuclei formation. The defect in nuclear shape of NPM-depleted cells, which is clearly observed by live-cell imaging, is due to the distortion of cytoskeletal (alpha-tubulin and beta-actin) structure, resulting from the defects in centrosomal microtubule nucleation. These results indicate that NPM is an essential protein not only for the formation of normal nucleolar structure, but also for the maintenance of regular nuclear shape in HeLa cells.     

In situ real-time imaging of the satellite cells in rat intact and injured soleus muscles using quantum dots

The recruitment of satellite cells, which are located between the basement membrane and the plasma membrane in myofibers, is required for myofiber repair after muscle injury or disease. In particular, satellite cell migration has been focused on as a satellite cell response to muscle injury because satellite cell motility has been revealed in cell culture. On the other hand, in situ, it is poorly understood how satellite cell migration is involved in muscle regeneration after injury because in situ it has been technically very difficult to visualize living satellite cells localized within skeletal muscle. In the present study, using quantum dots conjugated to anti-M-cadherin antibody, we attempted the visualization of satellite cells in both intact and injured skeletal muscle of rat in situ. As a result, the present study is the first to demonstrate in situ real-time imaging of satellite cells localized within the skeletal muscle. Moreover, it was indicated that satellite cell migration toward an injured site was induced in injured muscle while spatiotemporal change in satellite cells did not occur in intact muscle. Thus, it was suggested that the satellite cell migration may play important roles in the regulation of muscle regeneration after injury. Moreover, the new method used in the present study will be a useful tool to develop satellite cell-based therapies for muscle injury or disease.     

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.     

Applications of mesenchymal stem cells labeled with Tat peptide conjugated quantum dots to cell tracking in mouse body

Fluorescent quantum dots have great potential in cellular labeling and tracking. Here, PEG encapsulated CdSe/ZnS quantum dots have been conjugated with Tat peptide, and introduced into living mesenchymal stem cells. The Tat peptide conjugated quantum dots in mesenchymal stem cells were assessed by fluorescent microscopy, laser confocal microscope and. flow cytometry. The result shows that Tat peptide conjugated quantum dots could enter mesenchymal stem cells efficiently. The Tat-quantum dots labeled stem cells were further injected into the tail veins of NOD/SCID beta2 M null mice, and the tissue distribution of these labeled cells in nude mice were examined with fluorescence microscope. The result shows that characteristic fluorescence of quantum dots was observed primarily in the liver, the lung and the spleen, with little or no quantum dots accumulation in the brain, the heart, or the kidney.     

Cross-linking cell surface chemokine receptors leads to isolation, activation, and differentiation of monocytes into potent dendritic cells

Monocytes express on the cell surface several kinds of chemokine receptors that facilitate chemotaxis followed by differentiation in target tissues. In the present study, we found that a large number of monocytes from peripheral blood mononuclear cells (PBMCs) tightly adhered to plastic cell culture plates precoated with a monoclonal antibody (mAb, clone T312) specific for human CCR5 but not an isotype control after overnight incubation. Soluble T312 did not induce such adhesion, indicating that cross-linking of CCR5 is required for the enhanced adhesion of monocytes. The adhesion was blocked by a PI3-K inhibitor and an anti-CD18 blocking mAb. Following the cross-linking of CCR5, monocytes synthesized high levels of M-CSF, RANTES, MIP-1 alpha, and MIP-1 beta associated with a readily detectable down modulation of CD14, CD4, CCR5, and CXCR4 expression. The T312-enriched monocytes differentiated into dendritic cells (DCs) in the presence of interleukin-4 alone. After maturation with beta-interferon, the T312-induced DCs stimulated proliferation of allogeneic na?ve CD4(+) T cells accompanied by the synthesis of high levels of gamma-interferon in vitro. Furthermore, the T312-induced DCs were capable of stimulating antigen-specific human T- and B-cell immune responses in our hu-PBL-SCID mouse system. Finally, screening of other anti-chemokine receptor mAbs showed that select clones of mAbs against CXCR4 and CCR3 were also capable of facilitating enrichment of monocytes similar to T312. These results show that cross-linking of chemokine receptors on monocytes by appropriate mAbs leads to activation and differentiation of monocytes and that the method described herein provides an alternate simple strategy for adherence-based isolation of monocytes and generation of functional DCs.     

Knockout of Arfrp1 leads to disruption of ARF-like1 (ARL1) targeting to the trans-Golgi in mouse embryos and HeLa cells

ADP-ribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family of GTPases which operate as molecular switches in the regulation of intracellular protein traffic. Deletion of the mouse Arfrp1 gene leads to embryonic lethality during early gastrulation, suggesting that ARFRP1 is required for cell adhesion-related processes. Here we show that ARFRP1 specifically controls targeting of ARL1 and its effector Golgin-245 to the trans-Golgi. GTP-bound ARFRP1 (ARFRP1-Q79L mutant) is associated with Golgi membranes and co-localized with the GTPase ARL1. In contrast, the guanine nucleotide exchange defective ARFRP1 mutant (ARFRP1-T31N) clusters within the cytosol. ARFRP1-T31N or depletion of endogenous ARFRP1 by RNA interference disrupts the Golgi association of ARL1 and of the GRIP-domain protein Golgin-245 and alters the distribution of a trans-Golgi network marker, syntaxin 6. In contrast, the targeting of two other Golgi-associated proteins, GM130 and giantin, was unaffected. Furthermore, in Arfrp1^?/???embryos ARL1 dislocated from Golgi membranes whereas it was associated with intracellular membranes in wild-type embryos. These data suggest that lethality of Arfrp1 knockout embryos is due to a specific disruption of protein targeting, e.g., of ARL1 and Golgin-245, to the Golgi.     

