EDITORIALS: CELL CYCLE FEATURES Cell Cycle 14:24, 3773--3774; December 15, 2015; © 2015 Taylor & Francis Group, LLC
Mitotic spindle orientation: JAM-A can fix it €seyin Tuncay1, Benjamin F Brinkmann1,2, and Klaus Ebnet1,2,* Hu 1
University of M€ unster; M€ unster, Germany; 2Interdisciplinary Clinical Research Center (IZKF); University of M€ unster; M€ unster, Germany
Keywords: actin cortex, cell adhesion, cell-cell contacts, dynein, epithelial cells, JAM-A, mitosis, spindle orientation Epithelial cells form sheets of cells which are connected by intercellular junctions. When epithelial cells divide the mitotic spindle is aligned in the plane of the sheet, which ensures that both daughter cells are retained in the sheet. The planar orientation of the spindle apparatus must be a tightly regulated process as randomized spindle orientation would result in the delamination of cells and eventually in their transformation and metastasis. The alignment of the mitotic spindle requires a stable attachment of the astral microtubule plus ends to the lateral cortex. This attachment is mediated by the dynein - dynactin motor protein complex (hereafter referred to as dynein). At the beginning of mitosis dynein is associated with astral microtubules which dynamically scan the lateral cortex for dyneininteracting molecules. Cortical sites with accumulations of dynein receptors trigger dynein offloading from the microtubules and activation of its minus end-directed motor activity. Once immobilized at the cortex, activated dynein captures astral microtubule plus ends and generates pulling forces toward the centrosomes. These pulling forces align the spindle apparatus in the plane of the sheet and move the centrosomes toward the cortex. The stable association of dynein with the lateral cortex requires different dynein receptors. Among the most ubiquitously used dynein receptors is the NuMA LGN - Gai complex.1 NuMA directly interacts with dynein, Gai is myristoylated and inserted in the membrane, LGN interacts with both NuMA and Gai thereby linking dynein with the membrane. Besides the NuMA - LGN - Gai
complex, the cortical actin cytoskeleton is important for the stable cortical localization of dynein. The cortical actin cytoskeleton is reorganized during mitosis, which is probably necessary to regulate mitotic cell rounding and to render the cortex rigid enough to resist the forces associated with centrosome movement. The mitotic actin cortex might also serve as scaffold for band 4.1 proteins and ezrin - radixin moesin (ERM) family proteins which can interact with NuMA and microtubule plus ends, respectively.2,3 Finally, various phosphoinositide species such as PtdIns (3,4,5)P3, PtdIns(4,5,)P2, and PtdIns(4)P have been found to interact directly with NuMA.4 It has been known that intercellular junctions provide signals that regulate the planar alignment of the mitotic spindle. However, it has been unclear if specific adhesion receptors are involved in the cortical localization of dynein-interacting molecules or if intercellular junctions are only permissive for planar spindle orientation. We have identified Junctional Adhesion Molecule-A (JAM-A) as the first cell adhesion molecule that triggers an intracellular signaling cascade required for the localization of dynein at the cortex of mitotic cells.5 JAM-A is a member of the immunoglobulin superfamily and is localized at cell-cell contact of epithelial cells and endothelial cells. Its homophilic adhesive activity is weak, it acts predominantly as a scaffold for the recruitment and assembly of signaling complexes at cell-cell contact sites. JAM-A is enriched at the tight junctions but is localized along the entire lateral junction of epithelial cells.
