During cell division in Escherichia coli, the highly conserved tubulin homolog FtsZ polymerizes and assembles into a ring-like structure, called the Z-ring, at the site of septation early in the division pathway. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins. In E. coli, membrane recruitment and tethering of FtsZ are predominantly carried out by FtsA. FtsA shares structural homology with actin and, like actin, hydrolyzes ATP. Yeast actin detects nucleotide occupancy through a sensor region adjacent to the nucleotide binding site and adopts distinct conformations in monomeric and filamentous actin. Accordingly, bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and adopt a polymerized conformation. Here, we show that a cluster of amino acid residues in the central region of FtsA and proximal to the nucleotide binding site are critical for FtsA function in vitro and in vivo. Each of these residues are important for ATP hydrolysis, phospholipid (PL) binding, ATP-dependent vesicle remodeling, and recruitment to the divisome in vivo, to varying degrees. Notably, we observed that Ser 84 and Glu 14 are essential for ATP-dependent vesicle remodeling and magnesium-dependent membrane release of FtsA from vesicles in vitro, and these defects likely underlie the loss of function by FtsA(E14R) and FtsA(S84L) in vivo. Finally, we demonstrate that FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is defective for rapid ATP hydrolysis and ATP-dependent remodeling of PL vesicles in vitro. Together, our results show that loss of nucleotide-dependent activities by FtsA, such as ATP hydrolysis, ATP-dependent PL vesicle remodeling, and membrane release, lead to failed Z-ring assembly and division defects in cells.
20The normal embryogenesis of marine animals is typically confined to a species-specific range of 21 temperatures. Within that temperature range development results in a consistent, or canalized, phenotype, 22whereas above and below the range abnormal phenotypes are produced. This study reveals an abrupt high 23 temperature limit, occurring over a 1-2°C range, for normal embryonic development in C. intestinalis. 24Above that threshold morphological abnormalities in the notochord and other organs are observed, 25 beginning with cleavage and gastrula stages, and becoming more pronounced as embryogenesis proceeds. 26However, even in highly morphologically abnormal temperature disrupted (TD) embryos, cell type 27 specification, including muscle, endoderm, notochord, and sensory pigment cells is accomplished. An 28 explanation for this finding is that in C. intestinalis cell type specification occurs relatively early in 29 embryogenesis, due to cleavage stage segregation of maternal cytoplasmic determinants and short-range 30 cell interactions, which are largely intact in TD embryos. On the other hand, morphogenesis of the 31 notochord and other structures is dependent on precise cell movement and shape changes after the 32 gastrula stage, which appear to be disrupted above the high temperature threshold. These findings have 33 implications for the relationship between ecology and reproduction in C. intestinalis. More broadly they 34 point to mechanisms behind canalization in animals, such as ascidians, characterized by early, largely 35 autonomous, cell type specification. 36 37 38 39 42 temperature is a major determinant. This study seeks to establish which aspects of embryogenesis are the 43 most susceptible to high temperature in the model marine invertebrate Ciona. 44C. intestinalis, like many if not all animals, has a "normal" embryonic phenotype that is produced 45 over a range of environmental conditions. The ability for a developmental program to produce a 46 stereotyped outcome in spite of environmental variation has been termed "canalization" (Siegal and 47 Bergman, 2002; Waddington, 1942). 48While there has been much work done on the effects of water temperature on the overall life 49 history of marine invertebrates, a literature search reveals little work examining in detail water 50 temperature effects on embryogenesis itself. However, it is likely that the ability to develop to a 51 functional larval stage is an imporftant factor limiting the ranges of many marine invertebrates (Byrne et 52 al., 2009). It is also likely that embryologists historically have been more interested in how normal
During cell division in Escherichia coli, the highly conserved tubulin homolog FtsZ polymerizes and assembles into a ring-like structure, called the Z-ring, at the site of septation. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins, predominantly FtsA in E. coli. FtsA shares structural homology with actin and, like actin, hydrolyzes ATP. Yeast actin detects nucleotide occupancy through a sensor region adjacent to the nucleotide binding site and adopts distinct conformations in monomeric and filamentous actin. Bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and polymerize. Here, we show that several amino acid residues proximal to the nucleotide binding site in FtsA are critical for function in vitro and in vivo. Each of these residues are important for ATP hydrolysis, phospholipid (PL) binding, ATP-dependent vesicle remodeling, and recruitment to the divisome in vivo, to varying degrees. Notably, we observed that Ser 84 and Glu 14 are essential for ATP-dependent vesicle remodeling and magnesium-dependent membrane release of FtsA from vesicles in vitro, and these defects likely underlie the loss of function by FtsA(E14R) and FtsA(S84L) in vivo. Finally, we demonstrate that FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is defective for rapid ATP hydrolysis and ATP-dependent remodeling of PL vesicles in vitro. Together, our results show that loss of nucleotide-dependent activities by FtsA, such as ATP hydrolysis, membrane binding and release, and, most importantly, ATP-dependent PL remodeling, lead to failed Z-ring assembly and division defects in cells.
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