Septins regulate border cell surface geometry,shape, and motility downstream of Rho in Drosophila

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In vivo effects of pyrophosphate in Escherichia coli

Abstract Pyrophosphate (PP_i), a noncompetitive inhibitor of Rho poly(C)-dependent ATPase activity in vitro has been shown to relieve polarity in the lac operon. This indicates that PP_i could inhibit Rho activity in vivo too. An additional effect of PP_i on adenosine 3',5'-cyclic monophosphate (cAMP) synthesis during stationary phase of growth is also described.     

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

Summary Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)₁₀ to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. ¹H, ¹⁵N and ¹³C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.Abbreviations Rho116, Rho130 protein containing the first 116 (130) residues of rho - CSD cold shock domain - RRM RNA recognition motif - RBD RNA-binding domain - IPTG isopropyl -D-thiogalactopyranoside - EDTA ethylenediaminetetraacetic acid - NOE nuclear Overhauser enhancement     

A ROCK inhibitor promotes keratinocyte survival and paracrine secretion,enhancing establishment of primary human melanocytes and melanocyte–keratinocyte co‐cultures

Primary melanocytes isolated from skin and expanded in culture have been widely used for laboratory research and clinical applications. The conventional method to isolate primary melanocytes from skin usually requires about 3–4 weeks of culture for melanocytes to grow sufficiently to passage. Considering that melanocytes comprise only 3%–7% of epidermal cells in normal human skin, it would be extremely helpful to increase the isolation efficiency and shorten the initial culture time to quickly meet various application needs. Here, we report that adding Y‐27632, a Rho kinase inhibitor, into the initial culture medium for 2 days can dramatically increase the yield of melanocytes. We found that Y‐27632 can promote keratinocyte attachment and survival in the melanocyte culture system, resulting in not only better recovery, but also increased proliferation of melanocytes by a paracrine signaling pathway. More specifically, Y‐27632 significantly induced keratinocyte expression of stem cell factor, which played an important role in enhancing the growth of melanocytes. In summary, Y‐27632 could profoundly enhance the yield of primary melanocytes in the initial culture through paracrine effects on keratinocytes.     

Identification of 5H-chromeno[3,4-c]pyridine and 6H-isochromeno[3,4-c]pyridine derivatives as potent and selective dual ROCK inhibitors

A novel series of 5H-chromeno[3,4-c]pyridine, 6H-isochromeno[3,4-c]pyridine and 6H-isochromeno[4,3-d]pyrimidine derivatives as dual ROCK1 and ROCK2 inhibitors is described. Optimization led to compounds with sub-nanomolar inhibitory affinity for both kinases and excellent kinome selectivity. Compound 19 exhibited ROCK1 and ROCK2 IC₅₀ of 0.67 nM and 0.18 nM respectively.     

Requirement for Rho in Integrin Signalling

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.     

TOR under stress: Targeting TORC1 by Rho1 GTPase

In the yeast Saccharomyces cerevisiae, small GTPase Rho1 controls polarized actin distribution and cell wall expansion in response to many different environmental and intracellular stimuli. Its activity is essential for cell survival and adaptation under various stress conditions. A recent study identified the TOR complex 1 (TORC1), a central regulator in cell growth and metabolism, as a direct target of the small GTPase. This novel crosstalk extends the signaling network of Rho1 into many TORC1-dependent processes and sheds light on how yeast cells coordinate polarized spatial expansion with mass increase.     

Coordinated control of lumen size and collective migration in the salivary gland

Drosophila Smaug is a sequence-specific RNA-binding protein that can repress the translation and induce the degradation of target mRNAs in the early Drosophila embryo. Our recent work has uncovered a new mechanism of Smaug-mediated translational repression whereby it interacts with and recruits the Argonaute 1 (Ago1) protein to an mRNA. Argonaute proteins are typically recruited to mRNAs through an associated small RNA, such as a microRNA (miRNA). Surprisingly, we found that Smaug is able to recruit Ago1 to an mRNA in a miRNA-independent manner. This work suggests that other RNA-binding proteins are likely to employ a similar mechanism of miRNA-independent Ago recruitment to control mRNA expression. Our work also adds yet another mechanism to the list that Smaug can use to regulate its targets and here we discuss some of the issues that are raised by Smaug’s multi-functional nature.