Different CHD chromatin remodelers are required for expression of distinct gene sets and specific stages during development of Dictyostelium discoideum.

Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. Here, we identify three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, we show that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression.

RPLC of intact proteins using sub-0.5 µm particles and commercial instrumentation.

This paper addresses whether one can gain an improvement in speed or resolution with a silica colloidal crystal (SCC) of nonporous 470 nm particles when using a commercial nano-UHPLC. Compared to a capillary packed with nonporous 1.3 µm particles and the same C4 bonded phase, the peak width for BSA is decreased by a factor of 6.8 for the SCC. Some of this improvement is attributable to slip flow since the ratio of particle diameters is only 2.8. Resolution in protein separations was compared for a 2-cm capillary of SCC vs a 5-cm column of porous 1.7 µm particles. Both used a C4 bonded phase, and on-column fluorescence detection was used for the SCC. Split flow (5:1) before the SCC decreased the gradient delay time to 0.4 min and the injected volume to 0.4 nL. For variants from the labeling of BSA, the SCC had a 5-fold higher speed and 2-fold higher resolution than did the commercial column. For a monoclonal antibody and its aggregates, the SCC had a 3-fold higher speed and a 3-fold higher resolution compared to the commercial column. The SCC gave baseline resolution of the monomer, dimer and trimer in 5 min. The results show that a significant advantage can be gained using a commercial instrument with the SCC, despite the instrument not being designed for use with such small particles.

Insights from theory and experiments on slip flow in chromatography.

Slip flow has become a topic of interest in reversed-phase liquid chromatography because it gives a flow enhancement that facilitates the use of submicrometer particles, providing a large improvement in separation efficiency. Moreover, slip flow provides an additional improvement in efficiency by reducing the velocity distribution in the mobile phase. The phenomenon of slip flow in open tubes is described in chromatographically relative terms. A recent paper in this journal is discussed, as it provides the first theoretical study of slip flow in packed beds, in this case for face-centered cubic geometry. The theory paper reveals that the presence of the packed bed introduces a heterogeneity in fluid velocities that is absent in open tubes, reducing the additional improvement in efficiency from slip flow. The recent paper also suggests that there is yet another factor improving efficiency, which is size-exclusion of proteins from regions of stagnant flow. The latter is supported by recently published data on restricted protein diffusion in face-centered cubic silica colloidal crystals. Extremely low plate heights are enabled by use of submicrometer particles, and further improvement appears to be possible when the analyte size is on the order of 1% of the particle diameter or larger.

Obstructed diffusion in silica colloidal crystals.

The hindered diffusion in silica colloidal crystals was studied experimentally, both by fluorescence recovery after photobleaching and by measurement of ionic conductivity. Particle size was varied to include 120, 220, 470, and 1300 nm, and the porosities were determined by flow measurements. For fluorescein, the results showed that the obstruction factor, which is the ratio of the diffusion coefficients inside the media and in open solution, is equal to the porosity within experimental error. For proteins, the same conclusion is made after correction for size exclusion of the pores. The obstruction factors for these media are 2-fold lower than those measured for chromatographic media, 60% higher than theoretical predictions, and equal to what is assumed for electrophoretic sieving in random fibers.

Slip flow through colloidal crystals of varying particle diameter.

Slip flow of water through silica colloidal crystals was investigated experimentally for eight different particle diameters, which have hydraulic channel radii ranging from 15 to 800 nm. The particle surfaces were silylated to be low in energy, with a water contact angle of 83°, as determined for a silylated flat surface. Flow rates through centimeter lengths of colloidal crystal were measured using a commercial liquid chromatograph for accurate comparisons of water and toluene flow rates using pressure gradients as high as 10(10) Pa/m. Toluene exhibited no-slip Hagen-Poiseuille flow for all hydraulic channel radii. For water, the slip flow enhancement as a function of hydraulic channel radius was described well by the expected slip flow correction for Hagen-Poiseuille flow, and the data revealed a constant slip length of 63 ± 3 nm. A flow enhancement of 20 ± 2 was observed for the smallest hydraulic channel radius of 15 nm. The amount of slip flow was found to be independent of shear rate over a range of fluid velocities from 0.7 to 5.8 mm/s. The results support the applicability of the slip flow correction for channel radii as small as 15 nm. The work demonstrates that packed beds of submicrometer particles enable slip flow to be employed for high-volume flow rates.

Slip flow in colloidal crystals for ultraefficient chromatography.

Slip flow occurs in colloidal crystals made of 470 nm silica spheres that are chemically modified with hydrocarbon, giving enhanced volume flow rates and a narrower distribution of fluid velocities. Bovine serum albumin separates by pressure-driven flow with a zone that is 15-fold narrower than the theoretical limit for Hagen-Poiseuille flow. The zone variance, normalized for separation length, is 15 nm, which is 500-fold smaller than previous reports for pressure-driven protein chromatography. A colloidal crystal is shown to separate a monoclonal antibody from its aggregates in only 40 s, representing a 10-fold increase in speed. Slip flow, thus, has profound implications for protein chromatography.

Glycogen synthase kinase-3 is required for efficient Dictyostelium chemotaxis.

Glycogen synthase kinase-3 (GSK3) is a highly conserved protein kinase that is involved in several important cell signaling pathways and is associated with a range of medical conditions. Previous studies indicated a major role of the Dictyostelium homologue of GSK3 (gskA) in cell fate determination during morphogenesis of the fruiting body; however, transcriptomic and proteomic studies have suggested that GSK3 regulates gene expression much earlier during Dictyostelium development. To investigate a potential earlier role of GskA, we examined the effects of loss of gskA on cell aggregation. We find that cells lacking gskA exhibit poor chemotaxis toward cAMP and folate. Mutants fail to activate two important regulatory signaling pathways, mediated by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and target of rapamycin complex 2 (TORC2), which in combination are required for chemotaxis and cAMP signaling. These results indicate that GskA is required during early stages of Dictyostelium development, in which it is necessary for both chemotaxis and cell signaling.