Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant protein in photosynthesizing plant parts and the most abundant protein on earth. Amino acids deriving from its net degradation during senescence are transported to sinks (e.g. developing leaves, fruits). Rubisco catabolism is not controlled only by the overall sink demand. An accumulation of carbohydrates may also accelerate senescence and Rubisco degradation under certain conditions. Amino acids produced by proteolysis are rapidly redistributed in plants with proper source-sink relationships. In leaves of wheat plants with reduced sink capacity (e.g. sink removal, phloem interruption by steam girdling at the leaf base), Rubisco is degraded and free amino acids accumulate. They may be washed out in the rain during late senescence. In leaves of depodded soybeans, Rubisco is degraded and amino acids can be reutilized in these leaves for the synthesis of special vacuolar proteins in the paraveinal mesophyll (vegetative storage proteins). Nitrogen deriving from Rubisco degradation in older (senescing) leaves of annual crops is integrated to some extent again in newly synthesized Rubisco in younger leaves or photosynthesizing tissues of fruits. Finally, a high percentage of this nitrogen is accumulated in protein bodies (storage proteins). At the subcellular level, Rubisco can be degraded in intact chloroplasts. Reactive oxygen species may directly cleave the large subunit or modify it to become more susceptible to proteolysis. A metalloendopeptidase may play an important role in Rubisco degradation within intact chloroplasts. Additionally, the involvement of vacuolar endopeptidase(s) in Rubisco catabolism (at least under certain conditions) was postulated by various laboratories.     

The evolving biology of small molecules: controlling cell fate and identity

Small molecules have been playing important roles in elucidating basic biology and treatment of a vast number of diseases for nearly a century, making their use in the field of stem cell biology a comparatively recent phenomenon. Nonetheless, the power of biology-oriented chemical design and synthesis, coupled with significant advances in screening technology, has enabled the discovery of a growing number of small molecules that have improved our understanding of stem cell biology and allowed us to manipulate stem cells in unprecedented ways. This review focuses on recent small molecule studies of (i) the key pathways governing stem cell homeostasis, (ii) the pluripotent stem cell niche, (iii) the directed differentiation of stem cells, (iv) the biology of adult stem cells, and (v) somatic cell reprogramming. In a very short period of time, small molecules have defined a perhaps universally attainable naive ground state of pluripotency, and are facilitating the precise, rapid and efficient differentiation of stem cells into somatic cell populations relevant to the clinic. Finally, following the publication of numerous groundbreaking studies at a pace and consistency unusual for a young field, we are closer than ever to completely eliminating the need for genetic modification in reprogramming.     

Dynamin function is important for chemokine receptor‐induced cell migration

The HIV viral entry co‐receptors CCR5 and CXCR4 function physiologically as typical chemokine receptors. Activation leads to cytosolic signal transduction that results in a variety of cellular responses such as cytoskeletal rearrangement and chemotaxis (CTX). Our aim was to investigate the signalling pathways involved in CC and CXC receptor‐mediated cell migration. Inhibition of dynamin I and II GTPase with dynasore completely inhibited CCL3‐stimulated CTX in THP‐1 cells, whereas the dynasore analogue Dyngo‐4a, which is a more potent inhibitor, showed reduced ability to inhibit CC chemokine‐induced CTX. In contrast, dynasore was not able to block cell migration via CXCR4. The same activation/inhibition pattern was verified in activated T lymphocytes for different CC and CXC chemokines. Cell migration induced by CC and CXC receptors does not rely on active internalization processes driven by dynamin because the blockade of internalization does not affect migration, but it might rely on dynamin interaction with the cytoskeleton. We identify here a functional difference in how CC and CXC receptor migration is controlled, suggesting that specific signalling networks are being employed for different receptor classes and potentially specific therapeutic targets to prevent receptor migration can be identified. Copyright © 2015 John Wiley & Sons, Ltd.     

High cell‐autonomy of the anterior endomesoderm viewed in blastomere fate shift during regulative development in the isolated right halves of four‐cell stage Xenopus embryos

The isolated right half (RH) or left half (LH) of Xenopus embryos can undergo regulation so as to form well‐proportioned larvae. To assess how the combined actions of maternal determinants and cell–cell interactions contribute to form the well‐proportioned larvae, we quantitatively compared four‐cell stage blastomere fate between normal larvae and regulated larvae from RH embryos. In normal larvae, the clones of the right dorsal blastomere (RD) and right ventral blastomere (RV) were located unilaterally. In contrast, in regulated larvae: (i) the RD clone exclusively occupied the anterior endomesoderm (AE) derivatives, coinciding no RV progeny in those derivatives of normal larvae. The clone bilaterally populated tissues along the dorsal midline, which characteristically included the medial regions of both somites adjoining the notochord, with higher percentages on the right and anterior sides. (ii) The RV clone extensively compensated for the missing left side at the expense of its right side contribution, and bilaterally occupied the ventroposterior and also dorsal regions excluding the AE derivatives. This clone considerably populated, with altered orientations, the derivatives of the left half gastrocoel roof plate (GRP), the left half GRP being essential for laterality determination. These results show that the high cell‐autonomy in the AE constitutes a mechanism common to both normal and regulative development. In regulated larvae, cell–cell interactions shifted the midlines on the dorsal side slightly and the ventral side to a greater extent. The cell lineage difference in the left half GRP could result in a different utilization of maternal determinants in that area.     

