TED: Enhancing Translation Efficiency in Bacterial Expression Systems.

Genetic engineering enables the forced expression of desired products in bacteria, which can then be used for a variety of applications, including functional analysis and pharmaceuticals. Here, we describe a method for tuning translation in bacteria, including Escherichia coli and Rhodobacter capsulatus, based on a phenomenon known as TED (translation enhancement by a Dictyostelium gene sequence). This method promotes translation of mRNA encoded by downstream genes by inserting a short nucleotide sequence into the 5' untranslated region between the promoter and the Shine-Dalgarno (SD) sequence.

Enucleation of the embryo revealed dynein-dependent spacing between microtubule asters.

The intracellular positioning of the centrosome, a major microtubule-organizing center, is important for cellular functions. One of the features of centrosome positioning is the spacing between centrosomes; however, the underlying mechanisms are not fully understood. To characterize the spacing activity in embryos, a genetic setup was developed to produce enucleated embryos.

Dynamics of intracellular cGMP during chemotaxis in Dictyostelium cells.

Cyclic guanosine 3',5'-monophosphate (cGMP) is a ubiquitous important second messenger involved in various physiological functions. Here, intracellular cGMP (cGMPi) was visualized in chemotactic Dictyostelium cells using the fluorescent probe, D-Green cGull. When wild-type cells were stimulated with a chemoattractant, fluorescence transiently increased, but guanylate cyclase-null cells did not show a change in fluorescence, suggesting that D-Green cGull is a reliable indicator of cGMPi.

An improved molecular tool for screening bacterial colonies using GFP expression enhanced by a sequence.

During molecular cloning, screening bacterial transformants is a time-consuming and labor-intensive process; however, tractable tools that can be applied to various vectors for visual confirmation of desired colonies are limited. Recently, we reported that translational enhancement by a gene sequence (TED) boosted protein expression even without an expression inducer in . Here, we demonstrate a generally applicable molecular tool using the expression of green fluorescent protein enhanced by TED.

Cytokinesis D is Mediated by Cortical Flow of Dividing Cells Instead of Chemotaxis.

Cytokinesis D is known as the midwife mechanism in which neighboring cells facilitate cell division by crossing the cleavage furrow of dividing cells. Cytokinesis D is thought to be mediated by chemotaxis, where midwife cells migrate toward dividing cells by sensing an unknown chemoattractant secreted from the cleavage furrow. In this study, to validate this chemotaxis model, we aspirated the fluid from the vicinity of the cleavage furrow of a dividing cell and discharged it onto a neighboring cell using a microcapillary.

Choice between 1- and 2-furrow cytokinesis in embryos with tripolar spindles.

Excessive centrosomes often lead to multipolar spindles, and thus probably to multipolar mitosis and aneuploidy. In , ∼70% of the paternal mutant embryonic cells contained more than two centrosomes and formed multipolar spindles. However, only ~30% of the cells with tripolar spindles formed two cytokinetic furrows.

Aurora B but not rho/MLCK signaling is required for localization of diphosphorylated myosin II regulatory light chain to the midzone in cytokinesis.

Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II.

Phosphorylation of myosin II regulatory light chain controls its accumulation, not that of actin, at the contractile ring in HeLa cells.

During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing.

Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis.

Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis.

Enhancement of myosin II/actin turnover at the contractile ring induces slower furrowing in dividing HeLa cells.

Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression.