Jerome B. Rattner

Primary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors.

The collagenous structure of menisci is a complex network of circumferentially oriented fascicles and interwoven radially oriented tie-fibres. To date, examination of this micro- architecture has been limited to two-dimensional imaging techniques. The purpose of this study was to evaluate the ability of the three-dimensional imaging technique; optical projection tomography (OPT), to visualize the collagenous structure of the meniscus. If successful, this technique would be the first to visualize the macroscopic orientation of collagen fascicles in 3-D in the meniscus and could further refine load bearing mechanisms in the tissue. OPT is an imaging technique capable of imaging samples on the meso-scale (1-10 mm) at a micro-scale resolution. The technique, similar to computed tomography, takes two-dimensional images of objects from incremental angles around the object and reconstructs them using a back projection algorithm to determine three-dimensional structure.

DNA damaging agents, including those used in the clinic, activate cell cycle checkpoints, which blocks entry into mitosis. Given that checkpoint override results in cell death via mitotic catastrophe, inhibitors of the DNA damage checkpoint are actively being pursued as chemosensitization agents. Here we explored the effects of gemcitabine in combination with Chk1 inhibitors in a panel of pancreatic cancer cell lines and found variable abilities to override the S phase checkpoint. In cells that were able to enter mitosis, the chromatin was extensively fragmented, as assessed by metaphase spreads and Comet assay. Notably, electron microscopy and high-resolution light microscopy showed that the kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). Cell lines that were unable to override the S phase checkpoint were able to override a G2 arrest induced by the alkylator MMS or the topoisomerase II inhibitors doxorubicin or etoposide. Interestingly, checkpoint override from the topoisomerase II inhibitors generated fragmented kinetochores (MUGs) due to unreplicated centromeres. Our studies show that kinetochore and centromere fragmentation is a defining feature of checkpoint override and suggests that loss of cell viability is due in part to acentric genomes. Furthermore, given the greater efficacy of forcing cells into premature mitosis from topoisomerase II-mediated arrest as compared with gemcitabine-mediated arrest, topoisomerase II inhibitors maybe more suitable when used in combination with checkpoint inhibitors.

Background: Osteoarthritis (OA) and Rheumatoid arthritis (RA) are diseases which result in the degeneration of the joint surface articular cartilage. Matrix Metalloproteinases (MMPs) are enzymes that aid in the natural remodelling of tissues throughout the body including cartilage. However, some MMPs have been implicated in the progression of OA and RA as their expression levels and activation states can change dramatically with the onset of disease. Yet, it remains unknown if normal and arthritic joints demonstrate unique MMPs expression profiles, and if so, can the MMP expression profile be used to identify patients with early OA. In this study, the synovial fluid protein expression levels for MMPs 1, 2, 3, 7, 8, 9, 12 & 13, as well as those for the Tissue Inhibitors of MMPs (TIMPs) 1, 2, 3, & 4 were examined in highly characterized normal knee joints, and knee joints with clinically diagnosed OA (early and advanced) or RA. The purpose of this study was to determine if normal, OA, and RA patients exhibit unique expression profiles for a sub-set of MMPs, and if early OA patients have a unique MMP expression profile that could be used as an early diagnostic marker. Methods: Synovial fluid was aspirated from stringently characterized normal knee joints, and in joints diagnosed with either OA (early and advanced) or RA. Multiplexing technology was employed to quantify protein expression levels for 8 MMPs and 4 TIMPs in the synovial fluid of 12 patients with early OA, 17 patients diagnosed with advanced OA, 15 with RA and 25 normal knee joints. Principle component analysis (PCA) was used to reveal which MMPs were most influential in the distinction between treatment groups. K – means clustering was used to verify the visual grouping of subjects via PCA. Results: Significant differences in the expression levels of MMPs and TIMPs were observed between normal and arthritic synovial fluids (with the exception of MMP 12). PCA demonstrated that MMPs 2, 8 & 9 can be used to effectively separate individuals diagnosed with advanced arthritis from early osteoarthritic and normal individuals, however, these MMP profiles do not separate early OA from normal synovial fluid. An apparent separation between advanced OA and RA subjects was also revealed through PCA. K-means clustering verified the presence of 3 clusters: normal joints clustered with early OA, and separate clusters of advanced OA or RA. Conclusions: This study demonstrates that unique MMP and TIMP expression profiles are present within normal, advanced OA and RA synovial fluid. These MMP profiles can be used to distinguish advanced OA & RA synovial fluid from early OA & normal synovial fluid, and even between synovial fluid samples from OA and RA joints. Although this methodology cannot be used for the diagnosis of early OA, high throughput multiplex technology of MMPs and TIMPs in synovial fluid may prove useful in determining the severity of the disease state, and/or quantifying the response of individuals to disease interventions.

Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation.

Background: 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. Results: 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. Conclusions: 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.

Menisci help maintain the structural integrity of the knee. However, the poor healing potential of the meniscus following a knee injury can not only end a career in sports but lead to osteoarthritis later in life. Complete understanding of meniscal structure is essential for evaluating its risk for injury and subsequent successful repair. This study used novel approaches to elucidate meniscal architecture. The radial and circumferential collagen fibrils in the meniscus were investigated using novel tissue‐preparative techniques for light and electron microscopic studies. The results demonstrate a unique architecture based on differences in the packaging of the fundamental collagen fibrils. For radial arrays, the collagen fibrils are arranged in parallel into ∼10 μm bundles, which associate laterally to form flat sheets of varying dimensions that bifurcate and come together to form a honeycomb network within the body of the meniscus. In contrast, the circumferential arrays display a complex network of collagen fibrils arranged into ∼5 μm bundles. Interestingly, both types of architectural organization of collagen fibrils in meniscus are conserved across mammalian species and are age and sex independent. These findings imply that disruptions in meniscal architecture following an injury contribute to poor prognosis for functional repair.

The synovium is a thin connective tissue that lines the joint space of free moving articulations. In this report, the expression, structure, and composition of non-motile (primary) cilia in fibroblast-like synoviocytes (FLS) that populate the synovium have been studied. Primary cilia are non-motile, microtubule-based organelles that have been found in a variety of vertebrate cell types. We document that primary cilia are expressed in normal human synovium FLS, cultured human FLS, and FLS cells present in human synovial fluid, and that the cellular region occupied by the primary cilium shows a similar and highly defined architecture within these FLS. This architecture includes the presence of a unique structure that surrounds the lower portion of the cilium shaft. This structure, given the term cilium-pit, includes a space, the pit reservoir. Actin filament bundles surround the cilium-pit, and when these bundles are removed experimentally the volume of the cilium-pit and its continuity with the extracellular environment changes. Finally, this study documents that the cilium-pit is a site of endocytosis and is also the site for the localization of receptors (TNF receptors TNFR1 and TNFR2) associated with synoviocyte function. Taken together, the results of the present study suggest that the FLS cilium-pit functions to regulate the exposure of the primary cilium, both spatially and temporally to extracellular molecules and to couple primary cilium based signaling pathways with those linked to endocytosis.

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Introduction: A limited understanding of the cellular mechanisms governing bone mechanotransduction has inhibited the development of clinical treatments for a variety of bone disorders, including osteoporosis, osteoarthritis and microgravity-associated bone atrophy. The cytoskeleton is thought to play a role in cellular mechanotransduction, however the exact mechanism in bone cells has not yet been clearly elucidated. Studies involving cytoskeletal inhibitors have not generally considered secondary effects on cellular organelles such as the primary cilia. These cellular projections could account for the disparity between shear stresses predicted to occur in vivo and the minimum threshold of membrane deformation required to elicit a cellular response in vitro. Methods: MG-63 (human osteoblast-like) cells were cultured in vitro. Cultures were exposed to intermittent cyclic fluid flow shear stress (1 Pa amplitude), for 8 or 12 hrs. Some cultures were loaded in the presence of nocodazole (a microtubule inhibitor) or cytochalasin D (an actin filament inhibitor). The cellular response was analyzed through RT-PCR assessment of messenger RNA levels for specific molecules related to matrix metabolism. The effects of drug treatments on cytoskeletal disorganization and the primary cilia were assessed with immunocytochemistry and electron microscopy. Results: In untreated cultures, shear stress was associated with significant increases in mRNA levels for collagen I and matrix metalloproteinases 1 and 3, for both time points assessed. These increases were maintained in cultures loaded in the presence of cytochalasin D, but were almost completely abrogated in nocodazole-treated cultures. Cytoskeletal inhibitors exerted some dose-dependent effects on length and structure of primary cilia in MG-63 cells. Conclusions: The microtubule network appears to be necessary for some shear-induced responses of osteoblast-like cells. MG-63 cells possess primary cilia, organelles that could amplify fluid flow shear, accounting for some apparent contradictions between studies related to osteoblast mechanosensitivity. Since these structures are composed of microtubules, the observation that microtubule disruptors inhibit the shear response of osteoblast-like cells suggests the primary cilium may have a role in osteoblast mechanotransduction. The effects of cytoskeletal inhibitors on cilium structure may explain the conflicting results of earlier mechanotransduction studies.