Repression of GW/P body components and the RNAi microprocessor impacts primary ciliogenesis in human astrocytes

<p>Abstract</p> <p>Background</p> <p>In most cells, the centriolar component of the centrosome can function as a basal body supporting the formation of a primary cilium, a non-motile sensory organelle that monitors information from the extracellular matrix and relays stimuli into the cell via associated signaling pathways. Defects in the formation and function of primary cilia underlie multiple human diseases and are hallmarks of malignancy. The RNA silencing pathway is involved in the post-transcriptional silencing of > 50% of mRNA that occurs within GW/P bodies. GW/P bodies are found throughout the cytoplasm and previously published live cell imaging data suggested that in a malignant cell type (U2OS), two GW/P bodies reside at the centrosome during interphase. This led us to investigate if a similar relationship exists in primary cells and if the inhibition of the miRNA pathway impairs primary cilium formation.</p> <p>Results</p> <p>Two GW/P bodies as marked by GW182 and hAgo2 colocalized to the basal body of primary human astrocytes as well as human synoviocytes during interphase and specifically with the distal end of the basal body in the pericentriolar region. Since it is technically challenging to examine the two centrosomal GW/P bodies in isolation, we investigated the potential relationship between the global population of GW/P bodies and primary ciliogenesis. Astrocytes were transfected with siRNA directed to GW182 and hAgo2 and unlike control astrocytes, a primary cilium was no longer associated with the centrosome as detected in indirect immunofluorescence assays. Ultrastructural analysis of siRNA transfected astrocytes revealed that knock down of GW182, hAgo2, Drosha and DGCR8 mRNA did not affect the appearance of the earliest stage of ciliogenesis but did prevent the formation and elongation of the ciliary axoneme.</p> <p>Conclusions</p> <p>This study confirms and extends a previously published report that GW/P bodies reside at the centrosome in U2OS cells and documents that GW/P bodies are resident at the centrosome in diverse non-malignant cells. Further, our study demonstrates that repression of key effector proteins in the post-transcriptional miRNA pathway impairs primary cilium formation.</p

Mapping the assembly pathways that specify formation of the trilaminar kinetochore plates in human cells

We report the interactions amongst 20 proteins that specify their assembly to the centromere–kinetochore complex in human cells. Centromere protein (CENP)-A is at the top of a hierarchy that directs three major pathways, which are specified by CENP-C, -I, and Aurora B. Each pathway consists of branches that intersect to form nodes that may coordinate the assembly process. Complementary EM studies found that the formation of kinetochore trilaminar plates depends on the CENP-I/NUF2 branch, whereas CENP-C and Aurora B affect the size, shape, and structural integrity of the plates. We found that hMis12 is not constitutively localized at kinetochores, and that it is not essential for recruiting CENP-I. Our studies also revealed that kinetochores in HeLa cells contain an excess of CENP-A, of which ∼10% is sufficient to promote the assembly of normal levels of kinetochore proteins. We elaborate on a previous model that suggested kinetochores are assembled from repetitive modules (Zinkowski, R.P., J. Meyne, and B.R. Brinkley. 1991. J. Cell Biol. 113:1091–110)

Dose Dependent Effects on Cell Cycle Checkpoints and DNA Repair by Bendamustine

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 µM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway

Spectrum of centrosome autoantibodies in childhood varicella and post-varicella acute cerebellar ataxia

