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In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation (1), a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity (2,3), but it remains unclear if these markers are active regulators of transcription or just passive byproducts (4,5). This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells (2,5,6). Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response., To monitor the impact of histone acetylation on transcription, we used a mouse cell line harbouring an inducible tandem gene array and expressing a green fluorescent protein (GFP)-tagged version of [...]

Forms a preliminary theory from the shared reflections of Scottish teachers at a symposium on the teaching of investigative science. (Author/CCM)

The transition to multicellularity is a key feature of the Dictyostelium life cycle, and two genes, gbf and lagC, are known to play pivotal roles in regulating this developmental switch. lagC-null and gbf-null cells fail to induce cell-type-specific genes ordinarily expressed during multicellular development. The null routants also share a similar morphological phenotype: mutant cells repeatedly aggregate to form a loose mound, disperse, and reform a mound, rather than proceeding to form a tip. To characterize defects in morphogenesis in these mutants, we examined cell motion in the mutant mounds. In analogy with the failed transition in gene expression, we found that lagC-null and gbf-null mounds failed to make a morphogenetic transition from random to rotational motion normally observed in the parent strain. One reason for this was the inability of the mutant mounds to establish a single, dominant signaling-wave center. This defect of lagC-null or gbf-null cells could be overcome by the addition of adenosine, which alters cAMP signaling, but then even in the presence of apparently normal signaling waves, cell motility was still aberrant. This motility defect, as well as the signaling-wave defect, could be overcome in lagC-null cells by overexpression of GBF, suggesting that lagC is dispensable if GBF protein levels are high enough. This set of morphogenetic defects that we have observed helps define key steps in mound morphogenesis. These include the establishment of a dominant signaling-wave center and the capacity of cells to move directionally within the cell mass in response to guidance cues.

To examine the potential role of calcium in regulating Dictyostelium development, we reduced free cytosolic and total cell [Ca.sup.2+] in Dictyostelium cells by expressing a constitutively active form of a human erythrocyte plasma membrane calcium pump. The pump-expressing cells lacked a thapsigargin-mediated increase in cytoplasmic calcium, consistent with a reduced level of total cellular [Ca.sup.2+]. During aggregation, the cells initially formed a large number of aggregation centers, many of which coalesced to form mounds that were smaller than those of wild-type cells, and the cells did not exhibit the normal formation of elongated aggregation streams. The majority of the mounds either arrested at this stage with the formation of small protrusions or formed very aberrant finger-like structures, indicating an essential role for cellular calcium in morphogenesis. We used pump and wild-type cells differentially labeled by expressing different wavelength (green and blue) forms of green fluorescent protein and three-dimensional (3-D) reconstruction of serial fluorescent imaging to visualize the movement of pump and wild-type cells within the aggregate. The results showed that the pump cells exhibited very aberrant cell movement and sorting within the forming mound, suggesting that the reduced cytosolic calcium affects movement required for tip formation. When allowed to form chimeric organisms with wild-type cells, pump cells preferentially localized to two bands, one at the prestalk/prespore boundary and the other in the very posterior of the organism, suggesting that pump cells are unable to properly sort. Expression of the calcium pump had little effect on the induction of prestalk- or prespore-specific genes, whereas extended treatment with EGTA blocked induction of both classes of cell-type-specific genes. Our results suggest a role for intracellular [Ca.sup.2+] in controlling cell sorting and morphogenesis in Dictyostelium. Key Words: Dictyostelium; calcium; aequorin; GFP.

Examination of the three-dimensional trajectories of both normal axenic and myosin-II-null cells of Dictyostelium discoideum amebae by time-lapse computational optical-sectioning microscopy reveals that three-dimensional ameboid locomotion requires myosin-II. Locomotion of the amebae is regulated by various distinct, potentially competing cell-guide mechanisms.

Computational optical sectioning microscopy helps observe the transportation of cytoplasmic particles in Drosophila embryos, catalyzed by an unconventional myosin, the 95F myosin. This transport is actin-based and depends on ATP. The 95F myosin acts as a motor that transports cytoplasmic particles along action filaments and results in the redistribution of the particles in a cell cycle-based manner.

The kinetic modeling of H1 recovery curves obtained during fluorescence recovery after photobleaching (FRAP) experiments are utilized to determine the impact of core histone acetylation on the different variants of H1. The hyperacetylation of core histones are found to affect the cooperative nature of low-affinity H1 binding, with some variants undergoing a predicted decrease of almost 2 orders of magnitude.