Recruitment of the TolA Protein to Cell Constriction Sites in Escherichia coli via Three Separate Mechanisms, and a Critical Role for FtsWI Activity in Recruitment of both TolA and TolQ.

The Tol-Pal system of Gram-negative bacteria helps maintain the integrity of the cell envelope and ensures that invagination of the envelope layers during cell fission occurs in a well-coordinated manner. In Escherichia coli, the five Tol-Pal proteins (TolQ, -R, -A, and -B and Pal) accumulate at cell constriction sites in a manner that normally requires the activity of the cell constriction initiation protein FtsN. While septal recruitment of TolR, TolB, and Pal also requires the presence of TolQ and/or TolA, the latter two can recognize constriction sites independently of the other system proteins. What attracts TolQ or TolA to these sites is unclear. We show that FtsN indirectly attracts both proteins and that PBP1A, PBP1B, and CpoB are dispensable for their septal recruitment. However, the ß-lactam aztreonam readily interferes with the septal accumulation of both TolQ and TolA, indicating that FtsN-stimulated production of septal peptidoglycan by the FtsWI synthase is critical to their recruitment. We also discovered that each of TolA's three domains can separately recognize division sites. Notably, the middle domain (TolAII) is responsible for directing TolA to constriction sites in the absence of other Tol-Pal proteins and CpoB, while recruitment of TolAI requires TolQ and that of TolAIII requires a combination of TolB, Pal, and CpoB. Additionally, we describe the construction and use of functional fluorescent sandwich fusions of the ZipA division protein, which should be more broadly valuable in future studies of the E. coli cell division machinery. IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction will eventually occur. In the well-studied bacterium Escherichia coli, this machinery contains over 30 distinct proteins. We studied how two such proteins, TolA and TolQ, which also play a role in maintaining the integrity of the outer membrane, are recruited to the machinery. We find that TolA can be recruited by three separate mechanisms and that both proteins rely on the activity of a well-studied cell division enzyme for their recruitment.

Roles of the DedD Protein in Escherichia coli Cell Constriction.

Two key tasks of the bacterial septal-ring (SR) machinery during cell constriction are the generation of an inward-growing annulus of septal peptidoglycan (sPG) and the concomitant splitting of its outer edge into two layers of polar PG that will be inherited by the two new cell ends. FtsN is an essential SR protein that helps trigger the active constriction phase in Escherichia coli by inducing a self-enhancing cycle of processes that includes both sPG synthesis and splitting and that we refer to as the sPG loop. DedD is an SR protein that resembles FtsN in several ways. Both are bitopic inner membrane proteins with small N-terminal cytoplasmic parts and larger periplasmic parts that terminate with a SPOR domain. Though absence of DedD normally causes a mild cell-chaining phenotype, the protein is essential for division and survival of cells with limited FtsN activity. Here, we find that a small N-terminal portion of DedD (NDedD; DedD1-54) is required and sufficient to suppress ΔdedD-associated division phenotypes, and we identify residues within its transmembrane domain that are particularly critical to DedD function. Further analyses indicate that DedD and FtsN act in parallel to promote sPG synthesis, possibly by engaging different parts of the FtsBLQ subcomplex to induce a conformation that permits and/or stimulates the activity of sPG synthase complexes composed of FtsW, FtsI (PBP3), and associated proteins. We propose that, like FtsN, DedD promotes cell fission by stimulating sPG synthesis, as well as by providing positive feedback to the sPG loop.IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction eventually occurs. In the well-studied bacterium Escherichia coli, this machinery contains over 30 distinct proteins. We identify functionally important parts of one of these proteins, DedD, and present evidence supporting a role for DedD in helping to induce and/or sustain a self-enhancing cycle of processes that are executed by fellow septal-ring proteins and that drive the active constriction phase of the cell division cycle.

Roles for both FtsA and the FtsBLQ subcomplex in FtsN-stimulated cell constriction in Escherichia coli.

Escherichia coli FtsN is a bitopic membrane protein that is essential for triggering active cell constriction. A small periplasmic subdomain ((E) FtsN) is required and sufficient for function, but its mechanism of action is unclear. We isolated extragenic (E) FtsN*-suppressing mutations that restore division in cells producing otherwise non-functional variants of FtsN. These mapped to the IC domain of FtsA in the cytoplasm and to small subdomains of the FtsB and FtsL proteins in the periplasm. All FtsB and FtsL variants allowed survival without (E) FtsN, but many then imposed a new requirement for interaction between the cytoplasmic domain of FtsN ((N) FtsN) and FtsA. Alternatively, variants of FtsA, FtsB or FtsL acted synergistically to allow cell division in the complete absence of FtsN. Strikingly, moreover, substitution of a single residue in FtsB (E56) proved sufficient to rescue ΔftsN cells as well. In FtsN(+) cells, (E) FtsN*-suppressing mutations promoted cell fission at an abnormally small cell size, and caused cell shape and integrity defects under certain conditions. This and additional evidence support a model in which FtsN acts on either side of the membrane to induce a conformational switch in both FtsA and the FtsBLQ subcomplex to de-repress septal peptidoglycan synthesis and membrane invagination.

