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. In the aggregation stage, the responses of cGMPi propagated in a wave-like fashion from the aggregation center. The oscillation of the cGMPi wave was synchronized almost in phase with those of other second messengers, such as the intracellular cAMP and Ca2+. The phases of these waves preceded those of the oscillations of actomyosin and cell velocity, suggesting that these second messengers are upstream of the actomyosin and chemotactic migration. An acute increase in cGMPi concentration released from membrane-permeable caged cGMP induced a transient shuttle of myosin II between the cytosol and cell cortex, suggesting a direct link between cGMP signaling and myosin II dynamics.

Strategies for enhancing gene expression in Escherichia coli.

Regulation of gene expression is fundamental for cellular function. Upon manipulation of the mechanism of gene expression in Escherichia coli, various bioproducts have been developed that are valuable industrially and medically in the last four decades. To efficiently produce bioproducts, numerous molecular tools are used for enhancing expression at the transcriptional and translational levels. Our recent discovery identified a new approach that enhances the gene expression in E. coli using the gene sequence of the eukaryote, Dictyostelium discoideum. In this review, we highlight the current molecular strategies used for high-level gene expression techniques commonly utilized in basic and applied microbiology.

Translation enhancement by a Dictyostelium gene sequence in Escherichia coli.

Methods for heterologous protein production in Escherichia coli have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed "translation enhancement by a Dictyostelium gene sequence" ("TED") in E. coli. We found that protein expression was increased when a gene sequence of Dictyostelium discoideum was placed upstream of the Shine-Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was mlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3' end of the mlcR gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.

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. Various expression levels can be observed depending on the inserted sequence and its length, even with an identical promoter.

Enucleation of the C. elegans 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 Caenorhabditis elegans embryos, a genetic setup was developed to produce enucleated embryos. The centrosome was duplicated multiple times in the enucleated embryo, which enabled us to characterize the chromosome-independent spacing activity between sister and non-sister centrosome pairs. We found that the timely spacing depended on cytoplasmic dynein, and we propose a stoichiometric model of cortical and cytoplasmic pulling forces for the spacing between centrosomes. We also observed dynein-independent but non-muscle myosin II-dependent movement of centrosomes in the later cell cycle phase. The spacing mechanisms revealed in this study are expected to function between centrosomes in general, regardless of the presence of a chromosome/nucleus between them, including centrosome separation and spindle elongation.

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. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells.

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. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II.

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. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.

An improved molecular tool for screening bacterial colonies using GFP expression enhanced by a Dictyostelium 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 Dictyostelium gene sequence (TED) boosted protein expression even without an expression inducer in Escherichia coli. Here, we demonstrate a generally applicable molecular tool using the expression of green fluorescent protein enhanced by TED. By inserting a module related to TED into the cloning site in advance, we effectively screened E. coli colonies harboring the desired plasmid functions in a prokaryote (Magnetospirillum gryphiswaldense) or eukaryote (Dictyostelium discoideum). Thus, our system represents a user-friendly technique for cloning.

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

Excessive centrosomes often lead to multipolar spindles, and thus probably to multipolar mitosis and aneuploidy. In Caenorhabditis elegans, ∼70% of the paternal emb-27APC6 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. The rest formed one furrow, similar to normal cells. To investigate the mechanism via which cells avoid forming two cytokinetic furrows even with a tripolar spindle, we conducted live-cell imaging in emb-27APC6 mutant cells. We observed that the chromatids were aligned on only two of the three sides of the tripolar spindle, and the angle of the tripolar spindle relative to the long axis of the cell correlated with the number of cytokinetic furrows. Our numerical modeling showed that the combination of cell shape, cortical pulling forces, and heterogeneity of centrosome size determines whether cells with a tripolar spindle form one or two cytokinetic furrows.

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 Dictyostelium cell and discharged it onto a neighboring cell using a microcapillary. However, the neighboring cells did not show any chemotaxis toward the fluid. In addition, the cells did not manifest an increase in the levels of intracellular Ca2+, cAMP, or cGMP, which are expected to rise in chemotaxing cells. From several lines of our experiments, including these findings, we concluded that chemotaxis does not contribute to cytokinesis D. As an alternative, we propose a cortical-flow model, where a migrating cell attaches to a dividing cell by chance and is guided toward the furrow by the cortical flow on the dividing cell, and then physically assists the separation of the daughter cells.

Characterization of myosin II regulatory light chain isoforms in HeLa cells.

Myosin II regulatory light chain (MRLC) is canonically known as a subunit of conventional myosin (myosin II), which tunes cytoplasmic contractility in cells. Recent studies have also revealed the noncanonical functions of MRLC, such as engagement with other proteins including unconventional myosins. Three MRLC isoforms (MRLC1, MRLC2, and MRLC3) are known in humans. The characteristics of MRLC2 are well known, but those of MRLC1 and MRLC3 are unclear; therefore, the properties of the three MRLC isoforms were investigated. The MRLCs were all phosphorylated at Thr18/Ser19, which is required for myosin II stimulation. MRLC mRNAs were expressed at the same level throughout the cell cycle in HeLa cells. The MRLCs colocalized with each other and their turnover rate was similar to that of myosin II heavy chain. Depletion of all the MRLCs perturbed cell spreading. The overproduction of MRLC2 or MRLC3, but not MRLC1, could effectively compensate for this defect, suggesting that MRLC2 and MRLC3 play dominant roles in cell spreading. Finally, computer simulations of the three-dimensional protein structures indicated that the location of the N-terminus of MRLC1 differs from that of MRLC2 or MRLC3, depending on its sequence. Thus, these MRLC isoforms have overlapping but distinct functions have been proposed.

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. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells.