J B Rattner’s research while affiliated with University of Calgary and other places

The tight, relative positioning of nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated if extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a -reversible- increase in the distance between centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that i) cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, ii) Epac1 is critically involved and iii) Rap1B is important for the relative positioning of centrosome and nucleus. Our results represent to our knowledge the first report demonstrating that pathophysiological conditions can impact the distance between centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process.

This study has evaluated the swelling of meniscal test samples associated with altered osmotic environments. Meniscal samples were cut and weighed, then placed in one of 3 solutions: deionized water, phosphate buffered saline (PBS) or 2× concentration PBS. The amount of swelling in meniscal samples was solution independent with average swelling greater than 20%. The effect of this swelling on the mechanical properties of the samples was evaluated under confined compression testing. Samples were measured using a photogrammetric technique at the time of sample preparation and again after 1h in PBS. Meniscal samples used for mechanical testing swelled on average 18% in thickness after 1h in isotonic PBS. Free-swollen tissue was 1/3 as stiff at equilibrium as those that were recompressed to their original thickness prior to testing. Secant moduli at peak stress were nine times greater in the recompressed samples than the free-swollen samples. Relaxation times were faster in swollen samples, indicating increased permeability compared to recompressed specimen. Swelling pressure in the tissue averaged 14.4kPa in isotonic PBS, identifying that the menisci are pre-stressed structures within the knee joint. Histological analysis identified that the quantity of swelling is related to both the osmotic pressure generated by proteoglycans and the local collagen architecture in the sample. This is the first study to quantify the amount and swelling in meniscal test samples. This swelling behavior significantly influences the properties of the tissue in compression and should be addressed in future mechanical testing and protocol development for the menisci. Copyright © 2015 Elsevier Ltd. All rights reserved.

The meniscal roots, or insertional ligaments, firmly attach the menisci to tibial plateau. These strong attachments anchor the menisci and allow for the generation of hoop stress in the tissue. The meniscal roots have a ligament-like structure that transitions into the fibrocartilagenous structure of the meniscal body. The purpose of this study was to carry out a complete analysis of the structure and tissue organization from the body of the meniscus through the transition region and into the insertional roots. Serial sections were obtained from the meniscal roots into the meniscal body in fixed juvenile bovine menisci. Sections were stained for collagen and proteoglycans (PG) using fast green and safranin-o staining protocols. Unstained sections were imaged used a backlit stereo microscope. Optical projection tomography (OPT) was employed to evaluate the three-dimensional collagen architecture of the root-meniscus transition in lapine menisci. Tie-fibres were observed in the sections of the ligaments furthest from the bovine meniscal body. Blood vessels were observed to be surrounded by these tie-fibres and a PG-rich region within the ligaments. Near the tibial insertion, the roots contained large ligament-like collagen fascicles. In sections approaching the meniscus, there was an increase in tie-fibre size and density. Small tie-fibres extended into the ligament from the epiligamentous structure in the outermost sections of the meniscal roots, while large tie-fibre bundles were apparent at the meniscus transition. The staining pattern indicates that the root may continue into the outer portion of the meniscus where it then blends with the more fibrocartilage-like inner portions of the tissue. In unstained sections it was observed that the femoral side of the epiligamentous structure surrounding the root becomes more fibrous and thickens in the inferior inner portion of the posterior medial root. This thickening changes the shape of the root to more closely resemble the meniscus wedge shape. These observations support the concept of root continuity with the outer portion of the meniscus, thereby connecting with the hoop-like structure of the peripheral meniscus. OPT identified continuous collagen organization from the root into the meniscal body in longitudinal sections. In the radial direction, the morphology of the root continues into the meniscal body consistent with the serially sectioned bovine menisci. Blood vessels were prevalent on the periphery of the root. These blood vessels then arborized to cover the anterior femoral surface of the meniscus. This is the first study of the structural transition between the insertional ligaments (roots) and the fibrocartilagenous body of the menisci. These new structural details are important to understanding the meniscal load-bearing mechanism in the knee. © 2015 Anatomical Society.

Background: 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. Methods: Seven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Results: We report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium. Conclusions: Primary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously observed in cell cultures. This is the first study to demonstrate that defects in cilia expression and function are a true hallmark of GBM tumors and correlate with their unrestrained growth. A review of the current ultrastructural profiles in the literature provides suggestions as to the best possible candidate protein that underlies defects in the early stages of ciliogenesis within GBM tumors.