Following drug uptake and reactions inside Escherichia coli cells by Raman microspectroscopy.

Raman microspectroscopy combined with Raman difference spectroscopy reveals the details of chemical reactions within bacterial cells. The method provides direct quantitative data on penetration of druglike molecules into Escherichia coli cells in situ along with the details of drug-target reactions. With this label-free technique, clavulanic acid and tazobactam can be observed as they penetrate into E. coli cells and subsequently inhibit ß-lactamase enzymes produced within these cells. When E. coli cells contain a ß-lactamase that forms a stable complex with an inhibitor, the Raman signature of the known enamine acyl-enzyme complex is detected. From Raman intensities it is facile to measure semiquantitatively the number of clavulanic acid molecules taken up by the lactamase-free cells during growth.

Growth of the developing mouse heart: an interactive qualitative and quantitative 3D atlas.

Analysis of experiments aimed at understanding the genetic mechanisms of differentiation and growth of the heart, calls for detailed insights into cardiac growth and proliferation rate of myocytes and their precursors. Such insights in mouse heart development are currently lacking. We quantitatively assessed the 3D patterns of proliferation in the forming mouse heart and in the adjacent splanchnic mesoderm, from the onset of heart formation till the developed heart at late gestation. These results are presented in an interactive portable document format (Suppl. PDF) to facilitate communication and understanding. We show that the mouse splanchnic mesoderm is highly proliferative, and that the proliferation rate drops upon recruitment of cells into the cardiac lineage. Concomitantly, the proliferation rate locally increases at the sites of chamber formation, generating a regionalized proliferation pattern. Quantitative analysis shows a gradual decrease in proliferation rate of the ventricular walls with progression of development, and a base-to-top decline in proliferation rate in the trabecules. Our data offers clear insights into the growth and morphogenesis of the mouse heart and shows that in early development the phases of tube formation and chamber formation overlap. The resulting interactive quantitative 3D atlas of cardiac growth and morphogenesis provides a resource for interpretation of mechanistic studies.

RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli.

The bacterial MreB actin cytoskeleton is required for cell shape maintenance in most non-spherical organisms. In rod-shaped cells such as Escherichia coli, it typically assembles along the long axis in a spiral-like configuration just underneath the cytoplasmic membrane. How this configuration is controlled and how it helps dictate cell shape is unclear. In a new genetic screen for cell shape mutants, we identified RodZ (YfgA) as an important transmembrane component of the cytoskeleton. Loss of RodZ leads to misassembly of MreB into non-spiral structures, and a consequent loss of cell shape. A juxta-membrane domain of RodZ is essential to maintain rod shape, whereas other domains on either side of the membrane have critical, but partially redundant, functions. Though one of these domains resembles a DNA-binding motif, our evidence indicates that it is primarily responsible for association of RodZ with the cytoskeleton.

Identification of Escherichia coli ZapC (YcbW) as a component of the division apparatus that binds and bundles FtsZ polymers.

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified in Escherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission process per se but contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring in E. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.

A caudal proliferating growth center contributes to both poles of the forming heart tube.

Recent studies have shown that the primary heart tube continues to grow by addition of cells from the coelomic wall. This growth occurs concomitantly with embryonic folding and formation of the coelomic cavity, making early heart formation morphologically complex. A scarcity of data on localized growth parameters further hampers the understanding of cardiac growth. Therefore, we investigated local proliferation during early heart formation. Firstly, we determined the cell cycle length of primary myocardium of the early heart tube to be 5.5 days, showing that this myocardium is nonproliferating and implying that initial heart formation occurs solely by addition of cells. In line with this, we show that the heart tube rapidly lengthens at its inflow by differentiation of recently divided precursor cells. To track the origin of these cells, we made quantitative 3D reconstructions of proliferation in the forming heart tube and the mesoderm of its flanking coelomic walls. These reconstructions show a single, albeit bilateral, center of rapid proliferation in the caudomedial pericardial back wall. This center expresses Islet1. Cell tracing showed that cells from this caudal growth center, besides feeding into the venous pole of the heart, also move cranially via the dorsal pericardial mesoderm and differentiate into myocardium at the arterial pole. Inhibition of caudal proliferation impairs the formation of both the atria and the right ventricle. These data show how a proliferating growth center in the caudal coelomic wall elongates the heart tube at both its venous and arterial pole, providing a morphological mechanism for early heart formation.