From mechanical stimulation to biological pathways in the regulation of stem cell fate

Mechanical stimuli are important in directing the fate of stem cells; the effects of mechanical stimuli reported in recent research are reviewed here. Stem cells normally undergo two fundamental processes: proliferation, in which their numbers multiply, and differentiation, in which they transform into the specialized cells needed by the adult organism. Mechanical stimuli are well known to affect both processes of proliferation and differentiation, although the complete pathways relating specific mechanical stimuli to stem cell fate remain to be elucidated. We identified two broad classes of research findings and organized them according to the type of mechanical stress (compressive, tensile or shear) of the stimulus. Firstly, mechanical stress of any type activates stretch‐activated channels (SACs) on the cell membrane. Activation of SACs leads to cytoskeletal remodelling and to the expression of genes that regulate the basic growth, survival or apoptosis of the cells and thus regulates proliferation. Secondly, mechanical stress on cells that are physically attached to an extracellular matrix (ECM) initiates remodelling of cell membrane structures called integrins. This second process is highly dependent on the type of mechanical stress applied and result into various biological responses. A further process, the Wnt pathway, is also implicated: crosstalk between the integrin and Wnt pathways regulates the switch from proliferation to differentiation and finally regulates the type of differentiation. Therefore, the stem cell differentiation process involves different signalling molecules and their pathways and most likely depends upon the applied mechanical stimulation. Copyright © 2014 John Wiley & Sons, Ltd.     

The C terminus of p53 family proteins is a cell fate determinant

The p53 tumor suppressor is the most commonly mutated gene in human cancers. The ability of p53 to induce cell cycle arrest, apoptosis, DNA repair, and other p53-dependent activities is well known; however, the mechanism by which p53 induces a specific activity over another is unclear. Here, we showed that stringent regulation of and by p53 family isoforms facilitates differential target gene expression and thus determines cell fate. Through the use of engineered deletion mutants, we found that activation domain 2 is required for induction of the proapoptotic target gene insulin-like growth factor binding protein 3 (IGFBP3) by p53 and that the basic domain inhibits induction of this gene by p53. Thus, for the first time we provide evidence that the basic domain of p53 is inhibitory in vivo as has been determined in vitro. We also showed that the in vivo inhibitory activity of the basic domain depends upon activation domain 1, such that combined deletion of activation domain 1 and the basic domain was required to alleviate the inhibition by the basic domain. Importantly, deletion of the inhibitory functional domains, namely N-terminal activation domain 1 and the C-terminal basic domain, is paralleled in nature. We found that the IGFBP3 promoter was activated by p53(DeltaNDeltaBD), which mimics a naturally occurring N- and C-terminally truncated human p53 isoform, and by p53AS, a C-terminally truncated murine p53 isoform generated through alternative splicing, but not by full-length human or murine p53. In addition, we found that the C termini of p63 and p73 inhibit the induction of IGFBP3, such that C-terminally truncated p63 and p73 isoforms induce the expression of IGFBP3, whereas full-length ones cannot. We also demonstrated that IGFBP3 is an important effector of the apoptosis induced by N- and C-terminally truncated p53, such that knockdown of IGFBP3 by using an IGFBP3 neutralizing antibody or IGFBP3 small interfering RNA partially rescues the cell death induced by N- and C-terminally truncated p53. In addition, we identified that histone deacetylase activity, not p53 DNA binding ability, governs the regulation of IGFBP3 by full-length p53 family proteins, as inhibition of histone deacetylases restores the induction of IGFBP3 by exogenous full-length p53, p63, and p73 proteins. Furthermore, we found that activation of p53 or inhibition of histone deacetylases alone was not sufficient to induce IGFBP3; however, combined treatment endowed endogenous p53 with this activity. To better understand the significance of this regulation, we performed a microarray study and identified several target genes differentially regulated by full-length p53 and p53 lacking the N-terminal activation domain 1 and the C-terminal basic domain. Taken together, our data suggest a novel mechanism by which p53 family proteins differentially regulate gene expression and provide an insight for designing a combined therapy for cancer treatment.     

Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail-chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-alpha are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2alpha, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.     

