Jonne A. Raaijmakers

Systematic dissection of dynein regulators in mitosis

A comprehensive survey of the roles of dynein subunits and adaptors in mitosis reveals that dynein forms distinct complexes requiring specific recruiters and activators to promote orderly progression through mitosis.

Switching Polo-like kinase-1 on and off in time and space

Polo-like kinase (Plk)1 executes several essential functions to promote cell division. These functions range from centrosome maturation in late G2 phase to the regulation of cytokinesis, which necessitates precise separation of Plk1-dependent substrate phosphorylation over time. Multiple levels of control are in place to ensure that Plk1-dependent phosphorylation of its various substrates is properly coordinated in time and space. Here, we review the current knowledge on the mechanisms that enforce the temporal and spatial control of Plk1 activity, and how this results in coordinated phosphorylation of its many different substrates. We also review a number of newly discovered functions of Plk1 that provide more insights into the spatiotemporal control of Plk1-dependent substrate phosphorylation.

RAMA1 is a novel kinetochore protein involved in kinetochore-microtubule attachment

During mitosis, kinetochores need to attach to microtubules emanating from spindle poles. Several protein complexes have been shown to mediate the kinetochore-microtubule interaction. However, with the continually growing number of newly identified kinetochore proteins, it is unclear whether all major components of the kinetochore-microtubule interface have been identified. We therefore performed a high-throughput RNAi screen to identify additional factors involved in kinetochore-microtubule attachment, and identified RAMA1 as a novel regulator of this process. Depletion of RAMA1 results in severe chromosome alignment defects and a checkpoint-dependent mitotic arrest. We show that this is due to reduced kinetochore-microtubule attachments. RAMA1 localizes to the spindle and to outer kinetochores throughout all phases of mitosis and is recruited to kinetochores by the core kinetochore-microtubule attachment factor Hec1. Interestingly, unlike Hec1, the association of RAMA1 with kinetochores is highly dynamic, suggesting that it is not a structural component of the kinetochore. Consistent with this, all other kinetochore proteins tested do not require RAMA1 for their kinetochore localization. Taken together, these results identify RAMA1 as a novel kinetochore protein and suggest that RAMA1 may have a direct role in mediating kinetochore-microtubule interactions.

Nuclear envelope-associated dynein drives prophase centrosome separation and enables Eg5-independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.

Function and regulation of dynein in mitotic chromosome segregation.

Cytoplasmic dynein is a large minus-end-directed microtubule motor complex, involved in many different cellular processes including intracellular trafficking, organelle positioning, and microtubule organization. Furthermore, dynein plays essential roles during cell division where it is implicated in multiple processes including centrosome separation, chromosome movements, spindle organization, spindle positioning, and mitotic checkpoint silencing. How is a single motor able to fulfill this large array of functions and how are these activities temporally and spatially regulated? The answer lies in the unique composition of the dynein motor and in the interactions it makes with multiple regulatory proteins that define the time and place where dynein becomes active. Here, we will focus on the different mitotic processes that dynein is involved in, and how its regulatory proteins act to support dynein. Although dynein is highly conserved amongst eukaryotes (with the exception of plants), there is significant variability in the cellular processes that depend on dynein in different species. In this review, we concentrate on the functions of cytoplasmic dynein in mammals but will also refer to data obtained in other model organisms that have contributed to our understanding of dynein function in higher eukaryotes.

p53 Prohibits Propagation of Chromosome Segregation Errors that Produce Structural Aneuploidies

The presence of an abnormal karyotype has been shown to be profoundly detrimental at the cellular and organismal levels but is an overt hallmark of cancer. Aneuploidy can lead to p53 activation and thereby prevents proliferation, but the exact trigger for p53 activation has remained controversial. Here, we have used a system to induce aneuploidy in untransformed human cells to explore how cells deal with different segregation errors. We show that p53 is activated only in a subset of the cells with altered chromosome content. Importantly, we find that at least a subset of whole-chromosome aneuploidies can be propagated in p53-proficient cells, indicating that aneuploidy does not always lead to activation of p53. Finally, we demonstrate that propagation of structural aneuploidies (gain or loss of part of a chromosome) induced by segregation errors is limited to p53-deficient cells.

Astral microtubules control redistribution of dynein at the cell cortex to facilitate spindle positioning.

Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pulling forces on astral microtubules to position the mitotic spindle. Recent work has shown that dynein displays a dynamic asymmetric cortical localization, and that dynein recruitment is negatively regulated by spindle pole-proximity. This results in oscillating dynein recruitment to opposite sides of the cortex to center the mitotic spindle. However, although the centrosome-derived signal that promotes displacement of dynein has been identified, it is currently unknown how dynein is re-recruited to the cortex once it has been displaced. Here we show that re-recruitment of cortical dynein requires astral microtubules. We find that microtubules are necessary for the sustained localized enrichment of dynein at the cortex. Furthermore, we show that stabilization of astral microtubules causes spindle misorientation, followed by mispositioning of dynein at the cortex. Thus, our results demonstrate the importance of astral microtubules in the dynamic regulation of cortical dynein recruitment in mitosis.

BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised

The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment.

Nuclear envelope-associated dynein cooperates with Eg5 to drive prophase centrosome separation

Eg5 (kinesin-5) is a highly conserved microtubule motor protein, essential for centrosome separation and bipolar spindle assembly in human cells. Using an “in vitro” evolution approach, we generated human cancer cells that can grow in the complete absence of Eg5 activity. Characterization of these Eg5-independent cells (EICs) led to the identification of a novel pathway for prophase centrosome separation, which depends on nuclear envelope (NE)-associated dynein. Here, we discuss our recent findings and elaborate on the mechanism by which dynein drives centrosome separation.

Chromosome misalignments induce spindle‐positioning defects

Cortical pulling forces on astral microtubules are essential to position the spindle. These forces are generated by cortical dynein, a minus‐end directed motor. Previously, another dynein regulator termed Spindly was proposed to regulate dynein‐dependent spindle positioning. However, the mechanism of how Spindly regulates spindle positioning has remained elusive. Here, we find that the misalignment of chromosomes caused by Spindly depletion is directly provoking spindle misorientation. Chromosome misalignments induced by CLIP‐170 or CENP‐E depletion or by noscapine treatment are similarly accompanied by severe spindle‐positioning defects. We find that cortical LGN is actively displaced from the cortex when misaligned chromosomes are in close proximity. Preventing the KT recruitment of Plk1 by the depletion of PBIP1 rescues cortical LGN enrichment near misaligned chromosomes and re‐establishes proper spindle orientation. Hence, KT‐enriched Plk1 is responsible for the negative regulation of cortical LGN localization. In summary, we uncovered a compelling molecular link between chromosome alignment and spindle orientation defects, both of which are implicated in tumorigenesis.