Self-enhanced accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR domain (DamX, DedD, and RlpA) in Escherichia coli cell constriction.

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an alpha-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain ((E)FtsN) of at most 35 residues that is centered about helix H2 in the periplasm. (E)FtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain ((S)FtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of (S)FtsN depended on the ability of cells to constrict and, thus, on the activity of (E)FtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.

Conditional lethality, division defects, membrane involution, and endocytosis in mre and mrd shape mutants of Escherichia coli.

Maintenance of rod shape in Escherichia coli requires the shape proteins MreB, MreC, MreD, MrdA (PBP2), and MrdB (RodA). How loss of the Mre proteins affects E. coli viability has been unclear. We generated Mre and Mrd depletion strains under conditions that minimize selective pressure for undefined suppressors and found their phenotypes to be very similar. Cells lacking one or more of the five proteins were fully viable and propagated as small spheres under conditions of slow mass increase but formed large nondividing spheroids with noncanonical FtsZ assembly patterns at higher mass doubling rates. Extra FtsZ was sufficient to suppress lethality in each case, allowing cells to propagate as small spheres under any condition. The failure of each unsuppressed mutant to divide under nonpermissive conditions correlated with the presence of elaborate intracytoplasmic membrane-bound compartments, including vesicles/vacuoles and more-complex systems. Many, if not all, of these compartments formed by FtsZ-independent involution of the cytoplasmic membrane (CM) rather than de novo. Remarkably, while some of the compartments were still continuous with the CM and the periplasm, many were topologically separate, indicating they had been released into the cytoplasm by an endocytic-like membrane fission event. Notably, cells failed to adjust the rate of phospholipid synthesis to their new surface requirements upon depletion of MreBCD, providing a rationale for the "excess" membrane in the resulting spheroids. Both FtsZ and MinD readily assembled on intracytoplasmic membrane surfaces, and we propose that this contributes significantly to the lethal division block seen in all shape mutants under nonpermissive conditions.

Regionalized sequence of myocardial cell growth and proliferation characterizes early chamber formation.

Increase in cell size and proliferation of myocytes are key processes in cardiac morphogenesis, yet their regionalization during development of the heart has been described only anecdotally. We have made quantitative reconstructions of embryonic chicken hearts ranging in stage from the fusion of the heart-forming fields to early formation of the chambers. These reconstructions reveal that the early heart tube is recruited from a pool of rapidly proliferating cardiac precursor cells. The proliferation of these small precursor cells ceases as they differentiate into overt cardiomyocytes, producing a slowly proliferating straight heart tube composed of cells increasing in size. The largest cells were found at the ventral side of the heart tube, which corresponds to the site of the forming ventricle, as well as the site where proliferation is reinitiated. The significance of these observations is 2-fold. First, they support a model of early cardiac morphogenesis in 2 stages. Second, they demonstrate that regional increase in size of myocytes contributes significantly to chamber formation.

Reconstruction of the patterns of gene expression in the developing mouse heart reveals an architectural arrangement that facilitates the understanding of atrial malformations and arrhythmias.

Firm knowledge about the formation of the atrial components and of the variations seen in congenital cardiac malformations and abnormal atrial rhythms is fundamental to our understanding of the normal structure of the definitive atrial chambers. The atrial region is relatively inaccessible and has continued to be the source of disagreement. Seeking to resolve these controversies, we made three-dimensional reconstructions of the myocardial components of the developing atrium, identifying domains on the basis of differential expression of myocardial markers, connexin40, and natriuretic precursor peptide A. These reconstructions, made from serial sections of mouse embryos, show that from the outset of atrial development, the systemic and pulmonary veins are directly connected to the atrium. Relative to the systemic junctions, however, the pulmonary venous junction appears later. Our experience shows that three-dimensional reconstructions have three advantages. First, they provide clear access to the combined morphological and molecular data, allowing clarification and verification of morphogenetic concepts for nonmorphological experts and setting the scene for further discussion. Second, they demonstrate that, from the outset, the myocardium surrounding the pulmonary veins is distinct from that clothing the systemic venoatrial junctions. Third, they reveal an anatomical and molecular continuity between the entrance of the systemic venous tributaries, the internodal atrial myocardium, and the atrioventricular region. All these regions are derived from primary myocardium, providing a molecular basis for the observed nonrandom distribution of focal right atrial tachycardias.

