| Abstract No.: | A-B1063 |
| Country: | Canada |
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| Title: | β1-INTEGRINS REGULATE CENTROSOME STABILITY IN ASTROCYTES |
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| Authors/Affiliations: | 1 Yen May Ong*; 1 Huashan Peng; 1 Paul Holland; 1 Salvatore Carbonetto;
1 McGill University, Montreal, QC, Canada
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| Content: | Objectives: Centrosomes are major microtubule organizing centres. Their main function is to arrange microtubules (MTs) in the cytosol and to create a scaffold that contributes to cell shape. Rearrangement of this radial MT array can be dictated by the centrosome and occurs during cell polarization events in astrocytic migration. Previous work has shown that disrupting centrosome function leads to cell polarity dysfunction. However, little is known about what regulates centrosome assembly. We show that centrosome formation in rat astrocytes occurs rapidly and is dependent on β1-integrin signalling. This gives us a starting point to elucidate the signalling pathway that controls centrosome assembly in astrocytes. Understanding centrosome assembly would enable us to study its precise role in cell polarity.
Materials and Methods: Cell adhesion assays were used primarily to study centrosome assembly upon astrocytic adhesion to extracellular matrix (ECM) proteins or polylysine. Glass coverslips were coated with different ECM proteins or polylysine. Primary rat astrocytic cultures from E17-18 embryos were trypsinized and resuspended in serum-free media before plated on pre-coated glass coverslips. Cells were allowed to adhere to coverslips for predefined periods of time and were fixed with paraformaldehyde. For antibody treatment of cells, astrocytes suspended in serum free media were incubated with the antibody prior to plating.
Using Triton-X-100 and bovine serum albumin, cells were then permeabilized and blocked. Staining with primary antibodies was then carried out for 1h, followed by secondary antibody staining for 1h. Major centrosomal proteins, i.e. pericentrin and γ-tubulin were stained and then tagged with Alexa Fluor 488 and Alexa Fluor 555. DAPI was used to stain cell nuclei. Samples were then inverted and mounted on glass slides with SlowFade Gold and sealed with nail varnish. Cells were then visualized using epifluorescence microscopy.
Results: Centrosomes were observed to form within 10 min after plating on collagen, fibronectin, laminin and polylysine. The rate of centrosome formation seemed to be independent of the substrate used. Formation of centrosomes was disrupted when function blocking β1-integrin antibody was incubated with rat astrocytes. Also, when plated on the E3 laminin fragment (E3), known to engage dystroglycan, centrosome assembly was significantly slower in the first 30 min, but caught up with control centrosome assembly after 1h.
Conclusions: From these results, we conclude that centrosome assembly occurs rapidly (within 5-10 min) on different substrates and is regulated by β1-integrin signalling. Cell adhesion alone appears to be insufficient to activate centrosome formation since cells that have β1-integrin signalling blocked adhere to polylysine while showing centrosome disruption. This indicates that β1-integrin signalling is necessary for proper centrosome formation. The significant decrease in centrosome assembly observed in cells on E3 at earlier timepoints further support this. ECM protein secretion by astrocytes or possible cis interactions between Dg and β1-integrin might explain why the rate of centrosome assembly on E3 eventually catches up with the control. These results give us insight to the possible signalling pathways involved in centrosome assembly in astrocytes and help |
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