PBP1 of Staphylococcus aureus has multiple essential functions in cell division

Bacterial cell division is a complex process requiring the coordination of multiple components, to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multi–stage PG architecture that develops during septation. Penicillin binding proteins (PBPs) are essential for the final steps of PG biosynthesis — their transpeptidase activity links together the peptide sidechain of nascent glycan strands together. PBP1 is required for cell division in S. aureus and here we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C–terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thus coordinate the cell division process. The transpeptidase activity of PBP1 is also essential but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1–specific β–lactam, meropenem. Together these results lead to a model for septal PG synthesis where PBP1 enzyme activity is responsible for the characteristic architecture of the septum and PBP1 protein molecules coordinate cell division allowing septal plate formation.

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation and final cellularization, it remains unknown when and how the cell fate is determined during development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of individual cell types to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the 1-nucleate stage to the 7-cell stage after cellularization using an in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar–chalazal (distal–proximal) axis. During cellularization, the polar nuclei migrated while associating with the forming edge of the cell plate, and then, migrated toward each other to fuse linearly. We also tracked the gene expression dynamics and identified that the expression of MYB98pro::GFP–MYB98, a synergid-specific marker, was initiated just after cellularization in the synergid, egg, and central cells and was then restricted to the synergid cells. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild-type and myb98 mutant revealed that the myb98 synergid cells had egg cell–like gene expression profiles. Although in myb98, egg cell–specific gene expression was properly initiated in the egg cells only after cellularization, but subsequently expressed ectopically in one of the 2 synergid cells. These results, together with the various initiation timings of the egg cell–specific genes, suggest complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell type–specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.

BIOMECHANICAL FACTORS IN RESIDUAL LIMB FORMATION AFTER AMPUTATION

The aim: To study the influence of biomechanical factors on the character of morphological disorders in the process of reparative regeneration at the end of the residual limb after amputation. Materials and methods: 10 series of experiments on 144 rabbits were conducted. We used myodesis with normal, insufficient and excessive muscle tension, their electrical stimulation, tight and leaky closure of the bone marrow canal. Terms of observation 1, 3, 6 months. The method of research – histological with the filling of vessels with inkgelatin mixture. Results: Dense closure of the meduallary cavity and uniform muscle tension during plasty in the first three series of experiments allow to obtain a cylindrical residual limb with preservation of the cortical diaphyseal plate, formation of the bone closing plate, normalization of intraosseous microcirculation, completion of the reparative process. In the majority of observations of the IV-X series there was a reparative regeneration disorder connected with the incorrect tension of muscles and the absence of normalization of intraosseous circulation, the reparative process was not observed to be complete, which led to the pathological reorganization of bone tissue with the formation of stumps of various shapes. Conclusions: Uneven muscle tension and lack of closure of the intramedually canal except for microcirculation disorders leads to increased periosteum bone formation, formation of periosteum cartilage exostases, clavate stumps, resorption and fractures of the cortical diaphyseal plate with curvature and stump axis disorders, formation of a conical stump.

Notch signaling is a critical initiator of roof plate formation as revealed by the use of RNA profiling of the dorsal neural tube

The factors underlying establishment of the definitive roof plate (RP) and its segregation from neural crest (NC) and interneurons are unknown. We performed transcriptome analysis at trunk levels of quail embryos comparing the dorsal neural tube at premigratory NC and RP stages. This unraveled molecular heterogeneity between NC and RP stages, and within the RP itself. By implementing these genes, we asked whether Notch signaling is involved in RP development. First, we observed that Notch is active at the RP-interneuron interface. Furthermore, gain and loss of Notch function in quail and mouse embryos, respectively, revealed no effect on early NC behavior. Constitutive Notch activation caused a local downregulation of RP markers with a concomitant development of dI1 interneurons, as well as an ectopic upregulation of RP markers in the interneuron domain. Reciprocally, in mice lacking Notch activity both the RP and dI1 interneurons failed to form and this was associated with expansion of the dI2 population. Collectively, our results offer a new resource for defining specific cell types, and provide evidence that Notch is required to establish the definitive RP, and to determine the choice between RP and interneuron fates, but not the segregation of RP from NC.Summary statementA new set of genes involved in Notch-dependent roof plate formation is unraveled by transcriptome analysis.

THEORIES OF CONTINENTAL DRIFT

Continental drift is the process of continental plates moving. The movement is very slow, and it might take even years to show the impact on the Earth. The movement depends on gravity, convection drift, and plate formation. Gravity impacts the movements as the mantle (The layer below the crust) is always spinning because of gravity and the plates are located on the mantle. Convection drift impacts on the movement because convection drift is a cycle of melting and cooling of rocks in the mantle which can slowly impact the movement of the plate. Plate formation also affects the way the plates move as the new plates formed pushes the nearby plates causing movement.