Marked differences in tissue-specific expression of chitinases in mouse and man.

Two distinct chitinases have been identified in mammals: a phagocyte-specific enzyme named chitotriosidase and an acidic mammalian chitinase (AMCase) expressed in the lungs and gastrointestinal tract. Increased expression of both chitinases has been observed in different pathological conditions: chitotriosidase in lysosomal lipid storage disorders like Gaucher disease and AMCase in asthmatic lung disease. Recently, it was reported that AMCase activity is involved in the pathogenesis of asthma in an induced mouse model. Inhibition of chitinase activity was found to alleviate the inflammation-driven pathology. We studied the tissue-specific expression of both chitinases in mice and compared it to the situation in man. In both species AMCase is expressed in alveolar macrophages and in the gastrointestinal tract. In mice, chitotriosidase is expressed only in the gastrointestinal tract, the tongue, fore-stomach, and Paneth cells in the small intestine, whereas in man the enzyme is expressed exclusively by professional phagocytes. This species difference seems to be mediated by distinct promoter usage. In conclusion, the pattern of expression of chitinases in the lung differs between mouse and man. The implications for the development of anti-asthma drugs with chitinases as targets are discussed.

The transcriptional repressor Tbx3 delineates the developing central conduction system of the heart.

OBJECTIVE: The molecular mechanisms that regulate the formation of the conduction system are poorly understood. We studied the developmental expression pattern and functional aspects of the T-box transcription factor Tbx3, a novel marker for the murine central conduction system (CCS). METHODS: The patterns of expression of Tbx3, and of Cx40, Cx43, and Nppa, which are markers for atrial and ventricular chamber-type myocardium in the developing heart, were analyzed in mice by in situ hybridization and three-dimensional reconstruction analysis. The function of Tbx3 in regulating Nppa and Cx40 promoter activity was studied in vitro. RESULTS: In the formed heart, Tbx3 is expressed in the sinoatrial node (SAN), atrioventricular node (AVN), bundle and proximal bundle branches (BBs), as well as the internodal regions and the atrioventricular region. Throughout cardiac development, Tbx3 is expressed in an uninterrupted myocardial domain that extends from the sinoatrial node to the atrioventricular region. This expression domain is present in the looping heart tube from E8.5 onwards. Expression of the chamber-type myocardial markers is specifically absent from the Tbx3 expression domain. Tbx3 is able to repress Nppa and Cx40 promoter activity and abolish the synergistic activation of the Nppa promoter by Tbx5 and Nkx2.5. CONCLUSION: We identified the T-box transcription factor Tbx3 as a novel and accurate marker for the central conduction system. Our analysis implicates a role for Tbx3 in repressing a chamber-specific program of gene expression in regions from which the components of the central conduction system are subsequently formed.

Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube.

OBJECTIVE: After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and quantitative analyses of cardiogenesis in vivo and in vitro. METHODS: Gene expression profiles were made by in situ hybridisation and real-time PCR and electrophysiological profiles by patch clamp analyses of cardiomyocytes derived from time series of differentiating HM1 mouse embryonic stem cells and from embryonic and adult mouse hearts. RESULTS: In embryoid bodies the in situ patterns of expression of alpha-myosin heavy chain, myosin light chain 2a and sarcoendoplasmic reticulum calcium ATPase 2a were similar to that of the heart muscle-specific marker gene cardiac troponin I. Myosin light chain 2v was expressed in part of the cardiac troponin I-expressing area, indicating heterogeneity within the cardiac cell population. Atrial natriuretic factor expression, indicative of the chamber-type program, could only very occasionally be detected by in situ hybridisation. Quantitative reverse transcriptase PCR showed that all cardiac genes, most notably atrial natriuretic factor, were expressed at relatively low levels, similar to those in embryonic hearts at embryonic day 8.75-9. Analysis of the electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes showed an increase of the upstroke velocity and a shorter duration of the action potential during prolonged differentiation in vitro. When embryonic mouse heart compartments of embryonic day 12.5 were used as a reference, the electrophysiological characteristics of a substantial part of the embryonic stem cell-derived cardiomyocytes were most reminiscent to those observed in the embryonic outflow tract. CONCLUSION: Together, these data suggest that most cardiomyocytes acquired by differentiation of embryonic stem cells maintain a phenotype reminiscent of that of the cardiomyocytes of the primary heart tube, and hardly any myocytes develop a chamber myocardial phenotype.