The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene

Bacterial microcompartments (MCPs) are complex organelles that consist of metabolic enzymes encapsulated within a protein shell. In this study, we investigate the function of the PduJ MCP shell protein. PduJ is 80% identical in amino acid sequence to PduA and both are major shell proteins of the 1,2‐propanediol (1,2‐PD) utilization (Pdu) MCP of Salmonella. Prior studies showed that PduA mediates the transport of 1,2‐PD (the substrate) into the Pdu MCP. Surprisingly, however, results presented here establish that PduJ has no role 1,2‐PD transport. The crystal structure revealed that PduJ was nearly identical to that of PduA and, hence, offered no explanation for their differential functions. Interestingly, however, when a pduJ gene was placed at the pduA chromosomal locus, the PduJ protein acquired a new function, the ability to mediate 1,2‐PD transport into the Pdu MCP. To our knowledge, these are the first studies to show that that gene location can determine the function of a MCP shell protein. We propose that gene location dictates protein‐protein interactions essential to the function of the MCP shell.     

The use of gene function to identify the rate-limiting steps controlling cell fate

The size and complexity of the genomes of mammals in general, and humans in particular, is such that it will take many years to utilise this information to produce a genuine understanding of the control of cell behaviour. Since there are tens of thousands of genes to consider, the task of identifying those which play the most significant roles, biologically and medically, is both crucial and very demanding. Here we emphasise the importance of functional approaches to answering this question, i.e. the application of techniques which use the function of the gene itself in identifying the critical rate-limiting steps in biological processes. In this review, we use the functional analysis of one of the most important of these processes, the control of survival and apoptosis, to illustrate the power of a number of functional genomic strategies.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

A T cell clone's avidity is a function of its activation state

At present it is unclear how Ag dose-dependent T cell functions, such as cytokine production, reflect TCR affinity and how the signal strength afforded by the Ag dose affects the kinetics of cytokine production by the individual T cell. We used a computer-assisted ELISPOT approach to address these issues. IFN-gamma release by a clonal population of CD4 T cells was monitored on a clonal population of APC while titrating the nominal peptide. The frequency of cytokine-producing cells, the net per-cell output of cytokine, and the onset of cytokine production were each found to be functions of the signal strength. Sigmoidal dose-response curves were seen at the clonal population level, but the activation thresholds for the individual T cells followed a Gaussian distribution. Moreover, the overall dose-response curve of the T cell clone revealed cyclic changes, becoming increasingly shifted toward lower Ag concentrations with the duration of time that elapsed since the last restimulation with Ag. Therefore, responsiveness to Ag ("functional avidity") is not a constant parameter of a T cell clone but a function of the T cell's history of last Ag encounter. The implications of such shifting activation thresholds are discussed for autoimmune disease.     

Sialylation is involved in cell fate decision during development,reprogramming and cancer progression

Sialylation, or the covalent addition of sialic acid to the terminal end of glycoproteins, is a biologically important modification that is involved in embryonic development, neurodevelopment, reprogramming, oncogenesis and immune responses. In this review, we have given a comprehensive overview of the current literature on the involvement of sialylation in cell fate decision during development, reprogramming and cancer progression. Sialylation is essential for early embryonic development and the deletion of UDP-GlcNAc 2-epimerase, a rate-limiting enzyme in sialic acid biosynthesis, is embryonically lethal. Furthermore, the sialyltransferase ST6GAL1 is required for somatic cell reprogramming, and its downregulation is associated with decreased reprogramming efficiency. In addition, sialylation levels and patterns are altered during cancer progression, indicating the potential of sialylated molecules as cancer biomarkers. Taken together, the current evidences demonstrate that sialylation is involved in crucial cell fate decision.     

Mechanical modulation of osteochondroprogenitor cell fate

Mesenchymal cells are natural tissue builders. They exhibit an extraordinary capacity to metamorphize into differentiated cells, using extrinsic spatial and temporal inputs and intrinsic algorithms, as well as to build and adapt their own habitat. In addition to providing a habitat for osteoprogenitor cells, tissues of the skeletal system provide mechanical support and protection for the multiple organs of vertebrate organisms. This review examines the role of mechanics on determination of cell fate during pre-, peri- and postnatal development of the skeleton as well as during tissue genesis and repair in postnatal life. The role of cell mechanics is examined and brought into context of intrinsic cues during mesenchymal condensation. Remarkable new insights regarding structure function relationships in mesenchymal stem cells, and their influence on determination of cell fate are integrated in the context of de novo tissue generation and postnatal repair. Key differences in the formation of osteogenic and chondrogenic condensations are discussed in relation to direct intramembranous and indirect endochondral ossification. New approaches are discussed to elucidate and exploit extrinsic cues to generate tissues in the laboratory and in the clinic.     

植物的次生生长及其分子调控

植物的次生生长是一重要的生命活动, 特别是植物次生木质部的形成对人类生产和生活具有重大意义。次生生长包括维管组织形成、次生细胞壁形成、木质化、PCD以及心材形成等过程。生长素和多个转录因子家族如MYB、NAC、AP2/EREBP、homeobox、MADS等参与了这一过程的调控, 利用分子生物学手段特别是基因组学方法, 人们已分离到生长素信号途径基因以及转录因子家族中的相关调控基因。文章介绍了植物次生生长的活动过程, 并论述了次生生长过程中调控基因的研究情况。