Calcium regulates the expression of a Dictyostelium discoideum asparaginyl tRNA synthetase gene

In a screen for calcium-regulated gene expression during growth and development ofDictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. TheddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the firstD. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of theAsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution ofGlnRS andAsnRS in eukaryotes

Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface

Imaging single events at the cell membrane

The ability to sense and respond to the environment is a hallmark of living systems. These processes occur at the levels of the organism, cells and individual molecules. Sensing of extracellular changes could result in a structural or chemical alteration in a molecule, which could in turn trigger a cascade of intracellular signals or regulated trafficking of molecules at the cell surface. These and other such processes allow cells to sense and respond to environmental changes. Often, these changes and the responses to them are spatially and/or temporally localized, and visualization of such events necessitates the use of highresolution imaging approaches. Here we discuss optical imaging approaches and tools for imaging individual events at the cell surface with improved speed and resolution. The use of ensemble measurements, which report changes that affect an averaged outcome, has been crucial in our understanding of transmembrane signaling. Such studies have identified various cell surface receptors and signaling mechanisms that detect and respond to extracellular signals, such as nutrients, growth factors, pathogens and the extracellular matrix. However, many physiological and pathological events affect individual molecules or organelles. Because information is lost by averaging signals, determining important mechanistic details necessitates studying individual events. For example, if a microscopic event exists in two or more states, such as a signaling cascade that is either on or off or an ion channel that is open or closed, the average will represent a state that does not exist at the microscopic level; averaging the amplitude of response thus results in a loss of information about individual events. As a second example, ensemble measurements report the dominant population but miss responses that occur from a minority of spatially or temporally localized signals. A third example is the inability to temporally order single events on the basis of an averaged measurement. Ordering the steps of a multistep process by ensemble study requires the individual events to be tightly synchronized, which is difficult to achieve and even more difficult to maintain. However, observing individual events allows the order of each step to be unambiguously determined without a need for synchronization. A fourth example is the loss of important temporal information about individual events in ensemble averages. Each molecule might alter its state exponentially over time, or the change might be abrupt but the distribution of when the change occurs among individual molecules might be exponential. The temporal nature of such microscopic events often cannot be resolved from ensemble measurements. Many approaches have been developed to improve the spatial and temporal resolution with which we can study cellular and molecular responses at or near the cell surface. Approaches such as electron microscopy allow very high spatial resolution but are not suitable for imaging dynamic processes. Other approaches, such as scanning probe microscopy (which includes atomic force microscopy) and near-field scanning optical microscopy, offer spatial resolution at the nanometer scale. However, when scanning an area even as big as the surface of a cell, the temporal resolution becomes poor. Further, these methods do not allow simultaneous measurement of activities all across the cell surface, thus limiting their applicability for monitoring cell surface events in real time. The use of electrical measurements, such as voltage-clamp recording, allows membrane events to be studied at submillisecond temporal resolution. However, when performed at the whole-cell level, electrophysiology does not provide spatial information. Information on finer spatial detail can be gathered with patch-clamping to study single channels and transporters. However, this allows sampling at only a single spot. Since the introduction of patch-clamping three decades ago 1 , it has been applied widely to study cell surface processes. Excellent discussion of these approaches, their applications and comparison with optical imaging are available 2-5 and will not be discussed here. Many of the limits on spatial and temporal resolution can be overcome by optical imaging. Optical imaging permits monitoring from molecular to organismal scales and for time periods ranging from milliseconds to several days. In the first part of this review, we will discuss developments that have improved the speed and resolution of live cell imaging. In the second part, we will highlight the contributions and practical applications of these techniques to improve monitoring of single events at the cell membrane. Tools for optimizing spatial and temporal resolution Conventional optical microscopy allows an axial resolution of approximately 400 nm to be achieved. However, the cell membrane is two orders of magnitude smaller, at a thickness of approximately 4-6 nm (refs. 6-8) Several approaches allow the reduction or elimination of out-ofplane fluorescence, and a few of these can surmount the physical limit imposed by the wavelength of visible ligh

Bioremediation of Chlorpyrifos Contaminated Soil by Microorganism

India is agricultural based country where 70% of the population survives on it. In order to increase the production of field various pesticides are used. Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) is an organophosphate pesticide which is widely used as insecticide for crop protection. But due to its persistent nature into the environment, it is leading to various hazards including neurotoxic effects, cardiovascular diseases and respiratory diseases. Bioremediation is a technology to eliminate chlorpyrifos efficiently from the environment. In bioremediation of chlorpyrifos the potential degradative microorganisms possess opd (organophosphate degrading) gene which hydrolyses the chlorpyrifos and utilizes it as a sole carbon source.Thus the present review discusses about how through bioremediation the pesticide chlorpyrifos can be degraded using potential soil microorganisms

Use of quantitative membrane proteomics identifies a novel role of mitochondria in healing injured muscles.

