BMC Cell Biology最新文献

Background: Staufen2 (STAU2) is an RNA-binding protein involved in the post-transcriptional regulation of gene expression. This protein was shown to be required for organ formation and cell differentiation. Although STAU2 functions have been reported in neuronal cells, its role in dividing cells remains deeply uncharacterized. Especially, its regulation during the cell cycle is completely unknown.

Results: In this study, we showed that STAU2 isoforms display a mitosis-specific slow migration pattern on SDS-gels in all tested transformed and untransformed cell lines. Deeper analyses in hTert-RPE1 and HeLa cells further indicated that the slow migration pattern of STAU2 isoforms is due to phosphorylation. Time course studies showed that STAU2 phosphorylation occurs before prometaphase and terminates as cells exit mitosis. Interestingly, STAU2 isoforms were phosphorylated on several amino acid residues in the C-terminal half via the cyclin-dependent kinase 1 (Cdk1), an enzyme known to play crucial roles during mitosis. Introduction of phospho-mimetic or phospho-null mutations in STAU2 did not impair its RNA-binding capacity, its stability, its interaction with protein co-factors or its sub-cellular localization, suggesting that STAU2 phosphorylation in mitosis does not regulate these functions. Similarly, STAU2 phosphorylation is not likely to be crucial for cell cycle progression since expression of phosphorylation mutants in hTert-RPE1 cells did not impair cell proliferation.

Conclusions: Altogether, these results indicate that STAU2 isoforms are phosphorylated during mitosis and that the phosphorylation process involves Cdk1. The meaning of this post-translational modification is still elusive.

Background: Mesenchymal stem cells (MSCs) are multipotent stromal cells that have the ability to self-renew and migrate to sites of pathology. In vivo tracking of MSCs provides insights into both, the underlying mechanisms of MSC transformation and their potential as gene delivery vehicles. The aim of our study was to assess the ability of superparamagnetic iron oxide nanoparticles (SPIONs)-labeled Wharton's Jelly of the human umbilical cord-derived MSCs (WJ-MSCs) to carry the green fluorescent protein (GFP) gene to cutaneous injury sites in a murine model.

Methods: WJ-MSCs were isolated from a fresh umbilical cord and were genetically transformed to carry the GFP gene using lentiviral vectors with magnetically labeled SPIONs. The SPIONs/GFP-positive WJ-MSCs expressed multipotent cell markers and demonstrated the potential for osteogenic and adipogenic differentiation. Fifteen skin-injured mice were divided into three groups. Group I was treated with WJ-MSCs, group II with SPIONs/GFP-positive WJ-MSCs, and group III with SPIONs/GFP-positive WJ-MSCs exposed to an external magnetic field (EMF). Magnetic resonance imaging and optical molecular imaging were performed, and images were acquired 1, 2, and 7 days after cell injection.

Results: The results showed that GFP could be intensively detected around the wound in vivo 24 h after the cells were injected. Furthermore, we observed an accumulation of WJ-MSCs at the wound site, and EMF exposure increased the speed of cell transport. In conclusion, our study demonstrated that SPIONs/GFP function as cellular probes for monitoring in vivo migration and homing of WJ-MSCs. Moreover, exposure to an EMF can increase the transportation efficiency of SPIONs-labeled WJ-MSCs in vivo.

Conclusions: Our findings could lead to the development of a gene carrier system for the treatment of diseases.

Background: In Dictyostelium discoideum, vesicular transport of the adenylyl cyclase A (ACA) to the posterior of polarized cells is essential to relay exogenous 3',5'-cyclic adenosine monophosphate (cAMP) signals during chemotaxis and for the collective migration of cells in head-to-tail arrangements called streams.

Results: Using fluorescence in situ hybridization (FISH), we discovered that the ACA mRNA is asymmetrically distributed at the posterior of polarized cells. Using both standard estimators and Monte Carlo simulation methods, we found that the ACA mRNA enrichment depends on the position of the cell within a stream, with the posterior localization of ACA mRNA being strongest for cells at the end of a stream. By monitoring the recovery of ACA-YFP after cycloheximide (CHX) treatment, we observed that ACA mRNA and newly synthesized ACA-YFP first emerge as fluorescent punctae that later accumulate to the posterior of cells. We also found that the ACA mRNA localization requires 3' ACA cis-acting elements.

Conclusions: Together, our findings suggest that the asymmetric distribution of ACA mRNA allows the local translation and accumulation of ACA protein at the posterior of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signal during chemotaxis.

Background: Autophagy is an inducible autodigestive process that allows cells to recycle proteins and other materials for survival during stress and nutrient deprived conditions. The kinase ULK1 is required to activate this process. ULK1 phosphorylates a number of target proteins and regulates many cellular processes including the early secretory pathway. Recently, ULK1 has been demonstrated to phosphorylate Sec16 and affects the transport of serotonin transporter at the ER exit sites (ERES), but whether ULK1 may affect the transport of other cargo proteins and general secretion has not been fully addressed.

Results: In this study, we identified Sec23A, a component of the COPII vesicle coat, as a target of ULK1 phosphorylation. Elevated autophagy, induced by amino acid starvation, rapamycin, or overexpression of ULK1 caused aggregation of the ERES, a region of the ER dedicated for the budding of COPII vesicles. Transport of cargo proteins was also inhibited under these conditions and was retained at the ERES. ULK1 phosphorylation of Sec23A reduced the interaction between Sec23A and Sec31A. We identified serine 207, serine 312 and threonine 405 on Sec23A as ULK1 phosphorylation sites. Among these residues, serine 207, when changed to phospho-deficient and phospho-mimicking mutants, most faithfully recapitulated the above-mentioned effects of ULK1 phospho-regulation.

Conclusion: These findings identify Sec23A as a new target of ULK1 and uncover a mechanism of coordinating intracellular protein transport and autophagy.

Background: Biomaterials are widely used to regenerate or substitute bone tissue. In order to evaluate their potential use for clinical applications, these need to be tested and evaluated in vitro with cell culture models. Frequently, immortalized osteoblastic cell lines are used in these studies. However, their uncontrolled proliferation rate, phenotypic changes or aberrations in mitotic processes limits their use in long-term investigations. Recently, we described a new pluripotent-like subpopulation of dental pulp stem cells derived from the third molars (DPPSC) that shows genetic stability and shares some pluripotent characteristics with embryonic stem cells. In this study we aim to describe the use of DPPSC to test biomaterials, since we believe that the biomaterial cues will be more critical in order to enhance the differentiation of pluripotent stem cells.

Methods: The capacity of DPPSC to differentiate into osteogenic lineage was compared with human sarcoma osteogenic cell line (SAOS-2). Collagen and titanium were used to assess the cell behavior in commonly used biomaterials. The analyses were performed by flow cytometry, alkaline phosphatase and mineralization stains, RT-PCR, immunohistochemistry, scanning electron microscopy, Western blot and enzymatic activity. Moreover, the genetic stability was evaluated and compared before and after differentiation by short-comparative genomic hybridization (sCGH).

Results: DPPSC showed excellent differentiation into osteogenic lineages expressing bone-related markers similar to SAOS-2. When cells were cultured on biomaterials, DPPSC showed higher initial adhesion levels. Nevertheless, their osteogenic differentiation showed similar trend among both cell types. Interestingly, only DPPSC maintained a normal chromosomal dosage before and after differentiation on 2D monolayer and on biomaterials.

Conclusions: Taken together, these results promote the use of DPPSC as a new pluripotent-like cell model to evaluate the biocompatibility and the differentiation capacity of biomaterials used in bone regeneration.

Background: Obesity is associated with multiple diseases, but it is unclear how obesity promotes progressive tissue damage. Recovery from injury requires repair, an energy-expensive process that is coupled to energy availability at the cellular level. The satiety factor, leptin, is a key component of the sensor that matches cellular energy utilization to available energy supplies. Leptin deficiency signals energy depletion, whereas activating the Hedgehog pathway drives energy-consuming activities. Tissue repair is impaired in mice that are obese due to genetic leptin deficiency. Tissue repair is also blocked and obesity enhanced by inhibiting Hedgehog activity. We evaluated the hypothesis that loss of leptin silences Hedgehog signaling in pericytes, multipotent leptin-target cells that regulate a variety of responses that are often defective in obesity, including tissue repair and adipocyte differentiation.

Results: We found that pericytes from liver and white adipose tissue require leptin to maintain expression of the Hedgehog co-receptor, Smoothened, which controls the activities of Hedgehog-regulated Gli transcription factors that orchestrate gene expression programs that dictate pericyte fate. Smoothened suppression prevents liver pericytes from being reprogrammed into myofibroblasts, but stimulates adipose-derived pericytes to become white adipocytes. Progressive Hedgehog pathway decay promotes senescence in leptin-deficient liver pericytes, which, in turn, generate paracrine signals that cause neighboring hepatocytes to become fatty and less proliferative, enhancing vulnerability to liver damage.

Conclusions: Leptin-responsive pericytes evaluate energy availability to inform tissue construction by modulating Hedgehog pathway activity and thus, are at the root of progressive obesity-related tissue pathology. Leptin deficiency inhibits Hedgehog signaling in pericytes to trigger a pericytopathy that promotes both adiposity and obesity-related tissue damage.