Knockout of Arfrp1 leads to disruption of ARF-like1 (ARL1) targeting to the trans-Golgi in mouse embryos and HeLa cells

ADP-ribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family of GTPases which operate as molecular switches in the regulation of intracellular protein traffic. Deletion of the mouse Arfrp1 gene leads to embryonic lethality during early gastrulation, suggesting that ARFRP1 is required for cell adhesion-related processes. Here we show that ARFRP1 specifically controls targeting of ARL1 and its effector Golgin-245 to the trans-Golgi. GTP-bound ARFRP1 (ARFRP1-Q79L mutant) is associated with Golgi membranes and co-localized with the GTPase ARL1. In contrast, the guanine nucleotide exchange defective ARFRP1 mutant (ARFRP1-T31N) clusters within the cytosol. ARFRP1-T31N or depletion of endogenous ARFRP1 by RNA interference disrupts the Golgi association of ARL1 and of the GRIP-domain protein Golgin-245 and alters the distribution of a trans-Golgi network marker, syntaxin 6. In contrast, the targeting of two other Golgi-associated proteins, GM130 and giantin, was unaffected. Furthermore, in Arfrp1-/ - embryos ARL1 dislocated from Golgi membranes whereas it was associated with intracellular membranes in wild-type embryos. These data suggest that lethality of Arfrp1 knockout embryos is due to a specific disruption of protein targeting, e.g., of ARL1 and Golgin-245, to the Golgi.     

Transfer of defined numbers of chloroplasts into albino protoplasts using an improved subprotoplast/protoplast microfusion procedure: Transfer of only two chloroplasts leads to variegated progeny

Summary A procedure is described by which it is possible to perform controlled microfusion of microscopically selected protoplast fusion partners with high efficiencies. The procedure is applied to fusion of Nicotiana tabacum (line 92V37, N. undulata cytoplasm) plastid albino protoplasts as a recipient and spontaneously formed subprotoplasts of green N. tabacum (line SRI) as donor. Products of individual electrofusion events are cloned via single cell nurse culture and the derived cell lines are analysed for the occurrence of variegated or green regenerating shoots, which are indicative of the establishment of the transferred organelles in the cell progeny. The plastid population in green regenerants recovered after the transfer of only two chloroplasts was demonstrated to have originated from the donor subprotoplast organelles by restriction analysis of total DNA using a plastome-specific probe.Some of the results described in this paper have been presented as posters at scientific meetings (Eigel and Koop 1989b; Eigel and Koop 1990)     

An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data

Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G₁ and G₂ cells, was calculated. RM was then used to calculate DNA synthesis time (T_S), at all post-labelling times (t). Since labelled cells in G₂ and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G₂ and M (T_g2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of T_S yields results that are affected by t. We found that RM data can be corrected with regard to T_G2+M resulting in the derivation of a non-linear T_S formula. This non-linear T_S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G₁, S and G₂+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. T_S obtained in this way was in agreement with T_S obtained with the corrected RM method. In conclusion, we present a method to calculate T_s which theoretically first makes the determination of RM independent of T_G2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G₂+ M.     

The combined influence of surface modification, size distribution, and interaction time on the cytotoxicity of CdTe quantum dots in PANC-1 cells

Mercaptopropionic acid (MPA) and cysteamine (Cys) capped CdTe quantum dots (QDs) were successfully prepared and used to investigate the combined influence of surface modification, size distribution, and interaction time on their cytotoxicity in human pancreatic carcinoma (PANC-1) cells. Results indicated that the smaller the size of MPA-CdTe QDs, the higher the cytotoxicity, which could be partly due to the difference of their distribution inside cells. Comparing with MPA-CdTe QDs, Cys-CdTe QDs had better cellular metabolizability and lower cytotoxicity. These QDs' cellular distribution and cytotoxicity were closely related to their interaction time with cells. Their cytotoxicity was found to be significantly enhanced with the increase of incubation time in medium. After QD treatments, the influence of recover time on the final cell viability was also dependent on the concentration and surface modification of QDs used in pretreatment. The combined influence of these factors discussed here might provide useful information for understanding and reducing the cytotoxicity of QDs in future biomedical applications.     

Colloidal iron binding to the surface of HeLa cells, spreading in monolayer culture.

We have used the colloidal iron (CI) binding technique, adapted for transmission electron microscopy, for semiquantitative evaluation of the negative charge density at the surface of HeLa cells in monolayer culture. The surface area increases when HeLa cells spread on the substrate. This increase brings about a decrease in the thickness of the CI rim, indicating a decrease in negative surface charge density. This phenomenon implicates lowering of the electrostatic repulsion, and explains the formation of intercellular contacts at the level of spread parts of the cell. Because of lack of penetration, CI particles are absent in regions of cose apposition between cells and between cells and substrates. Absence of CI binding in broader intercellular or cell-substrate spaces was explained through masking of the anionic groups.     

Codon optimization of the BirA enzyme gene leads to higher expression and an improved efficiency of biotinylation of target proteins in mammalian cells

Biotinylation of proteins is an attractive alternative to 'epitope-tagging', due to the strong biotin-(strept)avidin interaction and to the wide commercial availability of reagents for detection and purification of biotinylated macromolecules. Enzymatic biotinylation of target proteins in vivo using short biotin acceptor domains was described previously. Their use in mammalian cell requires expression of the bacterial biotinylation enzyme BirA. Here we describe the construction of a humanized version of BirA, with most of the rare codons replaced by codons that are more frequently used in human cells. The humanized BirA is expressed better in mammalian cells, resulting in improved efficiency of biotinylation in vivo. We anticipate that the humanized BirA gene will find use in many applications that involve in vivo biotinylation.