Chromosome Segregation and Aneuploidy series
Rod–Zw10–Zwilch: a key player in the spindle checkpoint

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The spindle checkpoint assures the proper segregation of chromosomes during mitosis. The best-characterized components of the checkpoint were originally identified in budding yeast. But three proteins with no yeast homologs – Rod, Zw10 and Zwilch – also play a crucial, but poorly understood, role in the metazoan spindle checkpoint. Recent work has begun to reveal the function of these proteins. The three form a complex (the RZZ complex), which is required for the recruitment of two better-known components of the kinetochore – the dynein–dynactin complex, and Mad1–Mad2. It has now been established that RZZ is directly or indirectly responsible for both Mad1–Mad2 recruitment to unattached kinetochores and its subsequent shedding from kinetochores following MT attachment, and thus is involved in both the activation and inactivation of the checkpoint. This review (which is part of the Chromosome Segregation and Aneuploidy series) covers recent developments in our understanding of RZZ dynamics and its function in the checkpoint.

Introduction

In the beginning, there were six: Bub1, Bub3, Mad1, Mad2, Mad3 and Mps1. These proteins of budding yeast are all required to delay the metaphase–anaphase transition in response to defects in spindle assembly, and their homologs in metazoan cells are also essential for proper regulation of the onset of anaphase. However in higher eukaryotes, several other proteins have been identified that also carry out essential aspects of the spindle checkpoint, suggesting that, in metazoan cells, the mechanism is more elaborate. Among these additional components are Rough Deal (Rod), Zeste-white 10 (Zw10) and Zwilch, the subjects of this review. They have been shown to function as a unit, and indeed can be isolated in a stable complex, called here ‘RZZ’. They have no obvious homologs among yeast proteins and no recognizable protein motif that might provide a hint as to their function. But they are essential to the checkpoint mechanism, and, like other components of the spindle checkpoint, mutations in their encoding genes are relatively frequent in colorectal cancers [1]. In the past few years, significant progress has been made in understanding their function, primarily from genetic and cell-biological studies in Drosophila, where they were first identified, but also from studies in vertebrate cells.

Section snippets

Recap of the checkpoint

The metaphase checkpoint (also called the spindle assembly checkpoint) is a surveillance system that delays anaphase onset and mitotic exit until all the chromosomes are ‘properly’ connected to the spindle by means of a specialized multiprotein complex called the kinetochore (Box 1). Normally, a cell with even a single unattached kinetochore cannot enter anaphase. Should the checkpoint fail, the ensuing anaphase would likely produce two aneuploid daughters, as the unattached chromatid would not

Identification of the RZZ complex

The genes zeste-white 10 (zw10) and rough deal (rod), originally identified in Drosophila, are conserved among multicellular eukaryotes 10, 11, 12, 13, 14. Null mutations in either gene provoke similar chromosome segregation defects: lagging chromatids, nondisjunction and anaphase bridges, leading to significant levels of aneuploidy in both mitotic and meiotic cells. When the homologs of Rod and Zw10 in Caenorhabditis elegans and vertebrate cells are depleted by RNAi or by antibody injection,

RZZ dynamics

The behavior of RZZ has been examined by immunostaining Drosophila spermatocytes and neuroblasts and in HeLa cells. More recently GFP–Rod has been followed in living Drosophila cells 20, 21. The basic description of RZZ dynamics is the same across tissues and species. During interphase, Rod Zw10 and Zwilch are cytoplasmic 11, 21, although it is not known whether they are already associated in a complex. In late prophase and during nuclear envelope breakdown, they enter the nucleus and begin

Recruitment of RZZ to kinetochores

What factors are required for RZZ recruitment to the kinetochore? The kinetochore recruitment of many checkpoint proteins is interdependent, whereas several studies in vertebrate cells and in Drosophila demonstrate that RZZ recruitment does not depend on Bub3, BubR1 [27], Mps1 [28], Cenp-I [29], Cenp-F [30], Mad2 (E. Buffin and R. Karess, unpublished) or CenpMeta (the fly homolog of CenpE) [19].

A novel protein called Zwint-1 was identified by yeast two-hybrid screening of human Zw10 [31]. This

The function of RZZ: recruiting dynein–dynactin

The identification of p50 dynamitin as a two-hybrid partner of Zw10 led to the important discovery that the RZZ complex is directly required for the recruitment of cytoplasmic dynein and dynactin to kinetochores [34]. The whole RZZ complex is required, as mutations in rod or zwilch abolish dynein recruitment even though Zw10 is still present in the cell 19, 34. Significantly, in such mutants, dynein is still found at spindle poles and other subcellular locations, demonstrating that RZZ is

The function of RZZ: recruiting Mad1–Mad2

So if it is not through dynein, how then does RZZ contribute to a functional spindle checkpoint? Very recent work has at last shed some light on this question. Two groups have now shown that depleting Rod or Zw10, either by siRNA, immunodepletion from extracts or with classical mutations, blocks Mad1 and Mad2 recruitment to unattached kinetochores 17, 20. These observations provide the first plausible explanation for the checkpoint defect in rod and zw10 mutants: without the Mad1–Mad2 complex

Functions beyond the kinetochore?

As so often occurs in experimental biology, the ‘specificity’ of a protein changes as a function of our knowledge. This appears to be the case for Zw10. Forgotten in the literature is evidence that Zw10 might participate in some cellular functions independently of Rod. zw10 mutant spermatocytes occasionally fail to undergo cytokinesis [22], a phenotype never seen in rod mutants. The fact that a small fraction of Zw10 (but not Rod) can be seen in the midbody of dividing cells, both in fly

Conclusions and perspectives

The big questions regarding the RZZ complex in the spindle checkpoint are beginning to be answered. It is now clear that RZZ is intimately involved with Mad1–Mad2. They are recruited together to kinetochores, and they are shed and migrate together along the KMTs; without RZZ, Mad1–Mad2 does not accumulate on unattached kinetochores, and presumably cannot generate the anaphase inhibitor. We are thus now in a position to predict that the mitotic phenotype of rod, zw10 and zwilch mutants should

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