In our recent study we observed that when MDCK cells that lack functional JAM-A are grown in a in 3-dimensional matrix develop multiluminal spheroids instead of spheroids with a single central lumen.5 Since single lumen formation depends on planar orientation of the mitotic spindle6 we hypothesized that JAM-A might regulate the alignment of the mitotic spindle in the plane of the monolayer. In fact, depleting JAM-A or expressing a dominant-negative JAM-A mutant almost randomized the orientation of the spindle apparatus. To understand the underlying molecular mechanism we focused on the small GTPase Cdc42. Cdc42 was described in HeLa cells to regulate the formation of a PtdIns(3,4,5)P3 gradient at the cortex as well as the reorganization of the cortical actin cytoskeleton during mitosis.7 We found that in polarized epithelial cells Cdc42 is activated at the onset of mitosis, peaks at metaphase and then gradually declines. This transient activation of Cdc42 was almost completely blunted in JAM-A knockdown cells. We also found that in the absence of JAM-A the PtdIns(3,4,5)P3 gradient at the cortex is disrupted and the cells fail to reorganize the cortical actin cytoskeleton during mitosis. Finally, we observed that in the absence of JAM-A less dynein is localized at the lateral cortex, explaining the randomization of the spindle orientation. Our study identifies the adhesion molecule JAM-A as a critical regulator of mitotic spindle orientation. JAM-A activates Cdc42 at the onset of mitosis which results in the formation of a
*Correspondence to: Klaus Ebnet; Email: [email protected] Submitted: 09/17/2015; Accepted: 09/25/2015 http://dx.doi.org/10.1080/15384101.2015.1105696 Feature to: Tuncay H, et al. JAM-A regulates cortical dynein localization through Cdc42 to control planar spindle orientation during mitosis. Nat Commun (2015); 6:8128; PMID: 26306570; http://dx.doi.org/10.1038/ncomms9128
www.tandfonline.com
Cell Cycle
3773
PtdIns(3,4,5)P3 gradient and the reorganization of the actin cytoskeleton at the cortex. JAM-A thus triggers the formation of 2 cortical components which are known to mediate the interaction of dynein with the cortex (Fig. 1). It is likely that JAM-A is not the only signal provider for dynein localization at the cortex, and it will be important to understand if different adhesion receptors act cooperatively or if they act consecutively during different phases of mitosis. Figure 1. JAM-A regulates the cortical localization of dynein. JAM-A at intercellular junctions between a mitotic cell (black line) and an interphase cell (gray line) activates Cdc42 in mitotic cells. Active Cdc42 regulates the formation of a PtdIns(3,4,5)P3 (PIP3) gradient and the reorganization of
References 1. Kotak S, Gonczy P. Curr Opin Cell Biol 2013; 25:741748; PMID:23958212; http://dx.doi.org/10.1016/j. ceb.2013.07.008 2. Kiyomitsu T, Cheeseman IM. Cell 2013; 154:391-402; PMID:23870127; http://dx.doi.org/10.1016/j. cell.2013.06.010
3774
3. Solinet S, et al. J Cell biol 2013; 202:251-260; PMID: 23857773; http://dx.doi.org/10.1083/jcb.201304052 4. Kotak S, et al. EMBO J 2014; 33:1815-1830; PMID: 24996901; http://dx.doi.org/10.15252/embj.201488147 5. Tuncay H, et al. Nat Commun 2015; 6:8128; PMID: 26306570; http://dx.doi.org/10.1038/ncomms9128
Cell Cycle
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
6. Jaffe AB, et al. The J Cell Biol 2008; 183:625-633; PMID:19001128; http://dx.doi.org/10.1083/jcb.200807121 7. Mitsushima M, et al. Mol Cell Biol 2009; 29:28162827; PMID:19273597; http://dx.doi.org/10.1128/ MCB.01713-08
Volume 14 Issue 24
Mitotic spindle orientation: JAM-A can fix it €seyin Tuncay1, Benjamin F Brinkmann1,2, and Klaus Ebnet1,2,* Hu 1
University of M€ unster; M€ unster, Germany; 2Interdisciplinary Clinical Research Center (IZKF); University of M€ unster; M€ unster, Germany
Keywords: actin cortex, cell adhesion, cell-cell contacts, dynein, epithelial cells, JAM-A, mitosis, spindle orientation Epithelial cells form sheets of cells which are connected by intercellular junctions. When epithelial cells divide the mitotic spindle is aligned in the plane of the sheet, which ensures that both daughter cells are retained in the sheet. The planar orientation of the spindle apparatus must be a tightly regulated process as randomized spindle orientation would result in the delamination of cells and eventually in their transformation and metastasis. The alignment of the mitotic spindle requires a stable attachment of the astral microtubule plus ends to the lateral cortex. This attachment is mediated by the dynein - dynactin motor protein complex (hereafter referred to as dynein). At the beginning of mitosis dynein is associated with astral microtubules which dynamically scan the lateral cortex for dyneininteracting molecules. Cortical sites with accumulations of dynein receptors trigger dynein offloading from the microtubules and activation of its minus end-directed motor activity. Once immobilized at the cortex, activated dynein captures astral microtubule plus ends and generates pulling forces toward the centrosomes. These pulling forces align the spindle apparatus in the plane of the sheet and move the centrosomes toward the cortex. The stable association of dynein with the lateral cortex requires different dynein receptors. Among the most ubiquitously used dynein receptors is the NuMA LGN - Gai complex.1 NuMA directly interacts with dynein, Gai is myristoylated and inserted in the membrane, LGN interacts with both NuMA and Gai thereby linking dynein with the membrane. Besides the NuMA - LGN - Gai
complex, the cortical actin cytoskeleton is important for the stable cortical localization of dynein. The cortical actin cytoskeleton is reorganized during mitosis, which is probably necessary to regulate mitotic cell rounding and to render the cortex rigid enough to resist the forces associated with centrosome movement. The mitotic actin cortex might also serve as scaffold for band 4.1 proteins and ezrin - radixin moesin (ERM) family proteins which can interact with NuMA and microtubule plus ends, respectively.2,3 Finally, various phosphoinositide species such as PtdIns (3,4,5)P3, PtdIns(4,5,)P2, and PtdIns(4)P have been found to interact directly with NuMA.4 It has been known that intercellular junctions provide signals that regulate the planar alignment of the mitotic spindle. However, it has been unclear if specific adhesion receptors are involved in the cortical localization of dynein-interacting molecules or if intercellular junctions are only permissive for planar spindle orientation. We have identified Junctional Adhesion Molecule-A (JAM-A) as the first cell adhesion molecule that triggers an intracellular signaling cascade required for the localization of dynein at the cortex of mitotic cells.5 JAM-A is a member of the immunoglobulin superfamily and is localized at cell-cell contact of epithelial cells and endothelial cells. Its homophilic adhesive activity is weak, it acts predominantly as a scaffold for the recruitment and assembly of signaling complexes at cell-cell contact sites. JAM-A is enriched at the tight junctions but is localized along the entire lateral junction of epithelial cells.