Nesprins are a multi-isomeric family of spectrin-repeat (SR) proteins, predominantly known as nuclear envelope scaffolds. However, isoforms that function beyond the nuclear envelope remain poorly examined. Here, we characterize p50(Nesp1), a 50-kD isoform that localizes to processing bodies (PBs), where it acts as a microtubule-associated protein capable of linking mRNP complexes to microtubules. Overexpression of dominant-negative p50(Nesp1) caused Rck/p54, but not GW182, displacement from microtubules, resulting in reduced PB movement and cross talk with stress granules (SGs). These cells disassembled canonical SGs induced by sodium arsenite, but not those induced by hydrogen peroxide, leading to cell death and revealing PB-microtubule attachment is required for hydrogen peroxide-induced SG anti-apoptotic functions. Furthermore, p50(Nesp1) was required for miRNA-mediated silencing and interacted with core miRISC silencers Ago2 and Rck/p54 in an RNA-dependent manner and with GW182 in a microtubule-dependent manner. These data identify p50(Nesp1) as a multi-functional PB component and microtubule scaffold necessary for RNA granule dynamics and provides evidence for PB and SG micro-heterogeneity.

The collagenous structure of the knee menisci is integral to the mechanical integrity of the tissue and the knee joint. The tie-fibre structure of the tissue has largely been neglected, despite previous studies demonstrating its correlation with radial stiffness. This study has evaluated the structure of the tie-fibres of bovine menisci using 2D and 3D microscopy techniques. Standard collagen and proteoglycan (PG) staining and 2D light microscopy techniques were conducted. For the first time, the collagenous structure of the menisci was evaluated using 3D, second harmonic generation (SHG) microscopy. This technique facilitated the imaging of collagen structure in thick sections (50-100 μm). Imaging identified that tie-fibres of the menisci arborize from the outer margin of the meniscus toward the inner tip. This arborization is associated with the structural arrangement of the circumferential fibres. SHG microscopy has definitively demonstrated the 3D organization of tie-fibres in both sheets and bundles. The hierarchy of the structure is related to the organization of circumferential fascicles. Large tie-fibre sheets bifurcate into smaller sheets to surround circumferential fascicles of decreasing size. The tie-fibres emanate from the lamellar layer that appears to surround the entire meniscus. At the tibial and femoral surfaces these tie-fibre sheets branch perpendicularly into the meniscal body. The relationship between tie-fibres and blood vessels in the menisci was also observed in this study. Tie-fibre sheets surround the blood vessels and an associated PG-rich region. This subunit of the menisci has not previously been described. The size of tie-fibre sheets surrounding the vessels appeared to be associated with the size of blood vessel. These structural findings have implications in understanding the mechanics of the menisci. Further, refinement of the complex structure of the tie-fibres is important in understanding the consequences of injury and disease in the menisci. The framework of meniscus architecture also defines benchmarks for the development of tissue-engineered replacements in the future.

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. Bovine meniscal samples were imaged from four locations (outer main body, femoral surface, tibial surface and inner main body) to determine the variation in collagen orientation throughout the tissue. Bovine stifles (n = 2) were obtained from a local abattoir and the menisci carefully dissected. Menisci were fixed in methanol and subsequently cut using a custom cutting jig (n = 4 samples per meniscus). Samples were then mounted in agarose, dehydrated in methanol and subsequently cleared using benzyl alcohol benzyl benzoate (BABB) and imaged using OPT. Results indicate circumferential, radial and oblique collagenous orientations at the contact surfaces and in the inner third of the main body of the meniscus. Imaging identified fascicles ranging from 80-420 mum in diameter. Transition zones where fascicles were found to have a woven or braided appearance were also identified. The outer-third of the main body was composed of fascicles oriented predominantly in the circumferential direction. Blood vessels were also visualized using this technique, as their elastin content fluoresces more brightly than collagen at the 425 nm wavelength used by the OPT scanner. OPT was capable of imaging the collagenous structure, as well as blood vessels in the bovine meniscus. Collagenous structure variability, including transition zones between structural regions not previously described in the meniscus, was identified using this novel technique.

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 G 2 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.

Inhibition of GSK-3β causes dephosphorylation of CRMP-2. RPE cells were analyzed for expression of total CRMP-2 (panel A), phosphorylated pCRMP-2 (panel B) and actin (panel C). RPE cell extracts were analyzed by western blotting using anti-CRMP-2 antibody measuring total CRMP-2 (panel A), anti-pCRMP-2(Thr514) antibody specific for phosphorylated CRMP-2 (panel B) and anti-actin (Panel C), either from untreated (lane 1) or cells treated with lithium (lane 2). Note that phosphorylated pCRMP-2 levels are decreased after lithium treatment (panel B), which has no effect on overall CRMP-2 levels (panel A). (TIF)