(D) Conversely, a high proportion of the cells cultured in square microwells aligned the spindle along the long axis of the cell. Cells were imaged for DNA (green), tubulin (red) and microwell outline (transmission, blue) and assessed at different stages during mitosis, specifically (A) NEBD, (B) late SEC inhibitor KL-2 prometaphase, (C) metaphase, (D) anaphase and (E) cytokinesis; bars: 10 m.(TIF) pone.0066918.s002.tif (1.5M) GUID:?F82091D3-ECDA-4CA1-801E-C7CF5EC13ACF Physique S3: Cell shape did not impact on centrosome separation and spindle formation. (ACB) HeLa cells (RFP-tubulin/GFP-H2B) were synchronized and cultured on the different cell culture platforms SEC inhibitor KL-2 and assessed for the position of the centrosomes at NEBD and subsequent spindle formation. Cells were imaged for DNA (green), tubulin (red) and cell outline (transmission, blue); bars: 10 m. Cells initiated the separation of their centrosomes either (A) during prophase, resulting in centrosomes orthogonal at NEBD or (B) during prometaphase, resulting in centrosomes at the same side of the nuclear envelope at NEBD. (C) Cells with the centrosomes positioned on opposite sides of the nuclear envelope at NEBD were quicker at forming the spindle, regardless of the substrate upon which the cells were cultured. Key: *** p<0.001.(TIF) pone.0066918.s003.tif (403K) GUID:?075CDCEA-D5A5-4C39-9820-CF7A467D54CF Video S1: Assessment of the ITGA3 effect of cell shape on mitosis in cells cultured on 2D substrates. HeLa (GFP-H2B/RFP-tubulin) cells were synchronized and cultured on 2D substrates for 10 hours before imaging for DNA (green) and tubulin (red) using time lapse microscopy (Delta Vision imaging system). Time points, comprised of 10 z sections 1 m apart, were acquired every 4 minutes.(AVI) pone.0066918.s004.avi (299K) GUID:?32B5EC14-2029-4937-83B1-95CF02AD7E5A Video S2: Assessment of the effect of cell shape on mitosis in cells cultured in square microwells. HeLa (GFP-H2B/RFP-tubulin) cells were synchronized and cultured in 3D square microwells for 10 hours before imaging for DNA (green), tubulin (red) and microwell outline (transmission, blue) using time lapse microscopy (Delta Vision imaging system). Time points, comprised of 10 z sections 1 m apart, were acquired every 4 minutes.(AVI) pone.0066918.s005.avi (196K) GUID:?DC710C83-E454-4668-83C3-65BFD8117E64 Video S3: Assessment of the effect of cell shape on mitosis in cells cultured in circular microwells. HeLa (GFP-H2B/RFP-tubulin) cells were synchronized and cultured in 3D circular microwells for 10 hours before imaging for DNA (green), tubulin (red) and microwell outline (transmission, blue) using time lapse microscopy (Delta Vision imaging system). Time points, comprised of 10 z sections 1 m apart, were acquired every 4 minutes.(AVI) pone.0066918.s006.avi (216K) GUID:?8D41E265-C8C0-4638-A021-64ED867B58E4 Video S4: Assessment of the SEC inhibitor KL-2 effect of cell shape on mitosis in cells cultured on 2D patterns. HeLa (GFP-H2B/RFP-tubulin) cells were synchronized and cultured on 2D square patterns for 10 hours before imaging for DNA (green) and tubulin (red) using time lapse microscopy (Delta Vision imaging system). Time points, comprised of 10 z sections 1 m apart, were acquired every 4 minutes.(AVI) pone.0066918.s007.avi (252K) GUID:?B7483355-2780-45F0-8BC0-C1096D0530CB Abstract The formation and orientation of the mitotic spindle is a critical feature of mitosis. The morphology of the cell and the spatial distribution and composition of the cells’ adhesive microenvironment all contribute to dictate the position of the spindle. However, the impact of the dimensionality of the cells’ microenvironment has rarely been studied. In this study we present the use of a microwell platform, where the internal surfaces of the individual wells are coated with fibronectin, enabling the three-dimensional presentation of adhesive ligands to single cells cultured within the microwells. This platform was used to assess the effect of dimensionality and cell shape in a controlled microenvironment. SEC inhibitor KL-2 Single HeLa cells cultured in circular microwells exhibited greater tilting of the mitotic spindle, in comparison to cells cultured in square microwells. This correlated with an increase in the time required to align the chromosomes at the metaphase plate due to prolonged activation of the spindle checkpoint in an SEC inhibitor KL-2 actin dependent process. The comparison to 2D square patterns revealed that this dimensionality of cell adhesions alone affected both mitotic timings and spindle orientation; in particular the role of actin varied according to the dimensionality of the cells’ microenvironment. Together, our data revealed that cell shape and the dimensionality of the cells’ adhesive environment impacted on both the orientation of the mitotic spindle and progression through mitosis. Introduction The orientation of the mitotic spindle along a predetermined axis during mitosis plays an important role in cell fate and organ development [1]C[5]. Misorientation of the mitotic spindle has been implicated as a contributing factor in tumor development and polycystic kidney disease [6], [7]. Cell shape dictates.