Background: External root resorption, commonly starting from cementum, is a severe side effect of orthodontic treatment. In this pathological process and repairing course followed, cementoblasts play a significant role. Previous studies implicated that parathyroid hormone (PTH) could act on committed osteoblast precursors to promote differentiation, and inhibit apoptosis. But little was known about the role of PTH in cementoblasts. The purpose of this study was to investigate the effects of intermittent PTH on cementoblasts and its influence after mechanical strain treatment.

Results: Higher levels of cementogenesis- and differentiation-related biomarkers (bone sialoprotein (BSP), osteocalcin (OCN), Collagen type I (COL1) and Osterix (Osx)) were shown in 1-3 cycles of intermittent PTH treated groups than the control group. Additionally, intermittent PTH increased alkaline phosphatase (ALP) activity and mineralized nodules formation, as measured by ALP staining, quantitative ALP assay, Alizarin red S staining and quantitative calcium assay. The morphology of OCCM-30 cells changed after mechanical strain exertion. Expression of BSP, ALP, OCN, osteopontin (OPN) and Osx was restrained after 18 h mechanical strain. Furthermore, intermittent PTH significantly increased the expression of cementogenesis- and differentiation-related biomarkers in mechanical strain treated OCCM-30 cells.

Conclusions: Taken together, these data suggested that intermittent PTH promoted cementum formation through activating cementogenesis- and differentiation-related biomarkers, and attenuated the catabolic effects of mechanical strain in immortalized cementoblasts OCCM-30.

Background: Environmental stress can affect the viability or fecundity of an organism. Environmental stressors may affect the genome or the proteome and can cause cellular distress by contributing to protein damage or misfolding. This study examines the cellular response to environmental stress in the germline of the nematode, C. elegans.

Results: Salt stress, oxidative stress, and starvation, but not heat shock, induce the relocalization of ubiquitin, proteasome, and the TIAR-2 protein into distinct subnuclear regions referred to as stress induced nuclear granules (SINGs). The SINGs form within 1 h of stress initiation and do not require intertissue signaling. K48-linked polyubiquitin chains but not K63 chains are enriched in SINGs. Worms with a mutation in the conjugating enzyme, ubc-18, do not form SINGs. Additionally, knockdown of ubc-20 and ubc-22 reduces the level of SING formation as does knockdown of the ubiquitin ligase chn-1, a CHIP homolog. The nuclear import machinery is required for SING formation. Stressed embryos containing SINGs fail to hatch and cell division in these embryos is halted. The formation of SINGs can be prevented by pre-exposure to a brief period of heat shock before stress exposure. Heat shock inhibition of SINGs is dependent upon the HSF-1 transcription factor.

Conclusions: The heat shock results suggest that chaperone expression can prevent SING formation and that the accumulation of damaged or misfolded proteins is a necessary precursor to SING formation. Thus, SINGs may be part of a novel protein quality control system. The data suggest an interesting model where SINGs represent sites of localized protein degradation for nuclear or cytosolic proteins. Thus, the physiological impacts of environmental stress may begin at the cellular level with the formation of stress induced nuclear granules.

Background: The murine double minute 2 (MDM2) is an oncogene and a negative regulator of the tumor suppressor protein p53. MDM2 is known to be amplified in numerous human cancers, and upregulation of MDM2 is considered to be an alternative mechanism of p53 inactivation. The presence of many splice variants of MDM2 has been observed in both normal tissues and malignant cells; however their impact and functional properties in response to chemotherapy treatment are not fully understood. Here, we investigate the biological effects of three widely expressed alternatively spliced variants of MDM2; MDM2-A, MDM2-B and MDM2-C, both in unstressed MCF-7 breast cancer cells and in cells subjected to chemotherapy. We assessed protein stability, subcellular localization and induction of downstream genes known to be regulated by the MDM2-network, as well as impact on cellular endpoints, such as apoptosis, cell cycle arrest and senescence.

Results: We found both the splice variants MDM2-B and -C, to have a much longer half-life than MDM2 full-length (FL) protein after chemotherapy treatment indicating that, under stressed conditions, the regulation of degradation of these two variants differs from that of MDM2-FL. Interestingly, we observed all three splice variants to deviate from MDM2-FL protein with respect to subcellular distribution. Furthermore, while MDM2-A and -B induced the expression of the pro-apoptotic gene PUMA, this effect did not manifest in an increased level of apoptosis.

Conclusion: Although MDM2-B induced slight changes in the cell cycle profile, overall, we found the impact of the three MDM2 splice variants on potential cellular endpoints upon doxorubicin treatment to be limited.

Background: In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure.

Results: The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits.

Conclusions: We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.