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The number of every enzyme is the result of many mechanisms of regulation, at transcriptional and translational levels. We asked if the presence of rare amino acids in the sequence of an enzyme could represent a limiting factor in the balance between enzymes synthesis and enzymes efficiency. To try to find a correlation we analyzed the amino acids sequences of different housekeeping and metabolic enzymes.
Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far tFTs were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing greenFPs, while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT towards slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for design of new tFTs.\n\nAbbreviationstFT - tandem fluorescent protein timer\n\nFP - fluorescent protein\n\ngreenFP - green fluorescent protein\n\nredFP - red fluorescent protein
A well-known DNA-damaging agent and carcinogen, ionizing radiation (IR) can also exert detrimental effects in cells not directly exposed to it, through \"non-targeted effects\" (NTE). Whilst NTE are known to contribute to radiation-induced damage, their mechanism of induction and propagation remains incompletely understood. To investigate the possible role of lysosomes, key subcellular organelles, in NTE we used acridine orange uptake and relocation methods to monitor lysosomal permeability in irradiated and bystander human fibroblasts. As a potential mediator of lysosomal changes, oxidative stress was measured using the H2DCFDA assay for total reactive oxygen species (ROS). IR was found to induce significant lysosomal permeability in the first hour post irradiation, with reduced permeability persisting up to 24 hours. This occurred in conjunction with an increase in ROS in directly irradiated cells, in contrast with a decrease in ROS in bystander cells. Based on these observations we constructed a simple mathematical model of ROS-induced lysosomal damage, based on a bistable mechanism where a sufficiently strong IR insult can shift a cell from a low ROS, high lysosome to a high ROS, low lysosome state. This has profound cellular implications in radiation response and advances our understanding for the sub-cellular involvement in non-targeted effects.
The environmental effects of ischemia on chromatin nanostructure were evaluated using single molecule localisation microscopy (SMLM) of DNA binding dyes. Short-term oxygen and nutrient deprivation (OND) of the cardiomyocyte cell-line HL-1induces a previously undescribed chromatin architecture, consisting of large, chromatin sparse voids interspersed between DNA-dense hollow helicoid structures of the order of 40 to 700 nm in dimension. OND induced chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a significantly more open structure than in untreated cells. We show that this compacted state of chromatin reduces transcription, while the open chromatin structure following recovery has a higher transcriptional rate than in untreated cells. Digestion of chromatin with DNAseI and DNA binding dye loading assays confirm that OND induces compaction of chromatin and a general redistribution of chromatin to the nuclear periphery. Mechanistically, chromatin compaction is associated with a depletion of intracellular ATP and a redistribution of the cellular polyamine pool into the nucleus. Additionally, Fluorescence Recovery After Photobleaching (FRAP) shows that core histones are not displaced from compacted chromatin and that the mobility of linker histone H1 is considerably reduced by OND treatment, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin. These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.
Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N=101,989), region (N=697), \"block\" (N=243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts -83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation - while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p=0.71). These results depict a strong component of epigenetic memory in cell culture from primary tissue, even after several generations of daughter cells, related to cell state and donor age.
In 2014, it was reported that strong external stimuli, such as a transient low-pH stressor, was capable of inducing the reprogramming of mammalian somatic cells, resulting in the generation of pluripotent cells (Obokata et al. 2014a, b). This cellular reprograming event was designated stimulus-triggered acquisition of pluripotency (STAP) by the authors of these reports. However, after multiple instances of scientific misconduct in the handling and presentation of the data were brought to light, both reports were retracted. To investigate the actual scientific significance of the purported STAP phenomenon, we sought to repeat the original experiments based on the methods presented in the retracted manuscripts and other relevant information. As a result, we have confirmed that the STAP phenomenon as described in the original studies is not reproducible.
This reports the results of an attempt by Haruko Obokata to replicate the phenomenon of stimulus-triggered acquisition of pluripotency (STAP), which was first reported in a pair of papers authored by Obokata and colleagues in 2014. The most conclusive evidence for the pluripotency of so-called STAP cells was their purported ability to contribute to chimera formation. In the follow-up trial presented here, putative STAP cells prepared by Obokata were injected into 1154 embryos, of which 671 were recovered. However, the injected cells made no significant contribution in any tissue in any of the embryos developed.
