Dynamic interaction of NtMAP65-1a with microtubules in vivo

Plant microtubules are intrinsically more dynamic than those from animals. We know little about the dynamics of the interaction of plant microtubule-associated proteins (MAPs) with microtubules. Here, we have used tobacco and Arabidopsis MAPs with relative molecular mass 65 kDa (NtMAP65-1a and AtMAP65-1), to study their interaction with microtubules in vivo. Using fluorescence recovery after photobleaching we report that the turnover of both NtMAP65-1a and AtMAP65-1 bound to microtubules is four- to fivefold faster than microtubule treadmilling (13 seconds compared with 56 seconds, respectively) and that the replacement of NtMAP65-1a on microtubules is by random association rather than by translocation along microtubules. MAP65 will only bind polymerised microtubules and not its component tubulin dimers. The turnover of NtMAP65-1a and AtMAP65-1 on microtubules is similar in the interphase cortical array, the preprophase band and the phragmoplast, strongly suggesting that their role in these arrays is the same. NtMAP65-1a and AtMAP65-1 are not observed to bind microtubules in the metaphase spindle and their rate of recovery is consistent with their cytoplasmic localisation. In addition, the dramatic reappearance of NtMAP65-1a on microtubules at the spindle midzone in anaphase B suggests that NtMAP65-1a is controlled post-translationally. We conclude that the dynamic properties of these MAPs in vivo taken together with the fact that they have been shown not to effect microtubule polymerisation in vitro, makes them ideally suited to a role in crossbridging microtubules that need to retain spatial organisation in rapidly reorganising microtubule arrays.

The Arabidopsis microtubule-associated protein AtMAP65-1: Molecular analysis of its microtubule bundling activity

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Investigations of DNA damage induction and repair resulting from cellular exposure to high dose-rate pulsed proton beams

Studies regarding the radiobiological effects of low dose radiation, microbeam irradiation services have been developed in the world and today laser acceleration of protons and heavy ions may be used in radiation therapy. The application of different facilities is essential for studying bystander effects and relating signalling phenomena in different cells or tissues. In particular the use of ion beams results advantageous in cancer radiotherapy compared to more commonly used X-rays, since the ability of ions in delivering lethal amount of doses into the target tumour avoiding or limiting damage to the contiguous healthy tissues. At the INFN-LNS in Catania, a multidisciplinary radiobiology group is strategically structured aimed to develop radiobiological research, finalised to therapeutic applications, compatible with the use of high dose laser-driven ion beams. The characteristic non-continuous dose rates with several orders of magnitude of laser-driven ion beams makes this facility very interesting in the cellular systems' response to ultra-high dose rates with non-conventional pulse time intervals cellular studies. Our group have projected to examine the effect of high dose laser-driven ion beams on two cellular types: foetal fibroblasts (normal control cells) and DU145 (prostate cancer cells), studying the modulation of some different bio-molecular parameters, in particular cell proliferation and viability, DNA damage, redox cellular status, morphological alterations of both the cytoskeleton components and some cell organelles and the possible presence of apoptotic or necrotic cell death. Our group performed preliminary experiments with high energy (60 MeV), dose rate of 10 Gy/min, doses of 1, 2, 3 Gy and LET 1 keV/µm on human foetal fibroblasts (control cells). We observed that cell viability was not influenced by the characteristics of the beam, the irradiation conditions or the analysis time. Conversely, DNA damage was present at time 0, immediately following irradiation in a dose-dependent manner. The analysis of repair capability showed that the cells irradiated with 1 and 2 Gy almost completely recovered from the damage, but not, however, 3 Gy treated cells in which DNA damage was not recovered. In addition, the results indicate the importance of the use of an appropriate control in radiobiological in vitro analysis.

The role of SRC-CAS interactions in cellular transformation:ectopic expression of the carboxy terminus of CAS inhibits SRC-CAS interaction but has no effect on cellular transformation

Several lines of evidence indicate that the adapter molecule p130CAS (crk-associated substrate (CAS)) is required for src-mediated cellular transformation. CAS has been shown to be heavily tyrosine-phosphorylated in src-transformed cells, and genetic variants of src that are deficient in CAS binding are also unable to mediate cellular transformation. In this report, we investigated whether CAS phosphorylation and/or its association with src are required elements of the transformation process. Expression of the carboxy-terminal src binding domain of CAS in Rat 1 fibroblasts expressing a temperature-sensitive allele of v-src inhibited the formation of src-CAS complexes and also inhibited tyrosine phosphorylation of CAS. However, expression of this protein had no effect on morphological transformation, src-mediated actin rearrangements, or anchorage-independent growth of these cells when grown at the src-permissive temperature. Thus, the ability of activated src to mediate cellular transformation is either largely independent of endogenous CAS phosphorylation and/or its association with CAS or, alternatively, the carboxy-terminus of CAS may substitute for endogenous CAS in the process of src-mediated transformation.

Histone deacetylase 3 unconventional splicing mediates endothelial-to- mesenchymal transition through transforming growth factor β2

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -β, -γ, and -Δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor β2 (TGFβ2) secretion and cleavage. TGFβ2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFβ2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.

Regulation of TGF-β1-Driven Differentiation of Human Lung Fibroblasts: Emerging Roles of Cathepsin B and Cystatin C

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF) and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover addition of Cat B generated 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation, but had no effect on p38 MAPK and JNK phosphorylation indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. While mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of IPF and CCD-19Lu fibroblasts. In addition cystatin C participated in the control of extracellular Cats, since its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.