BACKGROUND: Sera from children with post-varicella infections have autoantibodies that react with centrosomes in brain and tissue culture cells. We investigated the sera of children with infections and post-varicella ataxia and related conditions for reactivity to five recombinant centrosome proteins: γγ-enolase, pericentrin, ninein, PCM-1, and Mob1. METHODS: Sera from 12 patients with acute post-varicella ataxia, 1 with post-Epstein Barr virus (EBV) ataxia, 5 with uncomplicated varicella infections, and other conditions were tested for reactivity to cryopreserved cerebellum tissue and recombinant centrosome proteins. The distribution of pericentrin in the cerebellum was studied by indirect immunofluorescence (IIF) using rabbit antibodies to the recombinant protein. Antibodies to phospholipids (APL) were detected by ELISA. RESULTS: Eleven of 12 children with post-varicella ataxia, 4/5 children with uncomplicated varicella infections, 1/1 with post-EBV ataxia, 2/2 with ADEM, 1/2 with neuroblastoma and ataxia, and 2/2 with cerebellitis had antibodies directed against 1 or more recombinant centrosome antigens. Antibodies to pericentrin were seen in 5/12 children with post-varicella ataxia but not in any of the other sera tested. IIF demonstrated that pericentrin is located in axons and centrosomes of cerebellar cells. APL were detected in 75% of the sera from children with post-varicella ataxia and 50% of children with varicella without ataxia and in none of the controls. CONCLUSION: This is the first study to show the antigen specificity of anti-centrosome antibodies in children with varicella. Our data suggest that children with post-varicella ataxia have unique autoantibody reactivity to pericentrin

Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells

<p>Abstract</p> <p>Background</p> <p>Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines.</p> <p>Methods</p> <p>Cultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis.</p> <p>Results</p> <p>We show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis.</p> <p>Conclusions</p> <p>The recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first evidence that altered primary cilium expression and function may be part of some malignant phenotypes. Further, we provide the first evidence that ciliogenesis is not an all or none process; rather defects can arrest this process at various points, particularly at the stage subsequent to basal body association with the plasma membrane.</p

Regional variations in the cellular matrix of the annulus fibrosus of the intervertebral disc

The three-dimensional architecture of cells in the annulus fibrosus was studied by a systematic, histological examination using antibodies to cytoskeletal components, in conjunction with confocal microscopy. Variations in cell shape, arrangement of cellular processes and cytoskeletal architecture were found both within and between the defined zones of the outer and innerannulus. The morphology of three, novel annulus fibrosus cells is described: extended cordlikecells that form an interconnected network at the periphery of the disc; cells with extensive, sinuous processes in the inner region of the annulus fibrosus; and cells with broad, branching processes specific to the interlamellar septae of the outer annulus. The complex, yet seemingly deliberate arrangement of various cell shapes and their processes suggests multiple functional roles. Regional variations in the organization of the actin and vimentin cytoskeletal networks is reported across all regions of the annulus. Most notable is the continuous, strand arrangement of the actin label at the disc's periphery in contrast to its punctate appearance in all other regions. The gap junction protein connexin 43 was found within cells from all regionsof the annulus, including those which did not form physical connections with surrounding cells. These observations of the cellular matrix in the healthy intervertebral disc should contribute to a better understanding of site-specific changes in tissue architecture, biochemistry andmechanical properties during degeneration, injury and healing

The cellular networks of normal ovine medial collateral and anterior cruciate ligaments are not accurately recapitulated in scar tissue

The purpose of this study was to characterize the cellular organization of the ovine medial collateral ligament (MCL) and anterior cruciate ligament (ACL) and compare this organization with that found in ligaments undergoing healing. Indirect immunofluorescence microscopy, used in combination with antibodies to cytoskeletal proteins, was employed to visualize individual ligament cells. Normal ligaments contained fusiform cells arranged in rows, which were stacked at regular intervals across the body of the ligament forming a three-dimensional cellular lattice. Each cell exhibited prominent cytoplasmic processes that extended for long distances through the extracellular matrix to adjacent cells, and these processes contained gap junctions. Thus the cells in rows and between rows were interconnected. The cells of the MCL and ACL scars were also arranged in rows, but these rows were shorter, irregularly arranged and closely packed into bundles resulting in tissue with a higher cellular density. In addition, cells transiting the cell cycle were detected in the scar but not in normal ligament. While the rows of cells in the normal ligament extended along the long axis of the ligament, the bundles of rows of ligament scar cells had a random orientation with respect to one another and to the region outside the scar. Over time both the ACL and the MCL scars displayed discontinuities in their cellular rows. In contrast to the scars of the MCL, which contained discontinuities filled with cellular projections and gap junctions, ACL scars contained discontinuities that were devoid of cells and gap junctions. These discontinuities as well as the differences between normal and scar cytoarchitecture may represent features of an inadequate healing response and/or may provide the structural basis for the altered biomechanics of healing ligaments