In this study, we show that during late anaphase, the nucleus of budding yeast is extensively compartmentalized. Unlike in early stages, during which exchange between mother and bud is not limited in nucleoplasmic compartments, all nuclear spaces become strongly compartmentalized in late mitosis, when the nucleus adopts a dumbbell-like shape. This compartmentalization has clear functional consequences for the cell. By limiting the exchange of the transcription factor Ace2 between mother and bud parts of the nucleus, compartmentalization of the nucleoplasm supports the establishment of Ace2 asymmetry and promotes its inheritance by the daughter cell. Thus, when Ace2 asymmetry is important, nucleoplasmic compartmentalization is likely to confer a selectable advantage to the cell. In other words, mechanisms that ensure compartmentalization of the nucleoplasm could have emerged at least partially in coevolution with Ace2 asymmetry.
Using a combination of experimental and simulated FLIP data, together with mutants affecting spindle organization and nuclear morphology, we could distinguish between the contribution of geometry and the permeability of potential diffusion barriers to nuclear compartmentalization. These experiments establish the following three points.
First, compartmentalization of the nucleoplasm is well explained by the geometry of the nucleus alone. Accordingly, parameters such as the length and width of the bridge connecting the two future daughter nuclei determine the °CP.
Second, compartmentalization of nucleoplasmic proteins is increased for those that are able to bind DNA even with low affinity, such as TetR. This twofold increase in compartmentalization is likely to be mediated by three convergent processes: the separation of the segregating chromosomes into two disjoint masses, the reduction of the diffusion speed of the protein, and its increased retention in the nucleus, which limits exchange via the cytoplasm.
Third, in contrast to what is observed in the nucleoplasm, the compartmentalization of the nuclear membranes cannot be explained by their geometry alone. Already in early anaphase, the dynamics of markers in both the INM and ONM are best explained by the presence of a barrier, which limits lateral diffusion around the spindle midzone, in addition to geometry. Accordingly, in both membranes, the length of the internuclear bridge only modestly affected compartmentalization. Most remarkably, the width of the bridge, which had a strong impact on the compartmentalization of the nucleoplasm, made no clear contribution to the compartmentalization of the INM and ONM.
In a previous study, we found that specialized membrane structures are present in the cortical ER at the bud neck (Luedeke et al., 2005
). These structures contribute to the formation of lateral diffusion barriers at the mother–bud junction. Further studies will be needed to investigate whether membrane compartmentalization in the nuclear envelope follows the same rules. If this is the case, it will be interesting to investigate whether compartmentalizing structures are primarily dedicated to other functions, compartmentalization being a secondary consequence, or whether they are primarily dedicated to barrier formation. For example, functionally specializing the nuclear membranes in the connecting bridge of late anaphase nuclei might be more relevant for spindle stability, chromosome segregation, or karyofission rather than compartmentalization. However, the observation that the passive compartmentalization of the nucleoplasm plays an important role in promoting the asymmetry of cell division suggests that nuclear compartmentalization in its own right is an important function of the interconnecting bridge. Also, the observation that the bud6Δ
mutation, which does not appear to affect nuclear morphology, enhanced the exchange between the mother and bud part of the ONM in early, but not in late, anaphase suggests that several independent mechanisms contribute to ONM compartmentalization. Thus, compartmentalization of this membrane appears to be a tightly controlled process.
Altogether our data support the notion that compartmentalization of the nucleus is an important aspect of the process of closed mitosis, most probably because it serves as a support for the asymmetric segregation of nuclear components, such as daughter-specific transcription factors and nonchromosomal DNA (Shcheprova et al., 2008
). A corollary of this conclusion is that the dumbbell shape of the nucleus is not circumstantial but the result of a complex evolution leading to the adaptation of the process of nuclear division to biologically relevant constraints, beyond karyofission.
Accordingly, it is interesting to note that the morphological events associated with nuclear division are highly variable in fungi. For example, the dividing nucleus of the fission yeast Schizosaccharomyces japonicus
does not develop the dumbbell morphology typical of S. cerevisiae
and Schizosaccharomyces pombe
and undergoes semi-open mitosis instead (Aoki et al., 2011
; Yam et al., 2011
). Hence, it is very likely that this organism, which apparently divides highly symmetrically, shows no compartmentalization of the nucleoplasm. It will be interesting to determine whether it does establish barriers in the nuclear envelope. Investigating the biological consequences of the different forms of nuclear division will shed light on the origins and roles of compartmentalization mechanisms.
From that perspective, gaining a molecular understanding of the mechanisms controlling the morphology and compartmentalization of the dividing yeast nucleus is likely to provide also important information about the process of mitosis in a general sense. Little is known about how the nuclear envelope is shaped and how this process is controlled as the spindle elongates and the nucleus acquires its dumbbell morphology. Understanding the contribution of Ase1, Cin8, and Slk19 in this process will provide insights about how microtubules of the spindle midzone interact with the nuclear membrane in fungi as well as how the same structure interacts with the plasma membrane during the cytokinesis of animal cells. However, clearly Ase1 and the spindle midzone are not the only factors involved in shaping the anaphase nucleus. Further studies will be required to investigate the role of the other components of the nucleus, such as chromatin, and to identify the machineries involved in membrane dynamics, curvature, and cleavage during the highly sophisticated process of nuclear division.