SABRE populates ER domains essential for cell plate maturation and cell expansion influencing cell and tissue patterning

The SABRE protein, originally identified in plants, is found throughout eukaryotes. In plants, SABRE has been implicated in cell expansion, division plane orientation and planar polarity. However, how SABRE mediates these processes remains an open question. Here, we have taken advantage of the fact that the bryophyte Physcomitrium patens has a single copy of SABRE, is an excellent model for cell biology and is readily amenable to precise genetic alterations to investigate SABRE’s mechanism of action. We discovered that SABRE null mutants were stunted in both polarized growing and diffusely growing tissues, similar to reported phenotypes in seed plants. However, in polarized growing cells, we observed significant delays in cell plate formation and sometimes catastrophic failures in cell division. We generated a functional SABRE fluorescent fusion protein and determined that it forms dynamic puncta on regions of the endoplasmic reticulum (ER) both in the cytoplasm during interphase and at the new cell plate during division. In the absence of SABRE, ER morphology was severely compromised with large aggregates accumulating in the cytoplasm and abnormal buckling along the developing cell plate late in cytokinesis. In fact, SABRE and the ER maximally accumulated on the developing plate specifically during cell plate maturation, coincident with the timing of the onset of failures in cell plate formation in cells lacking SABRE. Further we discovered that callose deposition is delayed in Δsabre cells, and in cells that failed to divide, abnormal callose accumulations formed at the cell plate. Our findings demonstrated that SABRE functions by influencing the ER and callose deposition, revealing a surprising and essential role for the ER in cell plate maturation. Given that SABRE is conserved, understanding how SABRE influences cell and tissue patterning has profound significance across eukaryotes.

Quick and Abnormal Ossification Results in No Growth Plate Formation and Skeletal Dysplasia in Postnatal Mice Induced by Conditional Knockout of Indian Hedgehog

Objective: To observe the dynamic effect of conditional knockout of Indian hedgehog (Ihh) on ossification of postnatal growth plate, skeletal abnormalities and the related signaling factors in mice.Methods: P0 Col2a1-Cre ERT2; Ihhfl/fl mice were randomly divided into tamoxifen (TM) injection to delete Ihh and oil injection as control. Real-time PCR (RT-PCR) was used to detect Ihh knockout rate. X-ray and micro-CT were used to evaluate gross and knee joint morphology of 8-week-old mice. The hind knee joints were harvested at P2-P14 after the animals were euthanized to observe the dynamic evolution of the growth plate. Safranin-O and Von Kossa staining were performed to assess growth plate development and the mineralization of bone respectively. The costal cartilage cells of mice were cultured in vitro on postnatal day 6. Changes in the expression of parathyroid hormone-related protein (PTHrP) and bone morphogenetic protein 6 (BMP-6) were detected by immunohistochemistry and RT-PCR.Results: RT-PCR results confirmed that the knockout rate of Ihh was 76.83%. X-ray and micro-CT scans showed that the short-limb deformity of the experimental group was associated with abnormal development of the epiphysis. Saffron O staining showed a disorder of cell arrange in the growth-plate area of Ihhd/d mice; Von Kossa staining showed an early and premature growth-plate ossification that prevents the growth plate formation in Ihhd/d mice starting at P8. Immunohistochemistry and RT-PCR showed significantly decreased PTHrP expression (P<0.05) and significantly increased BMP-6 expression (P<0.05). Conclusion: Postnatal conditional knockout of Ihh inhibited the expression of PTHrP and promotes the expression of BMP-6, which leads to early and abnormal ossification of the growth plate, no growth plate formation, and skeletal dysplasia in mice.

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis

ABSTRACTThe female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation, and the final cellularization, it remains unknown when and how the cell fate is determined during their development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of its individual cell types, to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the one-nucleate stage to the seven-cell stage after cellularization, using the in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar–chalazal axis. During cellularization, the polar nuclei migrated while associating with forming edge of the cell plate. Then, each polar nucleus migrated to fuse linearly towards each other. We also tracked the gene expression dynamics and identified that the expression of the MYB98pro∷GFP, a synergid-specific marker, was initiated before cellularization, and then restricted to the synergid cells after cellularization. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild type and myb98 mutant, revealed that the myb98 synergid cells had the egg cell-like gene expression profile. Although in the myb98, the egg cell-specific gene expressions were properly initiated only in the egg cells after cellularization, but subsequently expressed ectopically in one of the two synergid cells. These results, together with the various initiation timings of the egg cell-specific genes suggest the complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell-type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.