Identification of serum stress biomarkers in pigs housed at different stocking densities

Eight Duroc × (Landrace × Large White) male pigs housed at a stocking rate of 0.50 m2/pig were subjected to a higher stocking rate of 0.25 m2/pig (higher density, HD) for two 4-day periods over 26 days. Using biochemical and proteomic techniques serum and plasma samples were examined to identify potential biomarkers for monitoring stress due to HD housing. HD housed pigs showed significant differences (P < 0.001) in total cholesterol and low density lipoprotein-associated cholesterol, as well as in concentrations of the pig-major acute phase protein (Pig-MAP) (P = 0.002). No differences were observed in serum cortisol or other acute phase proteins such as haptoglobin, C-reactive protein or apolipoprotein A–I. HD-individuals also showed an imbalance in redox homeostasis, detected as an increase in the level of oxidized proteins measured as the total plasma carbonyl protein content (P < 0.001) with a compensatory increase in the activity of the antioxidant enzyme glutathione peroxidase (P = 0.012). Comparison of the serum proteome yielded a new potential stress biomarker, identified as actin by mass spectrometry. Cluster analysis of the results indicated that individuals segregated into two groups, with different response patterns, suggesting that the stress response depended on individual susceptibility.

A coiled-coil-repeat protein 'Ccrp' in Bdellovibrio bacteriovorus prevents cellular indentation, but is not essential for vibroid cell morphology

Bdellovibrio bacteriovorus are small, vibroid, predatory bacteria that grow within the periplasmic space of a host Gram-negative bacterium. The intermediate-filament (IF)-like protein crescentin is a member of a broad class of IF-like, coiled-coil-repeat-proteins (CCRPs), discovered in Caulobacter crescentus, where it contributes to the vibroid cell shape. The B. bacteriovorus genome has a single ccrp gene encoding a protein with an unusually long, stutter-free, coiled-coil prediction; the inactivation of this did not alter the vibriod cell shape, but caused cell deformations, visualized as chiselled insets or dents, near the cell poles and a general 'creased' appearance, under the negative staining preparation used for electron microscopy, but not in unstained, frozen, hydrated cells. Bdellovibrio bacteriovorus expressing 'teal' fluorescent protein (mTFP), as a C-terminal tag on the wild-type Ccrp protein, did not deform under negative staining, suggesting that the function was not impaired. Localization of fluorescent Ccrp-mTFP showed some bias to the cell poles, independent of the cytoskeleton, as demonstrated by the addition of the MreB-specific inhibitor A22. We suggest that the Ccrp protein in B. bacteriovorus contributes as an underlying scaffold, similar to that described for the CCRP protein FilP in Streptomyces coelicolor, preventing cellular indentation, but not contributing to the vibroid shape of the B. bacteriovorus cells.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of PI 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella containing vacuolae (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not colocalize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the downregulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation which may contribute to Klebsiella pathogenesis.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Clathrin is spindle-associated but not essential for mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.

Low-grade malignant Triton tumor of the oral cavity:a case report

Malignant Triton tumor (MTT) is a malignant peripheral nerve sheath tumor showing rhabdomyoblastic differentiation. It is considered a high-grade neoplasm with poor outcome. This report describes an MTT appearing in the oral cavity. On histologic examination the encapsulated lesion was composed of interlacing fascicles of spindle cells and scattered, large, strap-like pleomorphic cells with abundant eosinophilic cytoplasm. No cross striations were seen. Examination of levels through the tissue showed a total of only 4 normal mitoses and no necrosis. Immunohistochemistry demonstrated diffuse S100 positivity in the spindle cells. The large pleomorphic cells were weakly positive for alpha-sarcomeric actin and myoglobin, although variably but strongly positive for desmin. Management involved a small en bloc resection of the maxilla. After 33 months there was no sign of recurrence or distant metastasis. It was concluded that low-grade variants of MTT occur that do not have an aggressive clinical course.

Reference gene selection in human periodontal ligament cells

Background: Real-time quantitative PCR (qPCR) is a highly sensitive and specific method which is used extensively for determining gene expression profiles in a variety of cell and tissue types. In order to obtain accurate and reliable gene expression quantification, qPCR data are generally normalised against so-called reference or housekeeping genes. Ideally, reference genes should have abundant and stable RNA transcriptomes under the experimental conditions employed. However, reference genes are often selected rather arbitrarily and indeed some have been shown to have variable expression in a variety of in vitro experimental conditions.
Objective: The objective of the current study was to investigate reference gene expression in human periodontal ligament (PDL) cells in response to treatment with lipopolysaccharide (LPS).
Method: Primary human PDL cells were grown in Dulbecco’s Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum, 100UI/ml penicillin and 100µg/ml streptomycin. RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The expression of a total of 19 reference genes was studied in the presence and absence of LPS treatment using the Roche Reference Gene Panel. Data were analysed using NormFinder and Bestkeeper validation programs.
Results: Treatment of human PDL cells with LPS resulted in changes in expression of several commonly used reference genes, including GAPDH. On the other hand the reference genes β-actin, G6PDH and 18S were identified as stable genes following LPS treatment.
Conclusion: Many of the reference genes studied were robust to LPS treatment (up to 100 ng/ml). However several commonly employed reference genes, including GAPDH varied with LPS treatment, suggesting they would not be ideal candidates for normalisation in qPCR gene expression studies.

A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome

Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.