Skeletal muscles are proficient at healing from a variety of injuries. Healing occurs in two phases, early and late phase. Early phase involves healing the injured sarcolemma and restricting the spread of damage to the injured myofiber. Late phase of healing occurs a few days postinjury and involves interaction of injured myofibers with regenerative and inflammatory cells. Of the two phases, cellular and molecular processes involved in the early phase of healing are poorly understood. We have implemented an improved sarcolemmal proteomics approach together with in vivo labeling of proteins with modified amino acids in mice to study acute changes in the sarcolemmal proteome in early phase of myofiber injury. We find that a notable early phase response to muscle injury is an increased association of mitochondria with the injured sarcolemma. Real-time imaging of live myofibers during injury demonstrated that the increased association of mitochondria with the injured sarcolemma involves translocation of mitochondria to the site of injury, a response that is lacking in cultured myoblasts. Inhibiting mitochondrial function at the time of injury inhibited healing of the injured myofibers. This identifies a novel role of mitochondria in the early phase of healing injured myofibers

Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles.

Repair and regeneration of the injured skeletal myofiber involves fusion of intracellular vesicles with sarcolemma and fusion of the muscle progenitor cells respectively. In vitro experiments have identified involvement of Annexin A1 (Anx A1) in both these fusion processes. To determine if Anx A1 contributes to these processes during muscle repair in vivo, we have assessed muscle growth and repair in Anx A1-deficient mouse (AnxA1-/-). We found that the lack of Anx A1 does not affect the muscle size and repair of myofibers following focal sarcolemmal injury and lengthening contraction injury. However, the lack of Anx A1 delayed muscle regeneration after notexin-induced injury. This delay in muscle regeneration was not caused by a slowdown in proliferation and differentiation of satellite cells. Instead, lack of Anx A1 lowered the proportion of differentiating myoblasts that managed to fuse with the injured myofibers by days 5 and 7 after notexin injury as compared to the wild type (w.t.) mice. Despite this early slowdown in fusion of Anx A1-/- myoblasts, regeneration caught up at later times post injury. These results establish in vivo role of Anx A1 in cell fusion required for myofiber regeneration and not in intracellular vesicle fusion needed for repair of myofiber sarcolemma

The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50). We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro. NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation

Annexin A7 is required for ESCRT III-mediated plasma membrane repair

The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells. Here, using invasive breast cancer cells we show that the Ca2+ - and phospholipid-binding protein annexin A7 is part of the plasma membrane repair response by enabling assembly of the endosomal sorting complex required for transport (ESCRT) III. Following injury to the plasma membrane and Ca2+ flux into the cytoplasm, annexin A7 forms a complex with apoptosis linked gene-2 (ALG-2) to facilitate proper recruitment and binding of ALG-2 and ALG-2-interacting protein X (ALIX) to the damaged membrane. ALG-2 and ALIX assemble the ESCRT III complex, which helps excise and shed the damaged portion of the plasma membrane during wound healing. Our results reveal a novel function of annexin A7 – enabling plasma membrane repair by regulating ESCRT III-mediated shedding of injured plasma membrane

The Pediatric Cell Atlas:Defining the Growth Phase of Human Development at Single-Cell Resolution

Single-cell gene expression analyses of mammalian tissues have uncovered profound stage-specific molecular regulatory phenomena that have changed the understanding of unique cell types and signaling pathways critical for lineage determination, morphogenesis, and growth. We discuss here the case for a Pediatric Cell Atlas as part of the Human Cell Atlas consortium to provide single-cell profiles and spatial characterization of gene expression across human tissues and organs. Such data will complement adult and developmentally focused HCA projects to provide a rich cytogenomic framework for understanding not only pediatric health and disease but also environmental and genetic impacts across the human lifespan