Lower Mobile Fraction Research Articles

RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the RAG2 PHD region.

Abstract Association of RAG2 with the epigenetic marker H3K4me3 through a plant homeodomain (PHD) in the RAG2 non core domain is important for relieving auto inhibition of the RAG recombinase activity for V(D)J recombination. However, little is known regarding the properties of RAG2 that regulate its PHD-dependent interactions with H3K4me3. Accordingly, we measured the localization and dynamics of an N-terminal GFP fusion protein of full length RAG2 (GFP-FL); a T490A mutant of full length RAG2 (GFP-T490A) that does not undergo a regulatory phosphorylation proximal to the PHD region of RAG2; and GFP-labeled core domain of RAG2 (GFP-Core). The proteins were expressed in RAG2−/− pro-B cells, and the H3K4me3 was labeled using monoclonal antibody. Cross-correlation analysis showed that GFP-FL and GFP-Core exhibited similar colocalization with H3K4me3, which was significantly less than colocalization of GFP-T490A with H3K4me3. Blocking H3K4 demethylation using 2,4-pyridinedicarboxylic acid (PDA) caused GFP-FL/H3K4me3 colocalization to approach that of GFP-T490A and H3K4me3, whereas GFP-Core/H3K4me3 colocalization was unchanged. Super resolution imaging showed that RAG2 interactions with H3K4me3 were restricted to interfaces with H3K4me3-enriched puncta, and that GFP-T490A exhibited greater overlap with the H3K4me3-enriched puncta than GFP-FL. Finally, measurement of fluorescence recovery after photobleaching demonstrated that GFP-T490A had a lower mobile fraction and rate of diffusion than GFP-FL in conditions where RAG2 interactions with H3K4me3 were enhanced. These data suggest that RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the PHD region, such as by a phosphoryation/dephosphorylation cycle.

High-Field Electron Paramagnetic Resonance Reveals a Stable Glassy Fraction up to Melting in Semicrystalline Poly(dimethylsiloxane)

The reorientation of the guest 4-methoxy-TEMPO (spin probe) in the disordered fraction of semicrystalline poly(dimethylsiloxane) (PDMS) is investigated by high-field electron paramagnetic resonance (HF-EPR) at 190 and 285 GHz. Accurate numerical simulations of the HF-EPR lineshapes evidence that the reorientation times of the spin probes are distributed between the melting temperature $$T_{\rm m}$$ and $$T_{\rm m}$$ —30 K. The distribution exhibits, in addition to a broad component, a narrow component with low mobility up to the PDMS melting point. It is shown that the temperature dependence of the reorientation time of the spin probes with low mobility is the same of the spin probes in glassy PDMS. The result suggests that the low-mobility fraction is localized in the so-called rigid amorphous fraction.

Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain.

PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9.

Lateral diffusion of PDGF beta-receptors in human fibroblasts.

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum or PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding "normal" and "starved" cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0 x 10(-10) cm2s-1 and greater than 90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor, and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery

Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.

Mobility of cytoplasmic and membrane-associated actin in living cells.

We have combined fluorescent analogue cytochemistry with fluorescence photobleaching recovery to measure the mobility of fluorescently labeled actin and other labeled test proteins microinjected into living amoebae. Bovine serum albumin, ovalbumin, and ribonuclease A have a cytoplasmic mobility, expressed as a diffusion coefficient, that is 1/2 to 1/3 of that observed in aqueous solution; 90% of the actin has a mobility 1/2 to 1/8 of that of G-actin in aqueous solution, and approximately equal to 10% of the actin has a mobility comparable to that of F-actin in aqueous solution. Therefore, no more than 10% of the actin in the cytoplasm of amoebae can exist as static filaments. Microinjection of phalloidin decreases the diffusion coefficient of the mobile component of cytoplasmic actin, and it also increases the low-mobility fraction to 50% but has no effect on the mobility of labeled ovalbumin. By comparing the mobility of actin in different parts of amoebae and by separating cytoplasm from plasmalemma-ectoplasm, we found the low-mobility fraction of actin to be enriched in the tail, along the plasmalemma-ectoplasm, and in contracted cytoplasm.