BackgroundThe endoplasmic reticulum (ER) is the site of folding for membrane and secreted proteins. Accumulation of unfolded or misfolded proteins in the ER triggers the unfolded protein response (UPR). The UPR can promote survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. However, when ER stress is acute or prolonged cells undergo apoptosis. In this study we sought to determine the effect of globally compromised microRNA biogenesis on the UPR and ER stress-induced apoptosis\n\nResultsHere we report the role of Dicer-dependent miRNA biogenesis during the UPR and ER stress-induced apoptosis. We show that ER stress-induced caspase activation and apoptosis is attenuated in Dicer deficient fibroblasts. ER stress-mediated induction of GRP78, the key ER resident chaperone, and also HERP, an important component of ER-associated degradation, are significantly increased in Dicer deficient cells. Expression of the BCL-2 family members BIM and MCL1 were significantly higher in Dicer-null fibroblasts. However, ER stress-mediated induction of pro-apoptotic BH3 only protein BIM was compromised in Dicer mutant cells.\n\nConclusionsThese observations demonstrate key roles for Dicer in the UPR and implicate miRNAs as critical components of UPR.
A mechanism for polarized transport of vesicles by means of osmotic propulsions is proposed and substantiated for tip-growing cells. An analysis is presented which shows that in pollen tubes the gradient of cytosolic water potential can drive vesicle movement either in the anterograde or retrograde direction, depending on the vesicle position, its radius and the phase of growth oscillation. The importance of transcellular water flow for cytoskeletal dynamics and cell motility is highlighted.
The article by Briley et al is the latest of several papers based on the use of oligonucleotide-capped gold nanoparticles (or Spherical Nucleic Acids, SNAs) to detect mRNA within cells (1). The technology was developed, published and patented by Prof Chad Mirkin and is commercialised under the trade name SmartFlare. Briefly, the SNAs are hybridised with fluorescent reporter strands (resulting in quenching of the fluorescence by the gold particles) and internalised into cells by endocytosis. The target RNA binds either with the SNA, releasing the reporter strand (SmartFlare) or, in the latest study, with the reporter strand itself, releasing the reporter-strand/target-RNA duplex (Sticky-flares).\n\nSince they enter cells by endocytosis, Sticky-flares (and SmartFlares) need to escape those compartments before they can report on mRNA levels in the cytosol. This essential step is currently not documented in the literature. To the contrary, we (2) ...
Successful segregation of chromosomes during mitosis requires that each sister chromatid is captured by microtubules emanating from opposite spindle poles. A multiprotein complex called the kinetochore provides an attachment site on chromosomes for microtubules. We have found that the prolylhydroxylase PHD2 is a critical regulator of the assembly of the kinetochore. PHD2 hydroxylates the kinetochore component CENP-N on P311 and is essential for CENP-N localization to kinetochores. Either depletion of PHD2, or expression of a hydroxylation-deficient mutant, results in loss of the histone H3 variant CENP-A from centromeres. Loss of CENP-N from chromatin bound protein complexes is not due to decreased protein stability but is a consequence of lowered affinity of CENP-N for binding CENP-L. Loss of hydroxylation also results in increased targeting of CENP-L to chromatin. Hydroxylation by PHD2 thus plays an important role in controlling the stoichiometry of mitotic kinetochore components.
The assembly of the first centrosome occurs upon fertilisation when the male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. While inaccuracy in the assembly of centrosomes leads to infertility and abnormal embryogenesis, the mechanism that ensures accurate assembly in vertebrate embryos remains obscure. Here we applied a CRISPR-Cas9-mediated gene knockout to show that Wdr8, a novel centrosomal protein, is maternally essential for PCM assembly during embryonic mitoses of medaka (Oryzias latipes). Maternal/zygotic Wdr8-null (Wdr8-/-) blastomeres exhibit severe defects in PCM assembly that cause them to divide asymmetrically and develop multipolar mitotic spindles and aneuploidy. We demonstrate that Wdr8 interacts via its WD40 domains with the centriolar satellite protein SSX2IP. Strikingly, exogenously provided Wdr8 fully rescues Wdr8-/- embryos to adulthood, except in variants with mutations in the WD40 domains. This combination of targeted gene inactivation and in vivo reconstitution of the maternally essential Wdr8-SSX2IP complex reveals an essential link between maternal PCM and the stability of the zygotic genome in the early vertebrate embryo.
Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast to this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we find that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport where peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.
Genomic variation across cancers scales with tissue stiffness: meta-analyses show tumors in stiff tissues such as lung and bone exhibit up to 100-fold more variation than tumors in soft tissues such as marrow and brain. Here, nuclear lamina damage and DNA double-strand breaks (DSBs) result from invasive migration of cancer cells through stiff constrictions. DSBs increase with lamin-A knockdown and require micro-pores sufficiently small for lamins to impede migration. Blebs in the vast majority of post-migration nuclei are enriched in lamin-A but deficient in lamin-B and an age-associated form of lamin-A. Validation of DSBs by an electrophoretic comet assay calibrates against a cancer line having nuclease sites engineered in chromosome-1, and DSB-bound repair factors in nuclei pulled into constrictions show folded chromatin orients, extends, and concentrates without fragmentation. Mobile repair proteins simultaneously segregate away from pore-condensed chromatin. Global squeeze-out of repair factors and loss with lamin-A-dependent rupture explains why overexpression of repair factors cannot rescue DSBs in migration through stiff constrictions, ultimately favoring genomic variation.
The transmembrane semaphorin, Sema6A, has important roles in axon guidance, cell migration and neuronal connectivity in multiple regions of the nervous system, mediated by context-dependent interactions with plexin receptors, PlxnA2 and PlxnA4. Here, we demonstrate that Sema6A can also signal cell-autonomously, in two modes, constitutively, or in response to higher-order clustering mediated by either PlxnA2-binding or chemically induced multimerisation. Sema6A activation stimulates recruitment of Abl to the cytoplasmic domain of Sema6A and phosphorylation of this cytoplasmic tyrosine kinase, as well as phosphorylation of additional cytoskeletal regulators. Sema6A reverse signaling affects the surface area and cellular complexity of non-neuronal cells and aggregation and neurite formation of primary neurons in vitro. Sema6A also interacts with PlxnA2 in cis, which reduces binding by PlxnA2 of Sema6A in trans but not vice versa. These experiments reveal the complex nature of Sema6A biochemical functions and the molecular logic of the context-dependent interactions between Sema6A and PlxnA2.
Sickle Cell Disease (SCD) is a serious recessive genetic disorder caused by a single nucleotide polymorphism (SNP) in the {beta}-globin gene (HBB). Sickle hemoglobin polymerizes within red blood cells (RBCs), causing them to adopt an elongated \"sickle\" shape. Sickle RBCs damage vasculature, leading to severe symptoms, ultimately diminishing patient quality of life and reducing lifespan. Here, we use codelivery of a pre-formed Cas9 ribonucleoprotein complex (RNP) and a singlestranded DNA (ssDNA) oligonucleotide donor to drive sequence replacement at the SCD SNP in human CD34+ hematopoietic stem/progenitor cells (HSPCs). Corrected HSPCs from SCD patients produce less sickle hemoglobin protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When injected into immunocompromised mice, treated HSPCs maintain editing long-term at therapeutically relevant levels. These results demonstrate that the Cas9 RNP/ssDNA donor approach can mediate efficient HSPC gene editing and could form the basis for treatment of SCD by autologous hematopoietic cell transplantation.
Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.
Mechanical cell forces play determinant roles in cell growth and tissue development. Cell migration is a relevant context to explore single cell forces, since mechanotransduction and cell morphology are dynamically and biochemically co-regulated in order to modulate cell shape changes minute to minute. In this work we present the design of a hybrid microdevice made from a set of parallel PDMS microchannels in which oil emulsion droplets are introduced to be used as biocompatible and deformable cell forces transducers upon HL-60 human leukemia cell line migration. We show that the mechanical stress exerted by the cells can be measured from the analysis of the droplets deformations at the single cell level. Finally we show that acto-myosin contraction plays a role in the cell ability to cross obstacles in confinement conditions.