Cell cycle: Choreographing the mitotic ballet
The generation of an intracellular gradient of the small guanosine triphosphatase (GTPase) Ran has been shown to regulate microtubule dynamics and mitotic spindle positioning.
During cell division, the chromosomal content is distributed to daughter cells by the mitotic spindle. Assembly of the mitotic spindle is tighly regulated and requires spatial cues. But what are the mechanisms that orchestrate this assembly? Using Xenopus laevis egg extracts — a system that is commonly used to study mitosis — Caudron et al. now show that spindle formation is coordinated by the generation of an intracellular gradient of the small GTPase Ran at the chromosomes.
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Spatial distribution of Alexa 488–Ran lifetime (top right) around a mitotic spindle (top left). Blue to green colouring reflects a decreasing interaction between RanGTP and importin- . Image courtesy of Philippe Bastiaens, EMBL, Heidelberg, Germany. | |
Two models have been put forward to explain how microtubules become organized in the mitotic spindle. The 'search and capture' model proposes that microtubules that grow from centrosomes are randomly captured and stabilized at the kinetochores on the chromosomes. However, an alternative model argues that chromatin changes the state of the mitotic cytoplasm in the surrounding area and promotes spindle assembly through a self-organization process. This model is based on the observation that Ran forms a chemical gradient from its GTP-bound active form (RanGTP), which is close to chromosomes to a GDP-bound inactive form (RanGDP) in the cytoplasm. The high activity of the cytoplasmic Ran-GTPase-activating protein (RanGAP) and a Ran-guanine nucleotide exchange factor (RanGEF or RCC1), which is localized on chromosomes, coordinate the two states of Ran. But is it possible that this gradient provides a positional signal that causes changes in microtubule dynamics and organizes the spindle around chromosomes?
To examine the formation of RanGTP-dependent gradients, the authors used a mathematical simulation to model the Ran gradient system. They showed that a long-range gradient of RanGTP that interacts with importin- and a short range gradient of free RanGTP occur around chromatin. This long-range gradient occurs because the RanGTP-importin- complex prevents RanGAP-induced GTP hydrolysis and diffuses away from the chromosomes. The authors argued that the long-range RanGTP-importin- gradient is physiologically relevant because it reflects the concentration gradient of released NLS-proteins that directly affect microtubule dynamics. Indeed, by analysing X. laevis egg extracts using fluorescence lifetime imaging microscopy (FLIM), they showed that RanGTP-dependent long-range gradients exist, such as the gradient of RanGTP-importin- and RanGTP-importin-–RanBP1. Furthermore, microtubule nucleation and stabilization of the growing ends, which take place at different distances from the chromosomes, occur at significantly different concentrations of RanGTP-importin- . Microtubule nucleation is restricted to a defined area around the chromosomes, whereas stabilization of the microtubule growing ends extends over longer distances.
The authors wanted to know whether the correct formation of a bipolar spindle around the chromosomes requires the RanGTP-importin- gradient. By experimentally varying the RanGTP-importin- gradient — using the addition of regulators of RanGTP production or RanGTP stability — they demonstrate that the correct gradient profile is important for proper spindle formation. A flattened gradient results in the loss of bipolar spindle assembly, the formation of random microtubule structures and a large decrease in microtubule–chromosome connections, indicating that the gradient has to operate within well defined dimensions for the proper coordination of spindle assembly.
This elegant study illustrates how an intracellular chemical gradient can provide spatial information within a cell. The authors propose that the Ran system does not simply signal where the chromosomes are, but that it functions as a control element that spatially coordinates the self-organization of the microtubule–chromosome system. Whether intracellular gradients are also required for the organization of other structures within the cells remains to be investigated.
Ekat Kritikou
References
- Caudron M. et al. Spatial coordination of spindle assembly by chromosome-mediated signaling gradients Science 309, 1373–1376 (2005) | Article | PubMed |
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