Electrophoretic separation of human lymphocytes by means of immunomicrospheres

AbstractImmuno‐labeling of human peripheral blood lymphocytes with either polyglutaraldehyde or polyacrolein microspheres was shown by analytical electrophoresis to generate two electrokinetically distinct sub‐populations of lymphocytes. By means of free‐flow electrophoresis, the high mobility fraction was found to consist predominantly of T lymphocytes using an anti‐T cell antibody, while the low mobility fraction consisted of lymphocytes labeled with immunomicrospheres, identifying these cells as B lymphocytes. The immunomicrosphere‐induced reduction of B‐cell mobility below that of the T cells thus allowed the enrichment and recovery of viable T cells in preparative quantities by free‐flow electrophoresis.

Separation of human bone marrow cell populations by density gradient electrophoresis: Differential mobilities of myeloid (CFU-C), monocytoid, and lymphoid cells

Human bone marrow cells from normal donors (purified by centrifugation on Ficoll-Hypaque density cushion) were separated by density gradient electrophoresis, on the basis of their surface charge. The separated cell fractions were analyzed by morphology, surface markers, and functional tests. Morphological examination revealed that immature cells of myeloid and erythroid lineage were significantly enriched in the high-mobility fractions, whereas mature cells in general, e.g., small lymphocytes, monocytes and granulocytes (few) were highly enriched in the low-mobility fractions. Ripley rosette-forming cells, as well as complement receptor and Fc (IgG)-receptor-bearing lymphocytes were relatively enriched in the high-mobility fractions, although they were present in small proportions and in the rest of the fractions. E-rosette-forming cells were enriched in the intermediate- and low-mobility fractions. T cells with Fc receptors for IgM (Tμ cells), determined by a double-rosetting technique, were found to be enriched in the intermediate-mobility fractions. In contrast T cells with Fc receptors for IgG (Tγ cells) were significantly enriched in the low-mobility fractions. Cells in the intermediate-mobility fractions (Tμ enriched) responded by proliferation to mitogens and to allogeneic cells in mixed lymphocyte culture. Cells in the very high or very low (Tγ enriched) mobility fractions responded poorly. Colony-forming units in vitro (CFU-C), determined using either the human leukocyte feeder or the placental CSA culture systems, were found to be significantly enriched in the high-mobility fractions. Low-mobility fractions were devoid of CFU-C. These studies demonstrate that density gradient electrophoresis can be successfully applied for the separation of human bone marrow cells of different lineage and studies of their functional properties.

Separation of human lymphocyte subpopulations by density gradient electrophoresis: I. Different mobilities of T (Tμ, Tα) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.

Density Gradient Electrophoresis of Mouse Spleen Lymphocytes

Preparative density gradient electrophoresis has been employed for the separation of BALB/c mouse spleen T and B lymphocytes, on the basis of their surface charge. The high mobility cells were found to be predominantly T lymphocytes, whereas the low mobility cells were B lymphocytes. One way mixed lymphocyte cultures were prepared using electrophoretically separated BALB/c lymphocytes, either as responding or stimulating cells and unfractionated CBA/H/T6j mouse spleen lymphocytes, in the appropriate combination. The responding lymphocytes were found only among the high mobility cells which are primarily T lymphocytes. In contrast, the stimulating cells were found in the intermediate and low mobility fractions which contain the B lymphocytes and macrophages. Minimal stimulation in mixed lymphocyte culture was observed by T cells in this allogeneic system. Ia-positive cells were found only in the intermediate and low mobility fractions, which exhibited the capacity to stimulate allogeneic cells in MLC. Macrophages, identified by nonspecific esterase staining, exhibited intermediate electrophoretic mobility, their peak height coinciding with the maximum observed stimulation.

