Flipping the switch: regulating MTOC location

Microtubules play essential roles in cell shape, polarity, transport, and division. These different cellular processes require distinct spatial arrangements of microtubules, which are achieved by switching the microtubule-organizing center (MTOC). In dividing cells, the centrosome functions as the MTOC, nucleating and anchoring microtubules within its pericentriolar material (PCM). MTOC activity at the centrosome is cyclical, with peak activity in metaphase and decreasing activity upon exiting mitosis. In differentiated cells, centrosome MTOC activity is further decreased or absent and MTOC function is assigned to alternative sites such as the plasma membrane.¹ In a recent report,² we use the developing C. elegans intestine as an in vivo model to investigate how the MTOC switches between the centrosome and the membrane as epithelial cells differentiate and divide.

A problem arises when cells with non-centrosomal MTOCs divide: MTOC location must switch back to the centrosome to build the mitotic spindle. To study this poorly understood problem, we asked (1) which MTOC state is dominant, membrane or centrosome, and (2) which factors regulate the MTOC location in coordination with cell cycle state. Our findings reveal that the centrosome MTOC state is dominant and that SPD-2 and cyclin-dependent kinases (CDKs) contribute to centrosome reactivation.²

We first asked what happens to the membrane MTOC as epithelial cells re-enter mitosis. We found that MTOC location switches from the membrane to the centrosome during mitosis and that MTOC location switches back to the membrane following cell division.² To investigate how this switch in MTOC location is regulated, we used cell fusion experiments to determine if either the centrosome or the membrane MTOC state is dominant. Upon fusing a mitotic cell to a differentiated cell, we found that the differentiated cell switched to a centrosome MTOC. This finding indicates that a diffusible component in mitotic cytoplasm is sufficient to (1) inactivate the membrane MTOC and (2) activate the centrosome MTOC. Importantly, this MTOC switch is rapid (<3 minutes), and is not accompanied by other markers of cell cycle progression, e.g. nuclear envelope breakdown or chromosome condensation, which eventually follow the fusion of interphase and mitotic cells.³ Thus, we propose that the MTOC switch is specifically activated by a factor in mitotic cytoplasm, and is not a secondary effect of induced cell cycle progression.

One possible way to achieve this rapid MTOC switch is through post-translational modifications like phosphorylation. Inhibiting CDK activity blocked MTOC activation at the centrosome, raising the possibility that mitotic CDKs control the switch. But what factors are regulated by the cell cycle to drive the MTOC switch? One possibility is that a centrosome component is modified in mitotic cells and activates the interphase centrosome as the MTOC. The centrosome proteins SPD-2/Cep192 and SPD-5 recruit PCM and γ-tubulin following phosphorylation by mitotic kinases,⁴ but it is unknown how SPD-2 and SPD-5 themselves are recruited to the centrosome upon activation. To determine if the mitotic cell contributes these proteins to the interphase cell upon fusion, we marked the origin of proteins adding onto the centrosome in the interphase cell: GFP-tagged proteins were photobleached in a mitotic cell, which was then fused to an interphase cell. With only fluorescence from the interphase cell remaining, we asked if interphase (GFP+) or mitotic (GFP−) SPD-2, SPD-5, or γ-tubulin are recruited to the activated centrosome. Our results show that mitotic SPD-2 is recruited to the interphase centrosome.² As SPD-2 levels are the same in interphase and mitotic cells, and SPD-2 overexpression does not activate the centrosome as the MTOC,² we propose that the SPD-2 recruitment to the centrosome involves differential SPD-2 modification.

Based on these findings, we propose a model for switching to a centrosome MTOC: CDK activity directly or indirectly modifies SPD-2 in mitotic cells, and modified SPD-2 promotes activation of the centrosome MTOC (Fig. 1). We speculate that CDK-1/CDK1 is the relevant CDK, since its activity is required in G2/M progression and early mitosis,⁵ the time when mitotic cytoplasm can activate the centrosome MTOC.² Interphase SPD-2 is not recruited to the interphase centrosome upon fusion, suggesting that the MTOC activator localizes to the mitotic centrosome, and its activated substrates such as SPD-2 can diffuse and activate the interphase centrosome. Consistent with this idea, recent reports show that centrosomal and cytoplasmic pools of SPD-2 exchange readily.^4,6 Future studies will reveal if CDKs directly modify SPD-2, if activated SPD-2 is sufficient to establish a centrosome MTOC, and how the membrane MTOC is switched off as the centrosome is switched on.

Figure 1.

Model for the MTOC switch. In a mitotic cell, SPD-2 is modified and centrosome reactivation as the MTOC is CDK-dependent. In an interphase or differentiated cell, SPD-2 is unmodified and the centrosome is inactivated as an MTOC. Upon fusing a mitotic and interphase/differentiated cell, mitotic SPD-2 is recruited to the interphase centrosome, which is then activated as the MTOC.

Regulating MTOC location is likely essential for many developmental processes, including tight control of proliferation. Recent work supports this hypothesis: in mammalian cardiomyocytes, the nuclear envelope becomes the MTOC and cells do not divide further; by contrast, centrosomes can be activated as MTOCs in zebrafish cardiomyocytes, correlating with their ability to further proliferate and regenerate.⁷ At the other extreme lies excessive proliferation, a defining feature of cancer. An important step in tumorigenesis may be inappropriate activation of centrosomes as MTOCs. Future investigations will reveal the exact mechanism underlying the coordination of the MTOC switch and cell cycle state, which will shed light on how proliferation is controlled in development and disease.