In our recent study we observed that when MDCK cells that lack functional JAM-A are grown in a in 3-dimensional matrix develop multiluminal spheroids instead of spheroids with a single central lumen.5 Since single lumen formation depends on planar orientation of the mitotic spindle6 we hypothesized that JAM-A might regulate the alignment of the mitotic spindle in the plane of the monolayer. In fact, depleting JAM-A or expressing a dominant-negative JAM-A mutant almost randomized the orientation of the spindle apparatus. To understand the underlying molecular mechanism we focused on the small GTPase Cdc42. Cdc42 was described in HeLa cells to regulate the formation of a PtdIns(3,4,5)P3 gradient at the cortex as well as the reorganization of the cortical actin cytoskeleton during mitosis.7 We found that in polarized epithelial cells Cdc42 is activated at the onset of mitosis, peaks at metaphase and then gradually declines. This transient activation of Cdc42 was almost completely blunted in JAM-A knockdown cells. We also found that in the absence of JAM-A the PtdIns(3,4,5)P3 gradient at the cortex is disrupted and the cells fail to reorganize the cortical actin cytoskeleton during mitosis. Finally, we observed that in the absence of JAM-A less dynein is localized at the lateral cortex, explaining the randomization of the spindle orientation. Our study identifies the adhesion molecule JAM-A as a critical regulator of mitotic spindle orientation. JAM-A activates Cdc42 at the onset of mitosis which results in the formation of a
*Correspondence to: Klaus Ebnet; Email: [email protected] Submitted: 09/17/2015; Accepted: 09/25/2015 http://dx.doi.org/10.1080/15384101.2015.1105696 Feature to: Tuncay H, et al. JAM-A regulates cortical dynein localization through Cdc42 to control planar spindle orientation during mitosis. Nat Commun (2015); 6:8128; PMID: 26306570; http://dx.doi.org/10.1038/ncomms9128
www.tandfonline.com
Cell Cycle
3773
PtdIns(3,4,5)P3 gradient and the reorganization of the actin cytoskeleton at the cortex. JAM-A thus triggers the formation of 2 cortical components which are known to mediate the interaction of dynein with the cortex (Fig. 1). It is likely that JAM-A is not the only signal provider for dynein localization at the cortex, and it will be important to understand if different adhesion receptors act cooperatively or if they act consecutively during different phases of mitosis. Figure 1. JAM-A regulates the cortical localization of dynein. JAM-A at intercellular junctions between a mitotic cell (black line) and an interphase cell (gray line) activates Cdc42 in mitotic cells. Active Cdc42 regulates the formation of a PtdIns(3,4,5)P3 (PIP3) gradient and the reorganization of
References 1. Kotak S, Gonczy P. Curr Opin Cell Biol 2013; 25:741748; PMID:23958212; http://dx.doi.org/10.1016/j. ceb.2013.07.008 2. Kiyomitsu T, Cheeseman IM. Cell 2013; 154:391-402; PMID:23870127; http://dx.doi.org/10.1016/j. cell.2013.06.010
3774
3. Solinet S, et al. J Cell biol 2013; 202:251-260; PMID: 23857773; http://dx.doi.org/10.1083/jcb.201304052 4. Kotak S, et al. EMBO J 2014; 33:1815-1830; PMID: 24996901; http://dx.doi.org/10.15252/embj.201488147 5. Tuncay H, et al. Nat Commun 2015; 6:8128; PMID: 26306570; http://dx.doi.org/10.1038/ncomms9128
Cell Cycle
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
6. Jaffe AB, et al. The J Cell Biol 2008; 183:625-633; PMID:19001128; http://dx.doi.org/10.1083/jcb.200807121 7. Mitsushima M, et al. Mol Cell Biol 2009; 29:28162827; PMID:19273597; http://dx.doi.org/10.1128/ MCB.01713-08
Volume 14 Issue 24