Application des méthodes préparatives en veine liquide à la séparation des cellules sanguines

Human blood cells can be separated using an apparatus described by Hanning and co-workers (Model FF4 Desaga Heidelberg). The method of free flow preparative electrophoresis uses a specific property of blood cells when subjected to an electric field: their electrophoretic mobility. Three regions of migration can be defined: --a high mobility region (HMR); --a low mobility region (LMR) and --an intermediate mobility region (IMR). After the electrophoretic migration, the various cell fractions are identified. If human leucocytes are subjected to an electric field (40 volts/cm) it is possible to isolate several populations which differ by their membranes electric charge. The repartition of the neutrophiles granulocytes is twofold: --one fraction of high mobility (HMR) and --one fraction of low mobility (LMR). The meaning of these two populations is being discussed. Between these teo fractions is located the lymphocyte fraction which is represented by a single peak of intermediate mobility (IMR). Eosinophiles and monocytes are concentrated in low mobility fraction (LMR). With appropriate migration parameters (80 volts/cm, various component buffers) it is possible to obtain from lymphocytes, purified by density gradient, an electrophoretic separation of T and B lymphocytes population. Histograms obtained from quantitative measures (on collected cell fractions) show an unimodal distribution. When separated cells are characterized by three membranes markers (E. Rosettes, EAC Rosettes and surface immunoglobulins) the electrophoretic heterogeneity of B and T cells is then demonstrated. With E Rosettes (T cells) the majority of T lymphocytes is distributed in HMR and IMR. With EAC Rosettes and membrane immunofluorescence (B cells markers) the majority of B lymphocytes is found in the LMR. Thus an important enrichment of T and B sub-population is obtained, but contamination from one another still remains too important. More selective methods should lead to an improvement of this cell separation.

Effects of hypophysectomy on the blood serum of male killifish, Fundulus heteroclitus, in salt water

Determinations were made of serum osmolality, major serum electrolytes, and selected organic constituents in microliter samples of the blood of parallel groups of hypophysectomized and mock-operated male killifish, Fundulus heteroclitus, maintained in salt water for 14–15 wk under standard conditions (ca. 20°C, 8 hr/day light, and daily feeding). Stress was minimized by “training” before autopsy. Although hypophysectomized killifish appeared to regulate the total serum osmolality, there was a marked decline in serum Na + (−7.9%), serum Cl − (−18.2%), and, among minor electrolytes, serum inorganic PO 4 2− (−45.3%). These decreases were offset, in part, by increases in serum Ca 2+ (9.7%), serum Mg 2+ (19.8%), and serum HCO 3 − (71.9%). Serum K + was variable, and the trend to increase was not significant. Hypophysectomy caused a significant decrease in serum glucose (−31.9%). Serum urea was more than doubled (129.2%), but the osmotic contribution remained small. Serum total protein levels rose significantly (46.4%), although only the low mobility fractions were affected. In spite of the decrease in serum inorganic PO 4 2− , serum organic phosphate increased greatly (104%), and there was a parallel, but even greater, increase in serum total cholesterol (155.2%). The estimated serum cholesterol:phospholipid ratio remained essentially the same as in the mock-operated controls (0.362 as compared with 0.352, respectively).

Cholinesterases in human spinal fluid and brain.

AbstractBy electrophoresis and chromatography on DEAE‐cellulose it was shown that human spinal fluid and brain tissue extracts contained three cholinesterases: Two butyrylcholinesterases and one acetylcholinesterase. One of the butyrylcholinesterases had the same electrophoretic mobility as serum cholinesterases. The other butyrylcholinesterase moved together with the γ‐globulin and the acetylcholinesterase together with the %‐globulin fraction of spinal fluid. These findings indicate that the acetylcholinesterase in spinal fluid derives from brain whereas it can not be decided whether the butyrylcholinesterases originate from plasma or from brain. The low mobility fraction of butyrylcholinesterase may arise from the action of sialidase.